WELCOME MESSAGE

3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
WELCOME MESSAGE
Dear Colleagues:
Welcome to the 3rd International Conference on Paraoxonases (ICP). Basic and clinical research
on the Paraoxonase (PON) family of proteins (PON1, PON2 and PON3) has increased
exponentially over the last five years following the initiation of paraoxonase conferences. We are
honored and pleased to host the 3rd ICP at the University of California Los Angeles, located in
beautiful, sunny Southern California. Based on recent publications and communications to this
conference, it is clear that PON proteins play important roles in inflammation, infection, and
toxicology. We sincerely hope that the 3rd ICP will help disseminate the latest aspects of
research on the genetics, biochemistry, structural biology, and regulation of PON genes. The
program includes expert lectures from 21 invited presentations, 20 short oral presentations, and
between 30-40 poster presentations. We are positive that the 3rd ICP will provide an excellent
environment for cross-fertilization of ideas among all the researchers in the field and help pave
road for translational research in the fight against inflammatory diseases, infectious diseases and
toxins.
~ Organizing Committee
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
SCIENTIFIC COMMITTEE
Michael Aviram, Israel
Jordi Camps, Spain
Lucio G. Costas, USA
Dragomir Draganov, USA
Clement E. Furlong, USA
Richard W. James, Switzerland
David Lenz, USA
Bharti Mackness, UK
Mike Mackness, UK
Mohamad Navab, USA
Srinu Reddy, USA
Ildiko Seres, Hungary
Diana Shih, USA
Dan S. Tawfik, Israel
LOCAL ORGANIZING COMMITTEE
Srinu Reddy
Diana Shih
Sumiji Takahashi
Tan Duong
Betty Morgan
CONTACT INFORMATION
Sumiji Takahashi
Email: [email protected]
Phone: 310-825-5280
Fax: 310-206-9133
Website: http://www.cardiology.med.ucla.edu/paraoxonases
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
SPONSORS AND EXHIBITORS
Sponsors:
Bristol-Myers Squibb
A pharmaceutical company whose mission is
to extend and enhance human life by developing
innovative medicines and health care.
Department of Medicine
David Geffen School of Medicine
University of California
Los Angeles
Division of Cardiology
Department of Medicine
David Geffen School of Medicine
University of California
Los Angeles
Defense Threat Reduction Agency
Consolidates a variety of US Defense
Department functions to deal more effectively
with the threats posed by nuclear,
chemical or biological weapons.
Exhibitors:
MEGA TIP IND.TRD.CO.LTD
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Los Angeles City Tour
Tuesday, September 9, 2008
"The first stop on the Los Angeles City Tour is Hollywood, world capital of the movie
industry: You will ride down Hollywood Boulevard (Walk of Fame), stopping at the legendary
Mann's Chinese Theatre, home of footprints and handprints of Movie Stars like Marilyn
Monroe, John Wayne, Liz Taylor and many more. Cruise Sunset Boulevard where the TV hit
"77 Sunset Strip" was filmed. Visit Beverly Hills and see the exclusive shops and boutiques on
Rodeo Drive, where Julia Roberts went shopping in the film "Pretty Woman". Then it is on to
Downtown Los Angeles where the first Highlight is a breathtaking panoramic view of its ultramodern skyscrapers. Visit the Music Center with The Dorothy Chandler Pavilion former
home of the Academy Awards (Oscars) and The Walt Disney Concert Hall, continuing through
L.A. Civic Center, Los Angeles Cathedral and Stopping at Olvera Street, the birthplace of
Los Angeles. Here you will browse through many shops, in a happy Mexican atmosphere. Finally
stop for shopping at the world famous Universal City Walk"
(Sign up at registration desk)
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3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Program
7th September 2008, Sunday
1300-1445
Registration
1445-1500
Srinu Reddy: Welcome Address
OPENING SESSION
Moderators – Mike Mackness and Bharti Mackness
1500-1530
Professor Alan M. Fogelman: The Bert N La Du Memorial Lecture – “An
approach to dysfunctional HDL”.
1530-1600
Clement Furlong: “Engineering Human Paraoxonase 1 (PON1) in an E. coli
Expression System for Therapeutic Applications”.
1600-1615
Hagai Tavori - Travelling Fellowship Presentation -- “Human carotid
atherosclerotic lesion promotes oxidation of macrophages and LDL”.
1615-1630
Birsen Can Demirdögen - Travelling Fellowship Presentation -- “PON1 -107TT
genotype is a risk factor and QRLMTC combined haplotype is protective for
ischemic stroke”.
1630-1645
Michal Efrat - Travelling Fellowship Presentation -- Di-Oleoyl Phosphatidylcholine
(PC-18: 1) Stimulates Paraoxonase 1 (PON1) Enzymatic and Biological Activities: In
vitro and in vivo studies
1645-1800
Reception
8th September 2008, Monday
PON STRUCTURE, FUNCTION AND CARDIOVASCULAR DISEASE
Moderators – Richard James and Dan Tawfik
0900-0930
Dan Tawfik: “Tinkering with PONs”.
0930-1000
Stanley Hazen: “Structural and Functional Studies of Nascent HDL and the HDL PON1 Complex”.
1000-1015
Thomas J. Magliery: “Engineering PON1 for bacterial expression, solubility and
drug-like properties”.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
1015-1030
Toby T. Sanan: “Molecular dynamics and binding studies on human paraoxonase 1:
in silico optimization of activity against chemical warfare agents”.
1030-1100
Coffee Break
1100-1130
Richard James: “HDL and PON1: mutual benefits from an intimate association”.
1130-1200
Mickey Aviram: “Paraoxonase 1 (PON1) and Diabetes”.
1200-1215
Philip W. Connelly: "Paraoxonase-1 mass and renal function in type 2 diabetes".
1215-1230
G.M. Anantharamaiah: “The ApoE mimetic peptide Ac-hE-18A-NH2 reduced
plasma lipid hydroperoxide content with a concomitant increase in HDL
paraoxanase activity and improves endothelial function”.
1230-1400
Lunch
PON STRUCTURE, FUNCTION AND CARDIOVASCULAR DISEASE
Moderators – Mickey Aviram and Mohamad Navab
1400-1430
Sampath Parthasarathy: “Induction of PON1 and ApoA-1 gene expression by
aspirin”.
1430-1500
Mohamad Navab: “Effect of ApoA-I mimetic peptides on PON1 activity in
atherosclerosis”.
1500-1530
Hieronim Jakubowski: “The role of paraoxonase 1 in the detoxification of
homocysteine-thiolactone”.
1530-1600
Coffee & Posters
1600-1630
Anatol Kontush: “Is paraoxonase 1 critical for antioxidative and antiinflammatory
activity of HDL?”
1630-1700
Sara Deakin: “Studies of the nature of the PON1-HDL association”.
1700-1715
György Paragh: “The human paraoxonase-1 phenotype modifies the effect of
statins on paraoxonase activity and lipid parameters”. (Presented by Ildiko Seres)
1715-1730
Daniel Rochu: “Parameters influencing stability and activity of human pon1: how
molecular environment/physiological location impact the promiscuity/moonlighting
of this enzyme”.
1745-2000
Conference Dinner (featuring special performance by Ajay Narasimha and
Avinash Anantharamaiah)
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
9th September, 2008, Tuesday
PON IN INFLAMMATION, IMMUNITY AND OTHER DISEASE
Moderators – Dragomir Draganov and Jordi Camps
0900-0930
Joseph Zabner: “PON and quorum sensing”.
0930-1000
Kim Janda: “New approaches to bacterial quorum quenching: from small molecules
to antibodies”.
1000-1015
Coffee and Poster
1015-1030
David Stoltz: “Transgenic expression of paraoxonase-1 in Drosophila protects from
Pseudomonas aeruginosa lethality”.
1030-1045
Bianca Fuhrman: “Macrophage paraoxonase 2 (pon2) expression is upregulated by
urokinase plasminogen activator (upa) in a redox-dependent pathway”.
1045-1100
Nicola Martinelli: “Novel serum paraoxonase activity assays are associated with
coronary artery disease”.
1100-1115
Noam Bourquard: “Insulin resistance in paraoxonase 2 deficient male mice”.
1115-1130
Break
1130-1145
Bełtowski J: “Liver x receptor agonist, to 901317, normalizes plasma PON1 activity
and reduces protein homocysteinylation in rats with experimental hyperleptinemia”.
1145-1200
Sebastian Altenhöfer: “Paraoxonase-2 reduces intracellular superoxide levels
independent of its lactonase activity”.
1200-1215
Ozcan Erel: “Fully automated determination of arylesterase, paraoxonase activities
and PON1 192Q/R phenotype”.
1215-1330
Lunch
1330
Site seeing (Tour of Hollywood, Beverly Hills, Los Angeles and Universal
City Walk)
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
10th September 2008, Wednesday
PON IN INFLAMMATION, IMMUNITY AND OTHER DISEASE
Moderators – Clement Furlong and Ildiko Seres
0900-0930
Dragomir Draganov: “Decreased serum paraoxonase (PON1) and apolipoprotein
A-I (apoA-I) levels in critically ill patients with sepsis: determinants or markers for
the severity and outcome”.
0930-1000
Jordi Camps: “Interrelationships between PON-1 and MCP-1 in the regulation of
hepatic inflammation”.
1000-1015
Ildiko Seres: “Alteration of PON1 activity in childhood obesity and its relation to
leptin and adiponectin levels”.
1015-1030
Lita A. Freeman: “PON1 and HDL in sickle cell disease”.
1030-1100
Coffee & Posters
PON AND TOXICOLOGY
Moderators – David Lenz and David Cerasoli
1100-1130
Antonio Hernandez-Jerez: “Interaction between Paraoxonase-1 and environmental
chemicals and its impact on health”.
1130-1200
Toby Cole: “Role of Paraoxonase (PON1) in Organophosphate Metabolisms and
Toxicity”.
1200-1215
Tamara C. Otto: “Progress in using recombinant forms of paraoxonase 1 to
develop a catalytic bioscavenger of nerve agents”.
1215-1230
JN Hofmann: “Biomarkers of sensitivity and exposure in Washington State
pesticide handlers”.
1230-1400
Lunch
PON2 AND PON3
Moderators – Srinu Reddy and Diana Shih
1400-1430
Mira Rosenblat: “Paraoxonase 2 (PON2) attenuates triglyceride biosynthesis and
accumulation in macrophages via inhibition of diacylglycerol acyltransferase 1
(DGAT1)”.
1430-1500
Diana Shih: “Increased susceptibility to diet-induced atherosclerosis and cholestasis
in PON3KO mice”.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
1500-1530
Srinu Reddy: “Role of PON2 in atherosclerosis and inflammation”.
1530-1545
Sven Horke: “Oppositional regulation of paraoxonase-2 expression by endoplasmic
reticulum stress and disturbance of calcium-homeostasis”.
1545-1600
Judit Marsillach: “Immunohistochemical analysis of paraoxonases-1, 2, and 3 in
human atheroma plaques”.
End of Program
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
POSTERS
Atherosclerosis
P1
Bayrak, Ahmet
P2
Bourquard, Noam
P3
Gugliucci, Alejandro
P4
Gupta, Nidhi
P5
P6
Kotur-Stevuljevic, Jelena
Marchegiani, Francesca
P7
Martinelli, Nicola
P8
Shih, Diana
Paraoxonase-1 gene Q192R polymorphism is not related to
the extent of atherosclerosis
Paraoxonase 2 deficiency aggravates atherosclerosis and
affects lipid metabolism in apoE null mice
Hypochlorous acid (HOCl) is a potent inactivator of human
proteins responsible for protection against thrombosis and
LDL oxidation: a comparative study on plasminogen and
paraoxonase 1
Study of serum paraoxonase (PON1) activity in coronary
artery disease patients with & without type 2 diabetes
mellitus in North Indians
PON1 activity decreasing in stroke patients
Relationship between paraoxonase2 C311S polymorphism
and acute coronary syndrome
PON1 activities in metabolic syndrome and risk of
coronary artery disease
The Pseudomonas autoinducer N-3-oxododecanoyl
homoserine lactone induces inflammatory response in the
human aortic endothelial cells
Biochemistry
P9
Bacchetti, Tiziana
P10 Ben-David, Moshe
P11 Camps, Jordi
P12 Camps, Jordi
P13 Cerasoli, Douglas
P14 Devarajan, Asokan
P15 Elias, Mikael
P16 Hugenmatter, Adrian
Paroxonase activity in high density lipoproteins
(HDL):guardian angel of cell membrane?
Structural analysis of rePON1 mutants and complexes
Distribution of the PON1, PON2 and PON3
immunohistochemical expressions in the tissues of normal
mice
Evaluation of a semi-automated method for the
measurement of the serum PON1 lactonase activity.
Influence of genotypes and application to the study of
patients with liver disease
Purification and Characterization of Functional Human
Paraoxonase 1 Expressed in Insect Larvae
Paraoxonase 2 localizes to and modulates function of
mitochondria in response to oxidative stress
Characterization and sequencing of a physiological partner
of HPON1: the Human Phosphate Binding Protein (HPBP)
Phylogeny-based directed Evolution of PON2 and PON3
for increased bacterial expression
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
POSTERS
Biochemistry (continued)
P17 Morrow, Dustin
P18 Muthukrishnan, Siva
P19 Suzuki, Stephanie
P20 Tavori, Hagai
P21 Teiber, John
Comparison of the Effects of Amino Acid Substitutions on
the Stereoselective Hydrolysis of Soman by Human and
Bacterially-Expressed PON1
Structure activity relationship studies towards the
understanding of active site residues in Paraoxonase-1:
interplay of experimental and computational studies
Recombinant Human Paraoxonase 1 (PON1) Engineered in
an E. coli Expression System
Characterization of the PON1 active site using modeling
simulation, in relation to PON1 lactonase activity
Paraoxonases hydrolyze the iodoeicosanoid deltaiodolactone and attenuate its toxicity in cell cultures
Diseases Other Than Atherosclerosis
P22 Bourquard, Noam
P23 Draganov, Dragomir
P24 Dronca, Maria
Role of Paraoxonase 2 in acute gram-negative microbial
infection
PON1 in Murine Cecum Ligation and Puncture (CLP)
Model of Sepsis
Investigation of Paraoxonase-1 Status In Autism Spectrum
Disorders
P25 Fulop, P
Association Between Human Paraoxonase 1 Activity and IntimaMedia Thickness in Subjects Under 55 Years of Age With
Carotid Artery Disease
P26 Gugliucci, Alejandro
Reduced paraoxonase 1 activity in sleep apnea-hypopnea
patients: effects of 6 month treatment with continuous
positive airway pressure treatment. A pilot study
Human PON1 During Development: Effects of Age and
Genetic Variability in a Latino Birth Cohort
The influence of oxidative stress to paraoxonase activity in
stroke patients
HIV-1 Infection Increases Human Paraxonase-2 Activity In
Vivo
PON1 Activity in a Brazilian Population Group of Healthy
and Dislipidemic Adults and Influence of Diet and
Lifestyle
The potential role of Paraoxonase-2 in oxidative stress
induced death
Paraoxonase 1 (PON1) Status as a Risk Factor for Disease
or Exposure
P27 Holland, Nina
P28 Joksic, Jelena
P29 Koka, Prasad
P30 Lima, Jaime de Silva
P31 Parsanejad, Mohammad
P32 Richter, RJ
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
POSTERS
Diseases Other Than Atherosclerosis (continued)
P33 Sözmen, Eser
P34 Sözmen, Eser
P35 Stoltz, David
Possible Influences of BMI, Menopause Period on the
Antioxidant Effects of Raloxifen Treatment in
Postmenopausal Osteoporosis
Red Wine Consumption is More Efficient on Antioxidant
System in Patients with Metabolic Syndrome
Paraoxonase-1 Attenuates Hyperoxia-induced Lethality in
Drosophila
Epidemiology
P36 Quintanilla-Vega, Betzabet
Comparison of PON1 –C108T and Q192R genotypes and
Phenotype in three Mexican mestizo populations
Toxicology
P37 Cole, Toby
P38 Connelly, Phillip
P39 Petrikovics, Ilona
P40 Quintanilla-Vega, Betzabet
Paraoxonase (PON1) Modulates the Toxicity of OP
Mixtures
Mouse serum paraoxonase-1 and ES-1 carboxylesterase
display distinct fatty acid lactonase activity
Optimal Liposomal Composition Determination and
Physicochemical Characterization of Stealth Liposomes
(SL) Encapsulating Organophosphorous Hydrolase (OPH)
Role of some PON1 genetic polymorphisms on semen
quality and sperm DNA integrity in farmers from southern
Mexico exposed to organophosphorous pesticides
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
ABSTRACTS
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
2008 Bert La Du Memorial Lecture
An approach to dysfunctional HDL
Alan M. Fogelman, M.D.
Bert La Du was a coauthor on manuscripts demonstrating that the ability of normal HDL to
inhibit the biologic activity of mildly oxidized LDL was partly due to paraoxonase activity (1).
He also contributed to work demonstrating that the acute phase response results in proinflammatory HDL that in part is due to a loss of paraoxonase activity (2). Patients with CHD, a
condition considered to be a “chronic acute phase response”, were shown to have proinflammatory HDL and statin therapy modestly but significantly improved their HDLinflammatory properties (3). HDL was also pro-inflammatory in other chronic inflammatory
conditions including patients with systemic lupus erythematous and patients with rheumatoid
arthritis (4). HDL inflammatory properties significantly predicted outcomes in patients on
chronic hemodialysis (5). An apoA-I mimetic peptide was shown to improve HDLinflammatory properties in mice (6) and humans (7) suggesting that this might be an approach to
the treatment of dysfunctional HDL.
1. Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M. Protective effect of HDL
associated paraoxonase-inhibition of the biological activity of minimally oxidized low density lipoprotein. J Clin
Invest 1995;96:2882-2891.
2. Van Lenten BJ, Hama SY, de Beer FC, Stafforini DM, McIntyre TM, Prescott SM, La Du BN, Fogelman AM,
Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. J Clin Invest
1995;96:2758-2767.
3. Ansell BJ, Navab M, Hama S, Kamranpour N, Fonarow G, Hough G, Rahmani S, Mottahedeh R, Dave R, Reddy
ST, Fogelman AM. Inflammatory/anti-inflammatory properties of high-density lipoprotein distinguish patients from
control subjects better than high-density lipoprotein cholesterol levels and are favorably affected by simvastatin
treatment. Circulation 2003;108:2751-2756.
4. McMahon M, Grossman J, FitzGerald J, Dahlin-Lee E, Wallace DJ, Thong BY, Badsha H, Kalunian K, Charles
C, Navab M, Fogelman AM, Hahn BH. Proinflammatory high-density lipoprotein as a biomarker for atherosclerosis
in patients with systemic lupus erythematous and rheumatoid arthritis. Arthritis and Rheumatism 2006;54:25412549.
5. Kalantar-Zadeh K, Kopple JD, Kamranpour N, Fogelman AM, Navab M. HDL-inflammatory index correlates
with poor outcome in hemodialysis patients. Kidney International 2007;72:1149-1156.
6. Navab M, Anantharamaiah GM, Reddy ST, Hama S, Hough G, Grijalva VR, Wagner AC, Frank JS, Datta G,
Garber D, Fogelman AM. Oral D-4F causes formation of pre-b high-density lipoprotein and improves high-density
lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null
mice. Circulation 2004; 109:3215-3220.
7. Bloedon LT, Dunbar R, Duffy D, Pinell-Salles P, Norris R, DeGroot BJ, Movva R, Navab M, Fogelman AM,
Rader DJ. Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk
cardiovascular patients. J Lipid Res. 2008; 49:1344-1352.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Engineering Human Paraoxonase 1 (PON1) in an E. coli Expression System
For Therapeutic Applications
CE Furlong, SM Suzuki, RC Stevens, TB Cole, RJ Richter
Depts. of Medicine - Division of Medical Genetics, and Genome Sciences, University of Washington,
Seattle, WA, USA 98195
Human PON1 (HuPON1) is the ideal protein candidate to engineer for treating cases of exposure
to toxic organophosphorus compounds. This protein is of human origin and it catalytically
hydrolyzes many different organophosphorus compounds (OPs) including the insecticide
metabolites chlorpyrifos oxon (CPO), diazoxon (DZO) and paraoxon (PO) as well as the nerve
agents soman, sarin and VX. While native human PON1 hydrolyzes DZO and CPO at rates
sufficient for use as a therapeutic, amino acid substitutions that enhance the catalytic efficiency
of hydrolysis of other OPs will be necessary for using rHuPON1 variants to treat other OP
exposures. It has often been stated that it is not possible to express active human PON1 in an E.
coli system. However, we have been able to express active recombinant HuPON1 in E. coli and
purify the expressed, active rHuPON1. Our rationale for design of the expression/ purification
protocols was to generate native rHuPON1 minus carbohydrate chains and purification tags to
minimize the immunogenicity of the rHuPON1 and to generate rHuPON1 variants with amino
acid substitutions that significantly enhance the catalytic efficiency of hydrolysis of specific OP
compounds. We knew from our earlier work that rabbit PON1, with lysine at position 192,
hydrolyzed CPO and PO much more efficiently than HuPON1 with glutamine or arginine at
position 192. Thus, the first variant that we expressed and purified was HuPON1K192. This
variant had approximately twice the catalytic efficiency of hydrolysis of CPO, DZO and PO.
The expressed rHuPON1K192 variant was purified with good yield, using 7 column
chromatographic steps. It was non-toxic, persistent over time and protected against DZO
exposure when injected into PON1-/- mice. Other rPON1s, rHuPON1Q192, rHuPON1R192 and
rHuPON1N192 have also been expressed and partially characterized.
Supported by: NIEHS ES09883, ES04696, ES09601/EPA RD-83170901.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Human carotid atherosclerotic lesion promotes oxidation
of macrophages and LDL
Hagai Tavori†,‡ Soliman Khatib†, Michael Aviram‡, Jacob Vaya†*,
† Laboratory of Natural Medicinal Compounds, MIGAL - Galilee Technology Center, P.O. Box 831, Kiryat
Shmona 11016, and Tel Hai College, Israel., ‡ Rappaport Family Institute for Research in the Medical Science,
Rambam Medical Center, Haifa 31096, Israel
Early atherosclerotic lesion is consisted of arterial macrophages filled with cholesterol, triglycerides,
phospholipids, and oxidized lipids. Paraoxonase 1 (PON1) is present in the lesion and was shown to
reduce lipid-peroxide content in the atherosclerotic plaque, yet the precise biological substrate for PON1
however has still remains elusive.
The main objective of the study was to investigate the ability of human atherosclerotic carotid plaque to
induce oxidative stress (OS) in macrophages and LDL, as well as to assess the possible anti-atherogenic
properties of PON1 in the reduction such OS induction by the plaques.
Carotid plaques were removed from patients undergoing routine carotid endatherectomy surgery, and
were pulverized under liquid nitrogen and extracted by organic solvents. Atherosclerotic lesion extracts
were incubated for 5-48 hours (at extract weight concentrations ranging from 0.07-1.4 mg/ml) with
J774A1 macrophage cell-line, mouse-peritoneal-macrophages (MPM), native LDL, or with an
exogenous OS-sensitive probes: either linoleoyl-tyrosine (LT), or linoleoyl-tyrosine2’dexoyguanosinolate (LTG).
All human carotid lesions were found to contain oxidized lipids. Upon incubation of the human carotid
plaque EtAc extracts at a concentration of 0.7 mg/ml for 24 h with the macrophages, the cellular lipids
became oxidized to a similar extent as that caused by the free radical generator 2,2'-azobis-2-methylpropan-imidamide,dihydrochloride (AAPH) action on macrophages. The oxidized carotid plaque extracts
were also able to induce LDL oxidation following lesion extract incubation with native LDL. Incubation of
the plaque extract with OS probes resulted in the oxidation of the linoleic acid residue to hydroperoxide
and epoxide, in a dose - and time - dependent manner. Correspondingly, the 2'-deoxyguanosine moiety of
the LTG probe was oxidized by the plaque extract to yield 8-oxo-2'-deoxyguanosine. Upon pre-incubation
of the atherosclerotic lesion extracts with PON1 for 24 hours, the above oxidative effects of the plaque
extracts on macrophages, and on the OS markers, were significantly reduced.
Human carotid lesion extract has the capacity to induce oxidation of the major components of the
atherosclerotic plaque such as macrophages and LDL unsaturated fatty acids. The HDL - associated
paraoxonase 1 has a protective anti - atherosclerotic effects, as a result of its ability to attenuate OS in the
lesion environment.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
PON1 -107TT genotype is a risk factor and QRLMTC combined haplotype is
protective for ischemic stroke
Birsen Can Demirdögen1, Aysun Türkanoglu1, Semai Bek2, Seref Demirkaya2, Orhan Adali1
1
Department of Biochemistry, Institute of Natural and Applied Sciences, Middle East Technical
University, 06531, Ankara, Turkey.
2
Department of Neurology, Gülhane Military Medical Academy, Ankara, Turkey.
Atherosclerosis in the carotid arteries represents a risk for ischemic stroke. PON1 might
have a protective role against the development of ischemic stroke due to its well recognized
function as an antiatherogenic enzyme. In this study, we aimed to determine the importance of
three PON1 genetic polymorphisms (−107T/C, 192Q/R and 55L/M) and two activities
(paraoxonase and arylesterase) as risk factors for ischemic stroke.
The study population was comprised of 172 unrelated adult Caucasian patients with acute
hemispheric ischemic stroke and 105 symptom-free controls. Serum PON1 activities towards
two substrates, paraoxon (paraoxonase activity; PON) and phenyl acetate (arylesterase activity;
ARE) were found to be lower in stroke patients compared to controls. Logistic regression
analysis revealed PON/ARE to be 1.3 times protective against stroke. The frequencies of the
risky alleles −107T, 192R and 55L were increased in the patient group. Frequency of the 55L
allele of PON1 was significantly increased among patients (0.690) compared to controls (0.628;
P=0.003). Logistic regression analysis revealed PON1 55LL genotype to be associated with a
1.8-fold increase in the risk of ischemic stroke versus control status. In addition, 55L allele was
associated with a 1.7 and 2.6 times increased risk of stroke in hypertensives and diabetics,
respectively. The low expressor genotype −107TT was associated with almost 2 times increased
risk for stroke in the elderly population (age >59). Prevalence of triple combined haplotype
QRLMTC was significantly lower in stroke patients (4.1%) when compared to controls (11.4%;
P=0.019). The combined heterozygote haplotype had around 7 times increased protective effect
against stroke in the overall population and 10 times protective effect in the elderly.
Key words: PON1, paraoxonase, arylesterase, stroke, genotypes, polymorphism.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Di-oleoyl Phosphatidylcholine (PC-18:1) Stimulates Paraoxonase 1 (PON1)
Enzymatic and Biological Activities: In vitro and in vivo studies
Michal Efrat1, Mira Rosenblat1, Saeed Mahmood2, Jacob Vaya2 and Michael Aviram1
1
The Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the Medical Sciences and
Rambam Medical Center, Haifa, Israel
2
The Laboratory of Natural Compounds for Medical Use (J.V., S.M.), Migal, Galilee Technological Center, Kiryat Shmona, Israel
Paraoxonase 1 (PON1) is an HDL associated esterase which is known to possess antioxidant and anti-atherogenic properties. It is associated with the HDL via the N-terminal region.
PON1 catalytic activities include paraoxonase, arylesterase and lactonase, and it has been shown
to stimulate HDL-induced macrophage cholesterol efflux (biological activity). As HDL
phospholipids are associated with PON1, we questioned their effect on PON1 catalytic and
biological activities.
Addition of different phosphatidylcholine (PC) containing different fatty acids (18:1,
18:2 or 14:0) to recombinant evolved PON1 (rePON1) demonstrated that dioleoyl
Phosphatidylcholine (PC-18:1) had the best stimulatory effect on PON1 catalytic activities.
We demonstrated that HDL isolated from serum enriched with PC-18:1 resulted in
significantly higher PC-18:1 levels and PON1 catalytic activities lactonase, arylesterase and
paraoxonase were increased by 20%,100% and 100% respectively, as compared to HDL from
control non-enriched serum. In addition, PON1 contribution to the HDL mediated cholesterol
efflux from J774A1 macrophages was higher in HDL enriched with PC-18:1, as compared to
control HDL.
In vivo, consumption of olive oil by 3 healthy subjects for 14d prior to HDL isolation
was shown to increase: HDL PC-18:1 levels (15%, 20% and 200%), PON1 ability to stimulate
HDL-mediated cholesterol efflux from macrophages and PON1 catalytic activities.
Upon using recombinant PON1 mutant that lacks the 20 amino acids at the N-terminal
region (Δ20-PON1) we demonstrated that PC-18:1 stimulatory effect on PON1 mutant catalytic
activities was decreased in comparison to PC-18:1 effect on wild type PON1, suggesting that the
N-terminal region is involved in PON1 stimulation by PC-18:1.
In conclusion, intervention to increase PON1 activities by HDL enrichment with PC-18:1
(including olive oil consumption) could be proven as a beneficial anti-atherogenic therapy.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Tinkering with PONs
Dan S. Tawfik
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76 100, Israel;
[email protected]
I will provide a brief review of our current understanding of PON1 at the structural and
biochemical level. I will then describe our ongoing efforts to engineer recombinant PON variants
that exhibit new and improved functions in the context of organophosphate detoxification and
treatment of aretriosclerois. These include PON1 variants exhibiting far higher stability than
human PON1 under a range of stress-related conditions, PON1 variants with improved
thiolactonase activities, and PON1 variants that target a range of toxic organophosphates (with a
reference to a lecture by Dr. Rinkoo D. Gupta). Finally, I will describe the engineering of new
recombinant PON2 and PON3 variants, and their preliminary analysis.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Structural and Functional Studies of Nascent HDL and
the HDL - PON1 Complex
Zhiping Wu1,2 , Shengqiang Gao1,2, Valentin Gogonea1,4, Xavier Lee1,2, Matthew A. Wagner4,
Xin-Min Li1,2, Joe Zaccai5, Roland P. May5, Michael Haertlein5, Martine Moulin5, Irina Gutsch6,
Dimitri Svergun7, Maxim Petouchov7, Joseph DiDonato1,2, Stanley L. Hazen1, 2, 3
Departments of 1 Cell Biology, 2 Center for Cardiovascular Diagnostics and Prevention, and 3Cardiovascular
Medicine, Cleveland Clinic, Cleveland, OH 44195
4
Department of Chemistry, Cleveland State University, Cleveland, OH 44115
5
Institut Laue-Langevin, 6, rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 9, France
6
EMBL, 6, rue Jules Horowitz, BP 156, 38042 Grenoble Cedex 9, France
7
EMBL Hamburg c/o DESY, Notkestrasse 85, 22603 Hamburg Germany
Recent data from human clinical studies confirms that the high density lipoprotein (HDL)
associated protein paraoxonase 1 (PON1) is genetically and mechanistically linked to oxidative
stress and the development of cardiovascular disease. Remarkably, both HDL and its associated
complexes, such as with PON1, have remained resistant to structural interrogation by traditional
high-resolution approaches. Using an integrative multimodal approach permitting for the first
time direct visualization of protein and lipid components individually within HDL, we
demonstrate that apolipoprotein A1 (apoA1) of nascent HDL exists as an anti-parallel double
helix. This surprising structure was obtained using a combination of contrast matching small
angle neutron scattering (SANS), isotope labeling with deuterium of apoA1, hydrogen-deuterium
exchange tandem mass spectrometry (HD-MS/MS) and novel computational tools. Direct
visualization of the lipid phase of nascent HDL shows a complex prolate ellipse that
interdigitates with the protein helix. 31P NMR and modeling suggest phospholipid adopts
micellar and pseudo-lamellar mesophase structures partially bounded by the hydrophobic
lipoprotein surface, filling the grove created by the open helical conformation of apoA1. The
remarkable protein and lipid structures observed accommodate SANS scattering data at multiple
D2O levels, apoA1 HD-MS/MS data, and reported distance constraints based upon chemical
cross-linking and resonance energy transfer studies. Subsequent structural studies of the HDLPON1 complex involving multiple distinct approaches identify sites on apoA1 of nascent HDL
as well as on PON1 that are involved in protein-protein, and protein - lipid interaction within the
HDL - PON1 complex. Site specific mutatgenesis studies of both apoA1 and PON1 reveal the
functional importance of these contact sites for preservation of PON1 activity and HDL
interactions. The present results have important implications for HDL particle genesis,
maturation/remodeling and PON1 interactions. They reveal HDL to be a highly dynamic and
adaptive structure capable of accommodating changes in particle shape, composition and
associated protein complex formation during HDL assembly, maturation, remodeling and PON1
complex formation.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Engineering PON1 for bacterial expression, solubility and
drug-like properties
Vivekanand Shete, Mohosin Sarkar, George Matic and Thomas J. Magliery*
Departments of Chemistry and Biochemistry, The Ohio State University, 100 W. 18th Ave., Columbus, OH 43210
*presenting author; [email protected]; phone +1 (614) 247-8425; fax +1 (614) 292-1685
Paraoxonase-1 (PON1) is a serum enzyme with native hydrolytic activity against toxic
organophosphorous agents. Consequently, PON1 has potential as a therapeutic agent against
pesticide and nerve agent poisoning. However, when expressed in E. coli, human PON1 is
insoluble and inactive, and lacks N-linked glycosylation present in human serum. Moreover,
three cysteines, two of which form a disulfide bond, complicate storage of the protein and may
lead to disulfide scrambling during folding. We sought to improve the drug-like properties of
human PON1 by removing or minimizing the number of cysteines, introducing surface cysteines
for chemical modification to mimic glycosylation, and generating rational and random mutants
with improved solubility but high homology to the human sequence. Working with the E. coli
expressible G2E6 variant of Tawfik and colleagues, we have found that mutation of the cysteines
has only a moderate effect on paraoxon hydrolytic activity, and in fact that some mutants that
remove the disulfide bond have similar or increased activity compared to wild-type. Mutation of
the two surface asparagine residues, believed to be glycosylated in the human form, to cysteine
causes a modest decrease in activity in G2E6 but provides a unique reactive handle for chemical
modification to mimic glycosylation. Fusion of G2E6 and human PON1 N-terminal to green
fluorescent protein accurately reports solubility by cellular fluorescence. Rational mutants of
human PON1 at the putative HDL binding site and at a subset of G2E6 mutation sites, and
random mutants of an N-terminal deletion mutant, have been shown to have increased solubility.
The prospects for integrating these kinds of modifications into an optimized therapeutic agent
will be discussed.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Molecular dynamics and binding studies on human paraoxonase 1: in silico
optimization of activity against chemical warfare agents
Toby T. Sanan, Peng Tao, Siva Muthukrishnan, Carrigan J. Hayes, Christopher M. Hadad*
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH 43210
[email protected], FAX: (614) 292-1685
Human Paraoxonase I (huPON1) is an endogenous human protein which has been identified as
having the ability to catalytically hydrolyze organophosphorous nerve agents, as well as a
number of other substrates including aryl esters and lactones. At present, the mechanism of
action of the enzyme is still under debate, and the majority of the available structural information
comes from the crystal structure of a gene-shuffled protein with 86% sequence homology with
the human enzyme, but with no substrate or inhibitor bound in the active site. Computational
models have been prepared for both the human wild-type enzyme, as well as the gene-shuffled
variant, and extensive (20 ns) molecular dynamics (MD) simulations were performed for these
models. Mutant proteins were prepared in silico to rationalize experimental mutagenesis studies,
and these models were also relaxed using MD techniques. Molecular docking simulations have
been performed to explore the nature of substrate binding with a variety of substrates, and critical
residues for binding and reactivity have been identified. Hybrid QM/MM simulations were
performed to analyze potential reaction mechanisms in conjunction with the binding studies.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
HDL and PON1; mutual benefits from an intimate association
Richard W. James, Department of Internal Medicine, Medical Faculty, University of Geneva,
Geneva, Switzerland.
Paraoxonase-1 (PON1) displays a marked preference for binding to HDL, and both
derive benefit from this union. The enzyme is proposed to be an important determinant of the
anti-oxidant capacity of HDL, whilst the latter provide a serum transport vector for the peptide,
concomitantly shielding its retained signal sequence from the aqueous milieu. A full description
of the nature of this interaction will allow not only a better understanding of the anti-oxidant
function of HDL, but should define the optimal conditions that allow PON1 to function. In
addition, it may indicate how pathophysiological modifications to HDL metabolism impact on
PON1 activity and its anti-oxidant capacity.
Three aspects of HDL structure merit attention with respect to its influence on PON1.
The lipid component, notably phospholipids, promotes PON1 secretion and can sustain, at least
in the short term, PON1 activity. Less is known about the impact of phospholipid subclasses or
individual phospholipids on the ability of HDL to complex the enzyme, and the consequences for
enzyme activity. The potential influence of the cholesterol component of HDL on PON1 activity
is virtually unknown. The principal peptide component of HDL, apoAI, appears important for
sustaining PON1 activity, even if it cannot promote PON1 secretion. Studies are beginning to
unravel the precise nature of its interaction with the enzyme, and how this may influence enzyme
activity. Conversely, the impact of the second major HDL peptide, apoAII, on PON1 is clear.
Recent data, indicating that a substantial fraction of HDL-associated PON1 occurs in apoAII
particles, emphasise the need to define the consequences for PON1 function, especially as
apoAII-containing HDL have an ambiguous relationship with risk of cardiovascular disease.
Finally, PON1 shows a particular affinity for the HDL complex, which has not been explained
with respect to the physico-chemical characteristics of the HDL particle. The latter has to take
into account the substantial, reversible modifications that occur during normal HDL metabolism,
and the more exaggerated alterations induced by perturbed blood lipid metabolism.
These considerations will be discussed with particular reference to the role played by
apoAII in binding of PON1 to HDL and the consequences for PON1 activity / function.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Paraoxonase 1 (PON1) and Diabetes
Michael Aviram, Berinic R. and Joseph Tenenbaum Chair in Preventive Medicine, Head, Lipid Research
Laboratory, Technion Faculty of Medicine, Director, Department of Laboraotory Medicine, Rambam
Medical Center, Bat-Galim, Haifa, Israel, 31096. We Site: www.aviramlipids.com; Tel: 972-4-8542970;
Fax: 972-4-8542130; [email protected]
According to the oxidative hypothesis of atherosclerosis, lipid peroxidation in lipoproteins and in cells of
the arterial wall, is a major contributor to accelerated atherogenesis. Antioxidants, both exogenous (such
as pomegranate, which is rich in the polyphenolic hydrolyzable tannin punicalagin), and endogenous
(such as paraoxonases and the glutathione system), act as a first and as a second line of defense against
oxidative stress respectively (inhibition of lipids peroxidation, and breakdown of oxidized lipids,
respectively). Paraoxonase 1 (PON1), the HDL-associated lactonase was shown (in PON1 KO and PON1
Tg mice) to reduce oxidative stress in serum and in macrophages, and to attenuate atherogenesis, through
its antioxidant and hydrolase /lactonase properties. However, under oxidative stress, PON1 undergoes
inactivation (either irreversible, or reversible- through glutationylation). In diabetes, increased oxidative
stress and dysfunctional HDL were shown. As Diabetes involves high oxidative stress and accelerated
atherogenesis, we questioned the relationship between PON1 and diabetes.
PON1 paraoxonase, arylesterase and lactonase activities, were all substantially decreased in diabetic
patients vs. healthy subjects, and this phenomenon could be related to decreased serum PON1 stability in
the patients group.
Similar PON1 inactivation could be seen upon HDL incubation with increasing glucose concentration,
and this effect was associated with an increase in the free PON1 levels. Indeed, in diabetic patients, we
observed PON1 dissociation from HDL to the lipoprotein deficient serum, and this free PON1 was found
to be less able to protect against lipid peroxidation, than the HDL- associated PON1, as PON1 association
to HDL stabilizes the enzyme.
We next questioned whether PON1 can protect against oxidative stress-induced diabetes development.
For this purpose, we analyzed first STZ-induced serum oxidative stress and diabetes development in C57
mice under antioxidant conditions (vitamin E supplementation, and NADPH oxidase dysfunction by
using P47 -/- mice). Under reduced oxidative stress, serum glucose levels increased following STZ
injection much slower and to a lesser extent than the levels observed in the control mice (not under low
oxidative state).
By using PON 1 KO and PON1 Tg mice, we could demonstrate the protective role of PON1 against
macrophage superoxide anion release and towards cellular glutathione elevation under diabetic
conditions. Similar protection by PON1 against oxidative stress in macrophages could be seen in cultured
cells that were incubated with high vs. low D-glucose concentration (30 vs. 5 mM).
Finally, as oxidative stress seems to stimulate diabetes development, and since PON1 attenuates oxidative
stress under diabetic conditions, we questioned the effect of PON1 on diabetes development. Using the
PON1 KO and PON 1 Tg mice we were able to demonstrate that PON1 overexpression in mice,
attenuated STZ induced diabetes development, as evident by a significant reduction in the rise in serum
glucose and insulin levels(in response to STZ injection), as well as in macrophage RAGE expression and
in mice mortality.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Paraoxonase 1 Mass and Renal Function in Type 2 Diabetes
Philip W. Connelly1, Bernard Zinman1, Graham Maguire1, Mary Mamakeesick2, Stewart B.
Harris3, Robert A. Hegele4, Ravi Retnakaran1, Anthony J.G. Hanley.1
1
University of Toronto, 2Sandy Lake Health and Diabetes Project, Sandy Lake First Nation, Ontario, 3Centre for
Studies in Family Medicine, University of Western Ontario, London, Ontario, 4Robarts Research Institute,
University of Western Ontario, London, Ontario
Objective: To evaluate the association of paraoxonase 1 (PON1) mass with renal function in 107
subjects with type 2 diabetes in a community-based study.
Subjects and Methods: Microalbuminuria was assessed using the urine albumin/creatinine ratio
(ACR); glomerular filtration rate was estimated using either creatinine (eGFR, Cockcroft-Gault)
or plasma cystatin C concentration. Cystatin C is a preferred measure of eGFR because plasma
creatinine concentrations are dependent on age, gender and lean body mass. Hemoglobin A1c,
blood pressure, lipids, waist circumference, and diabetes treatment and duration were assessed.
PON1 mass was measured by Western blot. PON1 Q192R, -161 and PON2 A148G genotypes
were determined.
Results and Conclusions: PON1 mass was inversely correlated with cystatin C (r = -0.2,
p=0.038) and positively correlated with HDL cholesterol (HDLC) (r=0.24, p=0.014). After
adjustment for age, sex, waist circumference, Type 2 Diabetes Mellitus (DM) duration,
hemoglobin A1c, HDLC, PON2 A148G genotype and DM treatment by multiple regression, the
inverse association of PON1 mass with cystatin C remained significant (p=0.008). Conversely,
after adjustment for age, sex, waist circumference, DM duration, and hemoglobin A1c, PON2
genotype A148G (p=0.0004), PON1 genotype -161 (p=0.009), cystatin C (p=0.008) and HDLC
(p=0.0002) were significant determinants of PON1 mass. Thus PON1 concentration is strongly
associated with cystatin C. The absence of an effect of PON1 and PON2 genotype on cystatin C
concentration suggests that renal function is a determinant of PON1 concentration in patients
with DM.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
The ApoE Mimetic Peptide Ac-hE-18A-NH2 Reduced Plasma Lipid
Hydroperoxide Content with a Concomitant Increase in HDL Paraoxanase
Activity and Improves Endothelial Function
G.M. Anantharamaiah, Himanshu Gupta, Geeta Datta, Mayaknond N. Palgunachari, Shaila P. Handattu,
David W. Garber and C.Roger White Department of Medicine, University of Alabama at Birmingham,
Birmingham, AL 35294.
Background: The apoE mimetic peptide possesses the heparin binding domain 141-150
(LRKLRKRLLR) of apoE, covalently linked to the class A amphipathic helical peptide 18A. It is
dramatically reduces plasma cholesterol in dyslipidemic mouse models and is expected to retain antiinflammatory properties. Recycling of ApoE mimetic peptide increases duration of preβ HDL
formation leading to extended atheroprotective and anti-inflammatory effects Objective: We
tested the hypothesis that the apoE mimetic peptide improves HDL quality and improves endothelial
function. Methods and Results: A single bolus (15mg/kg IV) of apoE mimetic peptide not only reduced
plasma cholesterol levels (from 562.0+29.0 mg/dl to 287.7+22.0 mg/dl at 18h) in Watanabe heritable
hyperlipidemic rabbits, but also significantly improved arterial endothelial function. This improvement
was associated with 1) reduction in plasma lipid hydroperoxide levels significantly, 2) a five fold increase
in paraoxonase (PON) activity. This was associated with a reduction in the formation of superoxide
anion, a scavenger of nitric oxide, in the arteries of these animals. Conclusion: The apoE mimetic peptide
retains both cholesterol reducing and anti-inflammatory properties. In particular, Peptide Ac-hE-18ANH2 modulated HDL properties to reduce plasma lipid hydroperoxide content with a concomitant
increase in HDL PON activity and improves endothelial function.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Induction of paraoxonase 1 and apolipoprotein A1 gene expression by aspirin
Sampath Parthasarathy, Division of Cardiothoracic Surgery, Ohio State University Medical
center, Columbus, OH, USA.
Low dose aspirin therapy has become a standard in the treatment of cardiovascular diseases.
Aspirin has also be shown to inhibit atherosclerosis in mouse models. In order to determine the
mechanisms by which aspirin might inhibit atherosclerosis, we incubated HEPG2 cells and rat
primary hepatocytes with aspirin or salicylic acid and noted an increase in paraoxonase 1(PON1)
activity in the medium, together with an induction of PON 1 and apolipoprotein A1 (APO A1)
gene expression. Mice treated with aspirin also showed a two fold increase in plasma PON 1
activity and a significant induction of both PON1 and APO A1 gene expression in the liver. The
induction of PON 1 gene in cell culture was accompanied by an increase in arylhydrocarbon
receptor (AhR) gene expression. Accordingly, aspirin treatment of AhR knockout animals failed
to induce PON 1 gene expression. We previously suggested that aspirin might be hydrolyzed by
serum PON 1 and could account for its short plasma half-life of 10 minutes. Taken together with
the current studies, we suggest the anti-atherosclerotic effects of aspirin might be mediated by its
hydrolytic product salicylate and the induction of PON 1 and APO A1 might be important in the
cardio-protective effects of aspirin.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Effect of apoA-I mimetic peptide 4F on PON1 activity
Mohamad Navab, Srinu Reddy, Greg Hough, Susan Hama, Ladan Vakili, Alan Fogelman.
Atherosclerosis Research Unit, University of California Los Angeles
Studies in animal models, have established the importance of apolipoprotein A-I (apoA-I) in
atherosclerosis and preliminary work in humans has confirmed its potential usefulness in
atherosclerosis. ApoA-I is a large protein comprising 243 amino acids, making venous
administration necessary. In addition, manufacture of apoA-I is expensive and relatively
complex. Investigation has, therefore, been directed towards finding small peptide mimetics that
produce similar effects to apoA-I, but that are simpler to manufacture and administer. The
earliest peptides did not prevent atherosclerosis in mice while mimicked some of the lipidbinding properties of apoA-I. Detailed studies of the physical–chemical characteristics of these
peptides resulted in the realization that in determining the peptide’s bioactivity it was essential to
focus on the hydrophobic region of the peptide. A potent peptide, 4F, significantly improved the
function of HDL in mice and monkeys. When 4F was administered in combination with a statin,
lesion size and macrophage content were reduced in apoE deficient mice with atherosclerosis,
and lesions regressed in older mice. I n other rodent studies endothelial sloughing and
vasoreactivity were also improved. Early human clinical trials have shown that 4F is efficacious
and does not result in any adverse effects. HDL from the subjects given 4F shows reduced
inflammatory properties and determination of PON 1 activity in the plasma of these subjects is in
progress.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
The role of paraoxonase (PON1) protein in the detoxification
of homocysteine-thiolactone
Jakubowski H1, Bełtowski J2, Shih DM3
1
Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, International
Center for Public Health, Newark, NJ 07101 and 2Department of Pathophysiology, Medical University,
20-090 Lublin, Poland, 3Department of Medicine, UCLA Medical School, Los Angeles, CA90095
Homocysteine (Hcy) and Hcy-thiolactone are intermediary metabolites that are implicated in
atherothrombotic and neurodegenerative diseases in humans (1). Hcy-thiolactone is formed as
result of en error-correcting reaction when Hcy is mistakenly selected in place of methionine by
methionyl-tRNA synthetase during protein biosynthesis. Hcy-thiolactone is harmful because of
its ability to form isopeptide bonds with protein lysine residues, which impairs or alters the
protein’s function.
Paraoxonase-1 (PON1) protein has a homocysteine (Hcy)-thiolactonase activity (2), which
protects against protein modification by Hcy thiolactone in cultured human cells (3) and serum in
vitro (4). PON1 knockout mice lack the serum Hcy-thiolactonase activity (2). Hcy is a negative
regulator of PON1 expression in the mouse (5), whereas the serum Hcy-thiolactonase activity is
negatively correlated with Hcy in humans (6). We will describe recent results of measurements
of Hcy-thiolactone and N-Hcy-protein levels in PON1-deficient mice fed a normal chow or a
high methionine diet, and in rats infused with Hcy-thiolactonase/PON1 inhibitors.
1. Jakubowski H. The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med
2007;45:1704-16
2. Jakubowski H. Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism
against protein N-homocysteinylation. J Biol Chem 2000;275:3957-62.
3. Jakubowski H et al. Homocysteine thiolactone and protein homocysteinylation in human endothelial cells:
implications for atherosclerosis. Circ Res 2000;87(1):45-51.
4. Jakubowski H, Ambrosius WT, Pratt JH. Genetic determinants of homocysteine thiolactonase activity in humans:
implications for atherosclerosis. FEBS Lett 2001;491:35-9.
5. Robert K et al. Altered gene expression in liver from a murine model of hyperhomocysteinemia. J Biol Chem
2003;278:31504-11.
6. Domagała TB et al. The correlation of homocysteine-thiolactonase activity of the paraoxonase (PON1) protein
with coronary heart disease status. Cell Mol Biol 2006;52:4-10.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Is paraoxonase 1 critical for antioxidative and
anti-inflammatory activity of HDL?
Anatol Kontush
Paraoxonase 1 (PON1) is a multifunctional hydrolytic enzyme which can metabolise a wide
array of substrates. Recent evidence suggests that the primary function of PON1 is hydrolysis of
biological lactones. In the circulation, PON1 is primarily associated with high-density
lipoprotein (HDL) particles and is thought to account for their antioxidative and antiinflammatory activities, which mechanistictically involve inactivation of lipid hydroperoxides.
PON1 possesses potent anti-atherosclerotic properties which are associated with reduced
systemic oxidative stress and have been long ascribed to the inactivation of atherogenic lipid
hydroperoxides in low-density lipoprotein (LDL). The fact that PON1 primarily functions as a
lactonase is however inconsistent with its proposed role in the antioxidative properties of HDL.
Our recent data reveal that PON1 is not essential for the metabolism of lipid
hydroperoxides by human plasma HDL and that HDL particles efficiently metabolise
lipid hydroperoxides in the absence of PON1. Consistent with these data, the capacity of
HDL to protect LDL from oxidative stress is independent of PON1. As a result of these
studies, we propose a novel two-step mechanism of antioxidative activity of HDL that
does not involve PON1. These data suggest that anti-atherosclerotic properties of PON1
are not related to the capacity of HDL to protect LDL from oxidation and rather involve
its major activity as a lactonase through a still unknown pathway upstream of the
regulation of systemic oxidative stress.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
STUDIES OF THE NATURE OF THE PON1-HDL ASSOCIATION
Sara P. Deakin, Silvana Bioletto and Richard W. James.
Clinical Diabetes Division, Department of Internal Medicine, Faculty of Medicine, University of Geneva,
Geneva, Switzerland.
While many studies exist pointing towards the athero-protective function of Paraoxonase1, little is known about its mode of action in vivo. To this end we are currently investigating the
mechanism of secretion of PON1 from hepatocytes and its association with HDL. We have
previously shown that PON1 is located in an enzymatically active form at the external surface of
hepatocyctes. In the presence of a suitable carrier lipoprotein, usually HDL it is released from the
membrane and associates with the carrier.
We hypothesized that PON1 may also be capable of transferring from HDL to the
membranes of cells with which it is brought into contact by circulating HDL. Here we show that
both recombinant PON1 and human HDL associated PON1 can be transferred from culture
medium to the membranes of non-PON1 producing CHO and HUVEC cells in vitro. It can
subsequently be removed from the membrane to re-associate with HDL indicating the existence
of an equilibrium between membrane and HDL bound PON1. The cell associated PON1 is active
and is able to both protect cells from oxidation and break down bacterial quorum sensing signals.
PON1 is known to be competed off HDL under certain circumstances, particularly during an
inflammatory response in mice and rabbits. Our results suggest that PON1 that has been
competed off HDL may be capable of associating with nearby cells and of protecting them from
any oxidative damage caused by the inflammation. This would be of particular relevance within
the vascular wall where an inflammatory response is thought to be responsible for the formation
and aggravation of atherosclerotic lesions. In support of this our data shows that PON1 can be
detected in atherosclerotic plaques in the absence of ApoAI (and hence HDL).
In conclusion we suggest that PON1 is readily transferred between cell membranes and
HDL and that membrane bound PON1 may represent an additional protective mechanism against
the development and progression of human atherosclerotic disease.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
THE HUMAN PARAOXONASE-1 PHENOTYPE MODIFIES
\THE EFFECT OF STATINS ON PARAOXONASE
ACTIVITY AND LIPID PARAMETERS
György Paragh, Mariann Harangi, Hossein Z. Mirdamadi, Ferenc Sztanek, Zoltan Derdak, Ildiko Seres
First Department of Medicine, University of Debrecen Medical and Health Science Center, Debrecen,
Hungary
Aims: Human serum paraoxonase-1 (PON1) protects lipoproteins against oxidation by
hydrolyzing lipid peroxides in oxidized LDL, therefore it may protect against atherosclerosis.
One of the two common PON1 gene polymorphisms within the PON1 gene is the Q192R, its
prevalence can be estimated by phenotype distribution analysis. The goal of this study was to
clarify the role of PON1 phenotypes on the effect of three different statins on paraoxonase
activity and lipid parameters.
Methods: 164 patients with type IIb hypercholesterolemia were involved in the study. We
examined the effect of 10 mg/day atorvastatin, 10/20 mg/day simvastatin and 80 mg/day
extended-release fluvastatin treatment on lipid levels and paraoxonase activity in patients with
different PON1 phenotypes. The phenotype distribution of PON1 was determined by the dual
substrate method.
Results: Three months of statin treatment significantly increased the paraoxonase activity in
every statin-treated group. In patients with AB+BB phenotype the statin treatment was
significantly more effective on paraoxonase activity than in the AA group. The statin treatment
more effectively decreased the triglyceride levels in the AB+BB group compared to the AA
group in the whole study population and in the simvastatin-treated group. The atorvastatin
treatment was significantly more effective on apoB levels in patients with AB+BB phenotype
than in the AA phenotype group.
Conclusions: Our results indicate that the PON1 phenotype may be a novel predictive factor for
the effectivity of statin treatment on PON1 activity and serum lipid levels; however, different
types of statins may exert different effects on these parameters.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
PARAMETERS INFLUENCING STABILITY AND ACTIVITY OF HUMAN PON1:
HOW MOLECULAR ENVIRONMENT/PHYSIOLOGICAL LOCATION IMPACT THE
PROMISCUITY/MOONLIGHTING OF THIS ENZYME
Daniel ROCHU1,2, Frédérique RENAULT1, Mikaël ELIAS3, Cécile CLÉRY-BARRAUD1,
Patrick MASSON1, Eric CHABRIÈRE3
1
Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, La Tronche, France
Institut für Pharmakologie und Toxikologie der Bundeswehr, München, Germany
3
Architecture et Fonction des Macromolécules Biologiques, Université de la Méditerranée, Marseille, France
2
Human serum paraoxonase (PON1) is an unorthodox protein, displaying distinct enzyme activities: a
native lipolytic lactonase activity and two promiscuous activities (phosphotriesterase and arylesterase) [1].
The protein is a six-bladed β-propeller with a “velcro” closure sealed by a disulfide bond, a fold known to
be associated with structural rigidity and functional diversity. PON1 plays a role in lipid metabolism and
prevention of atherosclerosis. As a result, clinical and biological interests first drove to toxicological
relationships of the enzyme, with special reference to chemical warfare medical defense and
environmental implications, then expanded to the field of cardiovascular medicine.
Actually, the anti-atherosclerotic activity and the detoxification role of PON1 are linked to and
modulated by its localization on HDL particles, a complex and dynamic molecular environment.
Unexpected turn of research occurred ensuing discovery and characterization of the human phosphate
binding protein (HPBP) [2], displaying a firm propensity to associate with PON1. This has steered new
directions for identifying PON1 functional states [3]. The association of PON1 with HPBP was
hypothesized to be essential for preserving the enzyme active conformation(s).
We investigated parameters influencing stability and activity of PON1. Complementary
methodological approaches were used to study HPBP-, phosphate- and calcium dependence of PON1
oligomeric state(s), thermal and storage stability of PON1, and environment-dependence of the three
catalytic activities of PON1, i.e. arylesterase, lactonase and phosphotriesterase activities.
The substrate promiscuity of PON1 is tightly linked to its conformational plasticity. It has to be
regarded through modular properties implying that the enzyme must minimally work within a modular
architecture. Deciphering the respective roles of PON1 partners in natural HDL environment is an
important requisite for understanding the true enzymatic activities of PON1 considered in temporal and
spatial constrains of protein-protein and protein-lipid interactions.
The aim of our approach is to provide useful data for defining stabilization conditions of PON1
mutants displaying enhanced phosphotriesterase efficacy [4]. Since HDL particles are in continuous
rearrangement with time, knowledge of conditions providing a stabilizing environment for active PON1 is
essential. Moreover, to understand PON1 in action, the fourth dimension, time, will be added to the
snapshots of proteins frozen in crystal structures. Finally, maintaining a favourable environment for a safe
administrable human rPON1 should preserve and/or enhance the enzyme anti-atherogenic activity.
1. Khersonsky O, Tawfik DS, Biochemistry 44 (2005) 6371-6382
2. Rochu D, Renault F, Cléry-Barraud C, Chabrière E, Masson P, Biochim Biophys Acta 1774
(2007) 874-883
3. Diemer H, Elias M, Renault F, Rochu D, Contreras-Martel C, Schaeffer C, Van Dorsselaer A,
Chabrière E Proteins (2008) DOI 10.1002/prot, in press
4. Rochu D, Chabrière E, Masson P, Toxicology 233 (2007) 47-59
D.R. is under contract with Bundesministerium der Verteidigung (M/SAB 1/7/A004). This work was
supported by Délégation Générale pour l’Armement (DGA/03CO-010-05) to P.M. and E.C., and Centre
National de la Recherche Scientifique to E.C.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
PON and quorum sensing
Joseph Zabner
Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to
regulate gene expression. The signaling molecule, N-3-oxododecanoyl homoserine lactone
(3OC12-HSL) is thought to play a central role in quorum sensing. Since paraoxonase (PON)
family members are lactonases that can degrade 3OC12-HSL, we hypothesized that PON
regulates P. aeruginosa virulence in vivo. Drosophila melanogaster has been a tractable model
system to investigate host-pathogen interactions. Importantly, Drosophila lacks PON. By using
quorum-sensing mutant deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression
of PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We
found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 flies had
arylesterase activity and were resistant to organophosphate toxicity. Moreover, PON1 flies were
protected from P. aeruginosa lethality and protection was dependent on PON1¹s lactonase
activity. Our findings show that PON can interfere with quorum sensing in vivo and provide
insight into a novel role for PON in the innate immune response to quorum-sensing dependent
pathogens. These results raise novel and intriguing possibilities about human-pathogen
interactions including potential roles for PON as a modifier gene, a regulator of normal bacterial
florae, a link between infection/inflammation and cardiovascular disease, and a potential
therapeutic modality.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
New Approaches to Bacterial Quorum Quenching: From
Small Molecules to Antibodies
Kim D. Janda
The Scripps Research Institute, The Skaggs Institute for Chemical Biology and Worm Institute
for Research and Medicine, Departments of Chemistry and Immunology, 10550 North Torrey
Pines Rd., La Jolla California 92037, [email protected]
Cell-to-cell communication via exchange of small molecules, 'autoinducers', is a widespread
phenomenon among Gram-negative and -positive bacteria. This intercellular signaling that
synchronizes population-wide gene expression in a cell-density-dependent manner is termed
'quorum sensing' (QS). The discovery that Gram-negative bacteria employ non-peptide
structures, N-acyl homoserine lactones, to globally regulate production of secondary metabolites
and proteins, initiated a new area of research. Subsequently, other quorum-sensing systems and
small signaling molecules have been identified including peptides and a universal autoinducer
termed AI-2. With the emergence of antibiotic-resistant bacteria, most prominently methicillinresistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, new approaches for
combating infections are needed. It has been shown that the blunting of QS results in attenuation
of virulence rather than direct killing of microbes. In this context we will highlight some of our
research strategies, which have focused on ways to “quench” QS either using active/passive
vaccines or small molecule antagonists. Our findings suggest that QS systems represent
attractive targets for discovery of novel anti-infective agents, including immunotherapeutic
strategies.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Transgenic Expression of Paraoxonase-1 in Drosophila
Protects From Pseudomonas aeruginosa Lethality
David A. Stoltz1, Peter J. Taft1, Egon A. Ozer2, Marilyn Barry3, Lei Liu1,
Peter Kiss1, Tom O. Moninger4, Matthew Parsek5, and Joseph Zabner1
1
Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa
City, IA; 2 Feinberg School of Medicine, Northwestern University, Evanston, IL; 3 School of Medicine, Tufts
University, Somerville, MA; 4 Central Microscopy Research Facility, Roy J. and Lucille A. Carver College of
Medicine, University of Iowa, Iowa City, Iowa; 5 Department of Microbiology, University of Washington, Seattle,
WA
Pseudomonas aeruginosa (PA), an opportunistic pathogen involved in sepsis, cystic fibrosis,
nosocomial pneumonia, and burn wound infections, uses a quorum-sensing system to control its
virulence. Drosophila melanogaster have been used extensively to study innate immune
mechanisms and bacterial virulence. We previously found that quorum sensing regulates PA
virulence in this model. Paraoxonase 1 (PON1) is a mammalian enzyme that degrades various
organophosphate molecules and 3OC12-HSL, the primary quorum-sensing signal in PA. Since
PON1 does not exist in flies we created PON1 transgenic flies and assayed their ability to
survive infection with PA. We hypothesized that PON1 transgenic flies would be protected from
a lethal challenge with PA. Western blot analysis confirmed that PON1 was expressed in the
flies. PON1 enzymatic activity was confirmed by lysate degradation of phenyl acetate. Under in
vivo conditions, PON1 flies demonstrated increased survival when fed chlorpyrifos. To test the
role of PON1 in PA infection, Drosophila were inoculated by pricking the abdomen with a
needle dipped in either PA (PAO1, 109 cfu/ml) or sterile medium. Fly survival and in vivo
bacterial growth were measured in all studies. We found that PA is lethal to wild type flies
within 72 hours. PON1 transgenic flies showed enhanced survival from infection. Thus,
paraoxonase disruption of PA quorum sensing improves survival in this in vivo model of
infection.
David A. Stoltz is a Parker B. Francis Fellow.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
MACROPHAGE PARAOXONASE 2 (PON2) EXPRESSION IS
UPREGULATED BY UROKINASE PLASMINOGEN ACTIVATOR (uPA)
IN A REDOX-DEPENDENT PATHWAY
Bianca Fuhrman, Jasmin Khateeb, Michael Aviram
The Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the
Medical Sciences, and Rambam Medical Center, Haifa, Israel.
Background and Aims: Atherosclerotic lesion macrophage foam cells are characterized by
increased oxidative stress. As macrophage uPA was shown to contribute to atherosclerosis
progression, we hypothesized that uPA atherogenicity could be related to its ability to increase
macrophage oxidative stress. Increased macrophage oxidative stress in turn, was shown to
enhance cellular PON2 expression and activity. Thus, in the present study we investigated the
effect of uPA on macrophage PON2 expression in relation to cellular oxidative stress.
Methods and Results: uPA increased PON2 mRNA, protein and activity in THP-1 macrophages,
in a dose-dependent manner. This effect required uPA/uPAR interaction, and was abolished by
cell treatment with antioxidants, suggesting a role for cellular oxidative stress. Indeed, uPA
increased macrophage oxidative stress, as measured by increased lipid peroxides content,
reactive oxygen species formation, superoxide anion release, and cell-mediated LDL oxidation.
These effects were related to uPA-mediated upregulation of NADPH oxidase cytosolic
component p47phox. A causal relationship between NADPH oxidase activation and the effects
of uPA on macrophage oxidative stress and PON2 expression was evident, as it could not be
reproduced in mouse peritoneal macrophages (MPM) harvested from p47phox-/- mice. Finally,
MPM from PON2-/- mice were more susceptible to uPA-induced cellular oxidative stress than
wild type MPM, suggesting a protective role for PON2 against uPA-stimulated macrophage
oxidative stress.
Conclusions: Upregulation of macrophage PON2 may provide a compensatory protective
mechanism against uPA-stimulation of macrophage oxidative stress during atherogenesis.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
NOVEL SERUM PARAOXONASE ACTIVITY ASSAYS ARE
ASSOCIATED WITH CORONARY ARTERY DISEASE
Nicola Martinelli1, Domenico Girelli1, Oliviero Olivieri1, Patrizia Guarini1, Antonella Bassi2, Elisabetta
Trabetti3, Simonetta Friso1, Francesca Pizzolo1, Claudia Bozzini1, Ilaria Tenuti1, Renzo Schiavon4, Pier
Franco Pignatti3, Roberto Corrocher1.
1
Department of Clinical and Experimental Medicine, University of Verona, Italy
Institute of Clinical Chemistry, University of Verona , Italy
3
Section of Biology and Genetics, Department of Mother and Child and Biology – Genetics, University of Verona,
Italy
4
Laboratory of Clinical Chemistry, Hospital of Legnago, Verona
2
Background: serum paraoxonase (PON1) exerts antiatherogenic effects. Novel PON1 enzymatic
tests have been recently developed: 5-thiobutyl,butyrolactone (TBBL) estimates PON1 lactonase activity,
whereas 7-O-diethyl phosphoryl,3-cyano,4-methyl,7-hydroxycoumarin (DEPCyMC) is considered a
surrogate marker of PON1 concentration. The TBBL to DEPCyMC ratio provides the normalized
lactonase activity (NLA), which may reflect the degree of PON1 lactonase catalytic stimulation.
Methods: the aim of this study was to evaluate for the first time TBBLase and DEPCyMCase
activity in patients with coronary artery disease (CAD). Thus, an angiography-based case-control study,
including 300 sex- and age-matched subjects (100 CAD-free, 100 CAD without myocardial infarction
(MI) and 100 CAD with MI), was carried out.
Results: a low DEPCyMCase activity (the lowest versus the highest tertile: OR 2.96; 95%CI
1.18-7.43) and an high NLA (the highest versus the lowest tertile: OR 3.25; 95%CI 1.28-8.26) were both
associated with CAD, independent of classical atherosclerosis risk factors, lipid-lowering therapy and
PON1 genotype.
Conclusions: novel PON1 activity assays may be associated with CAD. In this study, CAD
patients had low DEPCyMCase activity, a possible marker of low PON1 concentration, but presented an
high stimulation of PON1 lactonase activity.
ACKNOWLEDGEMENTS
We are very grateful to professor Dan Tawfik, Olga Khersonsky and Leonid Gaidukov
(Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel) for their ongoing
cooperation, for providing the substrates for TBBL and DEPCyMC assays, for their excellent technical
support and their very kind and useful suggestions.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Insulin resistance in paraoxonase 2 deficient male mice
Bourquard N1,2, Ng CJ, Pilouk P3, Reddy ST1,2
Department of Medicine1 and Molecular and Medical Pharmacology2, David Geffen School of
Medicine, UCLA, Los Angeles, California 90095
3
Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles,
California 90095
Objective. Insulin resistance, characterized by hyperinsulinemia and defective insulin signaling
in a variety of tissues, is a primary defect underlying the development of type 2 diabetes.
Monocyte/macrophages have recently been shown to play a critical role in provoking the
inflammatory response leading to insulin resistance. Paraoxonase 2 (PON2), a member of the
paraoxonase gene family, is a lactonase with anti-inflammatory and/or anti-oxidant properties,
and has been implicated in the pathogenesis of several inflammatory diseases including diabetes.
We have previously shown that peritoneal macrophages isolated from PON2-deficient (PON2def) mice are hyperresponsive to a number oxidative stress inducing agents, in terms of proinflammatory gene induction. The objective of this study was to determine if PON2 deficiency in
mice and primary macrophages contributes to an insulin resistant phenotype. Methods and
results. PON2-def mice maintained on a standard chow diet exhibit modest but significant
dyslipidemia, as characterized by increased levels of cholesterol, free fatty acids and
triglycerides. Fasting glucose and insulin levels were not different in PON2-def and control male
mice; however, PON2-def mice displayed significant abnormalities in glucose homeostasis as
determined by glucose (AUC p<0.05) and insulin (AUC p<0.01) tolerance tests. This phenotype
was observed only in males, as glucose metabolism was otherwise normal in the PON2-def
female counterparts. Administration of an atherogenic diet to PON2-def males, relative to
littermate controls, resulted in several characteristics consistent with an insulin resistant
phenotype in addition to abnormal glucose metabolism (AUC p<0.05), including increased
weight (26.3±1.8 vs. 29.4±1.5, p<0.01) and hyperinsulinemia (0.89±0.4 vs. 1.78±1.1 ng/mL,
p<0.05) with normal fasting glucose levels. Moreover, high glucose (30mM) led to an over twofold increase in the mRNA expression of TNFα and IL-1β in PON2-def peritoneal macrophages
relative to control macrophages (p<0.05). Conclusion. Our data suggests that, in addition to its
involvement in the pathogenesis of atherosclerosis, PON2 may also play a critical role in glucose
homeostasis. Inflammation is closely linked to the pathogenesis of both insulin resistance and
atherosclerosis thus PON2 may represent a potential biological link between these two diseases.
We are now further exploring the mechanism by which PON2 deficiency contributes to an
insulin resistant phenotype.
This work was supported by the NHLBI Grant 1RO1HL71776
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Evolving PON1 for improved organophosphate hydrolysis
Rinkoo D Gupta, Gavriel Mullokandov, Dan S Tawfik
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
The relatively high catalytic efficiency of human PON1 toward sarin and soman, as well as the
possibility to re-inject it in humans, renders PON1 a suitable candidate for medical
countermeasure against nerve agent poisoning. Since, neutral libraries of PON1 may have
increased potential of evolvability1; we examine PON1’s potential to drift towards the
evolutionary transition points while remaining neutral with respect to its primary function.
We performed three subsequent generations of random mutagenesis and purifying selection to
purge deleterious mutations. GFP was used as a C-terminal fusion protein of PON1 to measure
its expression level in crude lysate. Several hundred variants were characterized that are
apparently neutral with respect to WT PON1's level of expression and native lactonase activity1.
The neutral PON1 library was monitored for amiton, a V-type nerve agent analog and CMPMeCyC2, a G-type nerve agent analog. rePON1G3C9 does not hydrolyse amiton and the P(−)
isomer of CMP-MeCyC. Since the H115W mutant hydrolyses amiton very slowly3, we
introduced H115W mutation on the top of neutral library. In order to hydrolyse the more toxic
P(−) isomer of sarin/soman, we introduced the V346A mutation on top of neutral library which
produced several variants with improved hydrolyses of the P(−) isomer. Further, these variants
are being used as a starting point for directed evolution of PON1 for similar types of substrates.
Our results show that, neutral libraries of PON1 serve as a potent starting point for the evolution
of PON1. In oppose to wild-type PON1, H115W, together with two more active site amino acid
mutations gives measurable hydrolysis of amiton. Other mutants showed much improved
hydrolysis of the P(+), and P(-) isomers of CMP-MeCyC.
References:
1. Amitai, G., Gupta, R. D. & Tawfik, D. S. Latent evolutionary potentials under the neutral mutational drift of an enzyme.
HFSP J. 1, 67-78 (2007).
2. Amitai, G. et al. Asymmetric fluorogenic organophosphates for the development of active organophosphate hydrolases
with reversed stereoselectivity. Toxicology 233, 187-98 (2007).
3. Yeung, D. T., Lenz, D. E. & Cerasoli, D. M. Analysis of active-site amino-acid residues of human serum paraoxonase
using competitive substrates. FEBS J. 2225-2230 (2005).
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
LIVER X RECEPTOR AGONIST, TO901317, NORMALIZES PLASMA
PON1 ACTIVITY AND REDUCES PROTEIN HOMOCYSTEINYLATION
IN RATS WITH EXPERIMENTAL HYPERLEPTINEMIA
Beltowski J, Wójcicka G, Jamroz-Wiśniewska A, Marciniak A
Department of Pathophysiology, Medical University, Lublin, Poland
Background and aims. Recent studies indicate that elevated level of adipose tissue hormone,
leptin, contributes to the development of atherosclerosis in patients with obesity and metabolic
syndrome. Previously, we have demonstrated that experimental hyperleptinemia induced in the
rat by administration of exogenous leptin is associated with reduced plasma PON1 activity
(Atherosclerosis 2003; 170: 21). Agonists of liver X receptor (LXR) reduce atherosclerosis in
experimental models, and have been shown to up-regulate PON1 gene expression (Mol
Pharmacol 2003; 63: 945). Therefore, in this study we examined if LXR pathway is involved in
leptin-induced PON1 deficiency.
Methods. Leptin was administered at a dose of 0.25 mg/kg twice daily s.c. for 7 days in adult
male Wistar rats. Separate group of animals received synthetic LXR agonist, T0901317 (1
mg/kg/day). Subsequently, plasma PON1 activity was measured toward paraoxon, phenyl acetate
and homocysteine thiolactone (HTL). In addition, the amount of HTL linked to plasma proteins
(a marker of protein N-homocysteinylation) as well as total homocysteine were assayed.
Results. Leptin administration induced an almost 4-fold elevation of plasma leptin. PON1
activity toward paraoxon, phenyl acetate and HTL was lower in leptin-treated than in control
animals. Leptin increased the amount of HTL linked to plasma proteins by 57.8% but had no
effect on total homocysteine and lipid profile. Co-treatment with T0901317 prevented leptininduced decrease in PON1 and increase in protein homocysteinylation. In contrast, T0901317
had no effect on PON1 or protein homocysteinylation in animals not receiving leptin. Plasma
leptin, total homocysteine, lipid profile and markers of oxidative stress were not altered by
T0901317 in either leptin-treated or control group.
Conclusions. The results suggest that: (1) hyperleptinemia disrupts LXR-dependent regulation
of PON1 leading to enhancement of protein homocysteinylation, (2) LXR agonists may
normalize PON1 activity and reduce protein homocysteinylation in hyperleptinemic states such
as the metabolic syndrome.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Paraoxonase-2 reduces intracellular superoxide levels
independent of its lactonase activity
Sebastian Altenhöfer1, Ines Witte1, John F. Teiber2, Petra Wilgenbus1, Dennis Strand3, Ulrich
Förstermann1, and Sven Horke1*
From the (1)Departments of Pharmacology and (3)Internal Medicine I, University of Mainz, Obere
Zahlbacher Str. 67, 55131 Mainz, Germany, and (2)Department of Internal Medicine, Division of
Epidemiology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. *Send
correspondence to: [email protected]
Background- The paraoxonase (PON) family of enzymes comprises three proteins, PON1,
PON2 and PON3. Despite high homology at the amino acid level, PONs differ significantly in
their enzymatic activities, substrate specificities and localization. PON2 mainly localizes to the
endoplasmic reticulum (ER) and protects cells from ER stress-induced caspase-3 activation.
Moreover, all three PONs possess anti-atherogenic properties that may be a result of their antioxidative functions. Here we addressed which of the major reactive oxygen species (ROS)
found in diseased blood vessels is lowered by PON2. We further investigated if PON2’s
lactonase activity, its best established catalytic function, is fundamental to its anti-oxidative/antiapoptotic function(s).
Methods and Results- Chemiluminescence- and HPLC-based experiments revealed that PON2
reduces intracellular superoxide-, but no peroxynitrite levels. Furthermore, by introducing
multiple point mutations, we demonstrate that single amino acids, e.g. histidine-114, are essential
for PON2 lactonase activity. Lactonase activity was analyzed using N-(3-oxododecanoyl)-Lhomoserine lactone, which is the best PON2 substrate identified to date. Endothelial cells stably
overexpressing mutated PON2 were analyzed for both intracellular ROS and caspase-3
activation (the latter in response to ER stress). These studies revealed that single point mutations
abrogated PON2 lactonase activity, while leaving anti-oxidative and anti-apoptotic functions
intact. ER-targeting of mutated PON2 was controlled by confocal microscopy.
Conclusions- Our data thus provide evidence that PON2 reduces a major radical(s) involved in
cardiovascular damage independent of its predominant enzymatic function. Likewise, the
protection from ER stress-induced apoptosis did not require lactonase activity, hence indicating
at least two to three independent functions of the protein.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Fully automated determination of arylesterase, paraoxonase activities and
PON1 192Q/R phenotype*
Ozcan EREL, Sahbettin SELEK
[email protected]
Harran University, Medical Faculty Research Hospital, Sanliurfa, Turkey.
Background and aim: Paraoxonase-1 (PON1) is an high density lipoprotein (HDL)-associated
enzyme with antioxidant and antiatherogenic functions, protecting lipoproteins against oxidative
modification. It also catalyzes the hydrolysis of organophosphates such as paraoxon and
aromatic carboxylic acid esters of fatty acids. In this study, it was aimed that to develop a pair of
colorimetric and fully automated method, of which the used reagents are more stable and their
lifetime are too long, measuring arylesterase and paraoxonase activities of PON1. In addition,
fully automated phenotyping of PON1 192Q/R was also aimed.
Materials and Methods: Phenyl acetate, a substrate, was used for arylesterase activity
measurement and paraoxon, a substrate, was used for paraoxonase activity measurement.
Paraoxon and especially phenyl acetate exhibit fast spontaneous hydrolysis in aqueous solutions.
In the new assays more stable substrate solutions were developed. In the first assay, the phenol
producing by arylesterase activity was measured colorimetrically with end point reading mode.
In the second assay, the enzymatic hydrolyses of the substrate, paraoxon, was monitored with
kinetic reading mode. The assays were performed on an automated analyzer and analytical
performance characteristics of the assays were determined.
Results: There was a most significant correlation between new colorimetric automated
arylesterase activity measurement method and its reference method, which is based on the
determination of phenol at ultraviolet wavelength, (r= 0.98, p<0.0001). On the other hand, the
new stable paraoxonase activity measurement method showed a most important correlation with
its reference method too (r= 0.99 p<0.0001). All reagents of the both assays were stable for at
least 6 months on the automated analyzer. Their precision values were lower than 1%.
Results of fully automated phenotyping were same of the conventional method.
Conclusion: These easy, stable, reliable, sensitive, inexpensive, colorimetric and fully
automated methods described can be used to measure arylesterase and paraoxonase activities of
PON1 and beside of these automated measurements, PON1 QQ, QR and RR phenotype
distribution at positions 192 can also be determined automatically and simultaneously.
Keywords: arylesterase, paraoxon, paraoxonase, phenyl acetate, PON1.
*Granted by TUBITAK [Scientific and Technological Research Council of Turkey (107S167)].
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Decreased serum paraoxonase (PON1) and apolipoprotein A-I (apoA-I)
levels in critically ill patients with sepsis: determinants or
markers for the severity and outcome
Dragomir I. Draganov1,2, John F. Teiber1,3, Catherine E. Watson4, Charles L. Bisgaier1,4,
Theodore J. Standiford1, Bert N. La DU1
1
University of Michigan, Ann Arbor, MI 48105, USA; 2WIL Research Laboratories, LLC, Ashland, OH 44 805,
USA; 3The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; 4Esperion Therapeutics,
Division of Pfizer Global Research and Development, Ann Arbor, MI 48108, USA
This prospective observational study was designed to explore the relationship between serum
PON1 activity and the sepsis severity, progression and outcome. A total of 85 patients with
sepsis, severe sepsis or septic shock were recruited from the Critical Care Unit at the University
of Michigan Medical Center; 39 critically ill patients without sepsis were recruited as controls.
Among the clinical data recorded were Acute Physiology, Age and Chronic Health Evaluation
(APACHE) III scores, results of chest X-ray, ECG, ventilator parameters, urinary output, and
positive culture. Blood samples were collected at study entry and on 1, 3, 7 and 14 days postentry. Resultant plasma samples were analyzed for PON1 enzymatic activities, lipoprotein and
lipid profile, lipid peroxidation, standard clinical biochemistry and cytokine and chemokine
plasma levels. Compared to non-sepsis critically ill patients at study entry, sepsis patients had
significantly higher APAHE III scores, triglycerides, plasma lipid peroxides, myeloperoxidase
activity and MCP levels, and significantly lower HDL-cholesterol, apoA-I and PON1 levels.
The same trends were evident when comparing non-survivors versus survivors among the sepsis
patients. In the time course of observation, persistent low or further decreasing levels of apo-AI
and paraoxonase in the sepsis patients were associated with lethal outcome.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Interrelationships between PON-1 and MCP-1 in the
regulation of hepatic inflammation
Jordi Camps*, Judit Marsillach and Jorge Joven
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, C. Sant Joan s/n, 43201 Reus
(Catalunya) Spain
* E-mail: [email protected]
Background/Aims: PON-1 protects against liver impairment and, in endothelial cells, attenuates
the production of the pro-inflammatory monocyte chemoattractant protein-1 (MCP-1). In the
present study, we investigated the relationships between hepatic PON1 and MCP-1 expression in
rats with liver disease and explored the possible molecular mechanisms involved.
Methods: CCl4 was administered for up to 12 weeks to induce liver damage. Serum and hepatic
levels of PON1 and MCP-1, their gene and protein expression, nuclear transcription factors, and
histological and biochemical markers of liver impairment were measured.
Results: High levels of PON1 and MCP-1 expression occurred in the hepatocytes surrounding
the fibrous septa and inflammatory areas. CCl4-administered rats had an increased hepatic PON1
concentration that was the consequence of decreased gene transcription and inhibited protein
degradation. Decreased PON1 gene transcription was associated with PPARδ and Fra-2
expression. These changes were accompanied by increased hepatic MCP-1 concentration and
gene expression. There were significant direct relationships between hepatic PON1 and MCP-1
concentrations (P = 0.005) and between PON1 and the amount of activated stellate cells (P =
0.001).
Conclusion: Our results from this experimental model indicate a hepato-protective role for PON1
against inflammation, fibrosis and liver disease mediated by MCP-1.
Grant support: Supported by grants from the Instituto de Salud Carlos III (FIS 02/0430, 05/1607,
RCMN C03/08 and RD06), Ministerio de Sanidad, Madrid, Spain. J. M. is the recipient of a
post-graduate fellowship from the Generalitat de Catalunya (FI 05/00068).
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
ALTERATION OF PON1 ACTIVITY IN CHILDHOOD OBESITY AND ITS
RELATION TO LEPTIN AND ADIPONECTIN LEVELS
Ildiko Seres, Péter Koncsos, Ferenc Sztanek, Eva Varga, György Paragh
1st Department of Medicine, Medical and Health Science Centre, University of Debrecen, H4012 Debrecen, Nagyerdei krt. 98., Hungary, [email protected]
Introduction: Childhood obesity shows increasing tendency in the developed countries. This pathogenic
disorder means a predisposing factor concerning the cardiovascular morbidity and mortality in adulthood.
Our recent study investigated the alterations of two adipokines – leptin and adiponectin – and the human
paraoxonase-1 enzyme (PON1) in childhood obesity as influencing factors in atherosclerosis;
furthermore, we aimed to reveal whether there is any relation between these parameters in young subjects.
Patients and methods: 59 white, obese (obese group, OB; BMI corrected for age: 95.08±3.53 percentile,
age: 11.95±1.61 years) and 51 white, normal-weight children (control group, C; BMI corrected for age:
64.10±8.36 percentile, age: 12.00±3.91 years) were included into our study. We measured anthropometric
parameters, leptin, adiponectin levels and PON1 paraoxonase and arylesterase activities.
Results: Obese children had significantly higher leptin (OB: 43.61±26.64 ng/ml vs. C: 11.69±14.63
ng/ml, p<0.0001); lower adiponectin levels (OB: 8.59±4.39 µg/ml vs. C: 12.24±4.86 µg/ml, p<0.0001);
lower PON1 paraoxonase (OB: 104.77±56.55 U/l vs. C: 121.03±52.87 U/l, p<0.05) and arylesterase
activity (OB: 97.31±21.24 U/l vs. C: 111.44±23.52 U/l, p<0.01) compared to normal-weight subjects.
PON1 arylesterase activity showed inverse relation with leptin (r=-0.29, p<0.05) and positive relation
with adiponectin levels (r=0.39, p<0.01) in univariate correlation. In multiple regression analysis,
adiponectin levels proved to be independent predictor of PON1 arylesterase activity in obese subjects but
neither the leptin nor the other investigated factors, i.e. age, sex, body fat percentage, HDL-C.
Investigating PON 192 Q/R (A and B isoenzyme) polymorphism by phenotypic distribution we also
found significant correlation of PON1 arylesterase activity with leptin (r=-0.49, p<0.05) and adiponectin
levels (r=0.51, p<0.05) in obese children with PON1 192 B isoenzyme, moreover there were significant
inverse correlation between PON1 arylesterase activity and body fat percentage (r=-0.51, p<0.05). Obese
children with homozygous AA phenotype had significant correlation between PON1 arylesterase activity
and adiponectin levels (r=0.41, p<0.05).
Conclusion: Our results suggest that metabolic state in childhood obesity creates good conditions for the
development of the early atherosclerosis. Changed levels of leptin, adiponectin and PON1 activities, just
as 192 Q/R polymorphism determined by phenotypic distribution may be useful markers beside the
general risk factors.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
PON1 and HDL in sickle cell disease
Lita A. Freeman1 and Gregory J. Kato1,2, NHLBI1 and CCMD1,2, NIH, Bethesda, MD 20892
Sickle cell disease (SCD) is caused by a mutation in the
-chain of hemoglobin, leading to
sickle-shaped red blood cells that cause vasooclusion. Major clinical features include acute pain,
stroke, priapism, acute chest syndrome and chronic organ damage. Importantly, red blood cell
hemolysis is also increased in SCD patients, leading to increased free hemoglobin and arginase
in plasma and thus decreased NO bioavailability. Hemolysis in SCD has consequently been
linked to endothelial dysfunction and vasculopathy (Kato et al. Blood Reviews (2007) 21:31-47).
The pathways affecting endothelial function in SCD have not yet been fully elucidated. As HDL
and the HDL-associated lactonase PON1 are associated with vascular protection, we investigated
HDL and PON1 in sickle-cell disease. Here we report that plasma levels of both apoA-I and
PON1 are decreased in plasma of sickle-cell patients during pain crisis compared to their steadystate levels, as determined by Western analysis. We have also utilized the technique of nativenative 2D gel electrophoresis to identify specific apoA-I-containing HDL subpopulations
associated with PON1 and to demonstrate the presence of PON1 in additional particles that lack
apoA-I. In conjunction with previous reports from other investigators on the protective role of
PON1 and apoA-I towards the vasculature, our work suggests apoA-I and PON1 as potential
therapeutic targets against hemolytic vascular dysfunction in SCD patients.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
INTERACTION BETWEEN PARAOXONASE-1 AND ENVIRONMENTAL
CHEMICALS AND ITS IMPACT ON HEALTH
A.F. Hernández-Jerez. Department of Legal Medicine and Toxicology, University of Granada
Medical School, Avda. Madrid, 11. 18071-Granada. Spain ([email protected])
In addition to be an individual marker of susceptibility PON1 may also act as a biological
indicator of exposure to pesticides, since we have observed lower enzyme activity in individuals
occupationally exposed to these agents, particularly organophosphates. Furthermore, we have
shown that the PON1-192R allele was associated with certain biochemical markers of cytolysis
and oxidative stress. This allele was also linked with a lower risk of reporting either pesticiderelated symptoms or history of previous pesticide poisoning. However it failed to play any role in
changes observed in lung function tests of greenhouse workers regularly exposed to pesticides.
Besides, we have found that the PON1-55L allele also predicted a lower risk of pesticide-related
symptoms, while the promoter polymorphism at positions -108 and -909 had no effect. Given the
role of PON1 as a first line enzymatic defence against environmental toxicants (such as
organophosphorus pesticides and chemicals that cause oxidative stress), we sought any
association between serum PON1 activity (using paraoxon, phenylacetate, diazoxon and
dihydrocoumarin as substrates) and certain metal ions namely arsenic, mercury, lead, cadmium,
manganese and zinc in body fluids. Subjects from the general population of Andalusia (southern
Spain) with potential exposure to these agents through environmental or nutritional sources were
studied. PON1 showed a significant direct correlation with blood lead and urine mercury and an
inverse correlation with urine arsenic, blood cadmium and manganese, and serum zinc. Carriers
of the PON1 R isoform showed higher levels of blood lead and urine arsenic, near the
significance level, but lacked any difference as regards to the remaining metal ions. Although
mechanistic conclusions cannot be inferred from these data, an interaction between
environmental or nutritional metal ions and PON1 can be hypothesized. Thus, PON1 might play
a protective role against metal toxicity; nevertheless, this hypothesis has to be tested in furthers
studies.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
ROLE OF PARAOXONASE (PON1) IN ORGANOPHOSPHATE
METABOLISMS AND TOXICITY
Lucio G. Costa1,2, Toby B. Cole1,3, Karen Jansen1, Clement E. Furlong3
1
Dept. of Environmental and Occupational Health Sciences and 3Depts. of Medicine (Medical Genetics)
and Genome Sciences, University of Washington, Seattle, WA, USA. 2Dept. of Human Anatomy,
Pharmacology and Forensic Science, University of Parma, Italy.
Paraoxonase (PON1) owes its name to paraoxon (PO), the active metabolite of the
organophosphorus (OP) insecticide parathion. Ironically, though PO is a good in vitro substrate
for PON1, the enzyme does not appear to be of significance in PO’s metabolism and toxicity in
vivo. However, PON1 is an important determinant of the acute toxicity of other OPs, such as
diazoxon (DZO) and chlorpyrifos oxon (CPO) (active metabolites of diazinon and chlorpyrifos,
respectively), as shown by studies carried out in PON1 null mice and in mice expressing either
the human Q192 or R192 allele of PON1. Modulation by PON1 of the toxicity of OPs depends
on its catalytic efficiency toward a given substrate; thus, CPO is metabolized with high catalytic
efficiency, particularly by PON1R192, while PO (as well as other substrates such as the nerve
agent sarin) is metabolized with very low catalytic efficiency. In addition to influencing their
toxicity, PON1 status (encompassing both the Q192R polymorphism and the level of PON1
expression) also influences the interaction of these insecticides with other OPs. For example,
DZO and CPO, by virtue of their ability to inhibit carboxylesterase, potentiate the toxicity of
malaoxon, and the degree of potentiation is influenced by PON1 status. PON1 may have an
important role as a catalytic bioscavenger for OP intoxication. Indeed, huPON1 is a human
protein, thus minimizing possible immune responses, and protects animals against the toxicity of
OP insecticides such as CPO and DZO. For some substrates (e.g. PO, sarin), however, the
catalytic efficiency of PON1 has to be enhanced by one or two order of magnitude in order to
provide adequate protection. The recently achieved expression of active human PON1 in E.Coli,
will allow modifications of the protein to increase its catalytic efficiency. Such engineered
PON1s will be useful as a treatment for acute OP insecticide poisoning, and as a preventive
therapy for possible exposure to OP nerve agents, and may also find use in the treatment of
vascular disease.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Progress in Using Recombinant Forms of Paraoxonase 1 to Develop a
Catalytic Bioscavenger of Nerve Agents
Tamara C. Otto1, Christina K. Harsch2, Thomas J. Magliery2, Douglas M. Cerasoli1 and David E. Lenz1
1
US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD 210105400
2
Department of Chemistry, The Ohio State University, 100 W. 18th Avenue, Columbus, OH 43210-1185
Human paraoxonase 1 (HuPON1) is a naturally occurring plasma enzyme that exhibits catalytic
activity toward both G-type (GB and GD) and V-type (VX and VR) nerve agents. The catalytic
mechanism mediating this activity is, as yet, unresolved. The recent solution of the crystal
structure of a gene-shuffled chimeric (rabbit, human, rat, mouse) recombinant of PON1
(crePON1) has provided insight into the active site of this enzyme, and has led to a proposed
mechanism for hydrolysis of at least some classes of substrates. Utilizing crystallographic data,
mutants of PON1 have been made by rational design and expressed in either human 293T cells
or E. coli. While enzymes from both sources react with paraoxon and nerve agents, differences
in the kinetic properties of these enzymes have been observed. We have conducted a detailed
study comparing the ability of five mutants of HuPON1 and seven crePON1 variants to catalyze
the hydrolysis of VX, VR and five V agent structural analogs. We have also investigated the
stereospecificity of the hydrolysis of the nerve agent GD by HuPON1 and crePON1. The results
suggest that there are subtle but significant differences in the activity of these two forms of
PON1 with respect to nerve agent hydrolysis. We have indentified several residues that appear
to be involved in substrate binding to HuPON1, and we have gained insights into the overall
structural constraints of the active site binding pocket. This information will aid in the design of
additional mutants with enhanced catalytic activity against nerve agents.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Biomarkers of Sensitivity and Exposure in Washington State
Pesticide Handlers
JN Hofmann1, R Stevens2, M MacCoss3, D Goodlett4, AC Sherl4, MC Keifer5, H Checkoway1,5, JH
Kim2,6, RJ Richter2, SM Suzuki2, FM Farin5, AJ De Roos1,7, CE Furlong2,3.
Depts. of 1Epidemiology, 2Medicine - Division of Medical Genetics, 3Genome Sciences, 4Medicinal Chemistry,
5
Environmental and Occupational Health Sciences, and 6Anesthesiology, Univ. of Washington, Seattle, WA
7
The Fred Hutchinson Cancer Research Center, Seattle, WA
Since 2004, the State of Washington has implemented a program for monitoring serum
cholinesterase (BChE) activity in pesticide handlers who work in agriculture. Measurements are
made prior to and during the spraying season. If levels drop below 60% of the baseline levels for
an individual, they are removed from exposures until the level returns to above 80%.
Involvement in this process has provided an opportunity to examine the relationship between
PON1 status and organophosphate sensitivity. Pesticide handlers in Washington State were
recruited during the 2006 and 2007 spray seasons when they were seen by collaborating medical
providers as part of the statewide ChE monitoring program. Blood samples were collected from
163 participants and tested for PON1Q192R functional phenotype, PON1 genotype and plasma
levels of PON1 [arylesterase (AREase)]. Percent change in BChE activity from baseline levels
was evaluated relative to PON1 status. Participants with low AREase activity experienced a
significantly greater degree of BChE inhibition than participants with high AREase activity
(mean BChE inhibition of -8.44% and -3.27%, respectively; p=0.017). Greater BChE inhibition
was observed among PON1Q192 homozygous individuals relative to PON1R192 homozygous
individuals (mean BChE inhibition of -8.08% and -3.80%, respectively); this difference was
statistically significant after adjustment for AREase activity (p=0.028).
In addition to using BChE inhibition as a biomarker of exposure, we have been
developing protocols for analyzing OP modified protein biomarkers of exposure. The approach
involves the development of rapid protocols for extraction of the target biomarker protein from a
sample, digesting with the appropriate enzyme and identifying the OP modified peptide by mass
spectrometry. We are expressing active biomarker proteins in an E. coli system to provide heavy
isotope labeled standards to use in quantifying the degree of modification.
Supported by: NIOSH 1 U50 OH07544, NIEHS ES04696, ES09883, P30ES07033, ES09601/EPA: RD-83170901.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Paraoxonase 2 (PON2) attenuates triglyceride biosynthesis and accumulation
in macrophages via inhibition of diacylglycerol acyltransferase 1 (DGAT1)
Mira Rosenblat, Raymond Coleman*, Srinivasa T. Reddy** and Michael Aviram
The Lipid Research Laboratory, Technion Faculty of Medicine, the Rappaport Family Institute for Research in the
Medical Sciences, Rambam Medical Center,
* Department of Anatomy and Cell Biology, Technion Faculty of Medicine, Haifa, Israel, ** Atherosclerosis
Research Unit, Division of Cardiology, Department of Medicine, University of California Los Angeles, Los
Angeles, CA 90095-1679, United States.
Since triglycerides accumulate in atherosclerotic lesions, and as paraoxonase 2 (PON2) was
shown to inhibit atherosclerosis development, our aim was to examine the possible role and
mechanism of action of paraoxonase 2 (PON2), on macrophage triglyceride accumulation. In the
livers and aortas (unlike the serum) of PON2-deficient mice vs. the control C57BL/6 mice,
increased oxidative stress and elevated triglyceride levels were observed, whereas the cholesterol
levels were similar. Mouse peritoneal macrophages (MPM) harvested from PON2-deficient mice
vs. control C57BL/6 mice (5 months old), were found to be larger and filled with lipid droplets,
resembling foam cells. Triglyceride content and total peroxide levels in PON2-deficient vs.
control MPM were significantly increased by 4.6 fold and by 86%, respectively, whereas cellular
cholesterol content was similar. Macrophage triglyceride biosynthesis rate was substantially
increased, by 3.6 fold, in PON2-deficient vs. control MPM, whereas cellular triglyceride
degradation rate was almost similar in both groups. Microsomal acylCoA: diacylglycerol
acyltransferase 1(DGAT1), activity was significantly increased, by 4.4 fold, in PON2KO vs.
control MPM, while DGAT1 mRNA and protein levels were similar in both groups. We next
transfected PON2-deficient MPM with a vector containing the human PON2 (hPON2) plasmid,
or with an empty vector as a control. Three days after transfection, macrophage triglyceride
content, microsomal DGAT1 activity, and cellular oxidative stress (as measured by superoxide
anion release) in hPON2-transfected cells were all significantly decreased, by 53%, 60%, and
48%, respectively, as compared to control cells. Finally, incubation of PON2-deficient MPM
with increasing concentrations of recombinant PON2, also significantly decreased cellular levels
of lipid peroxides and DGAT1 activity, by up to 59% and 71%, respectively.
We conclude that PON2 attenuates triglyceride accumulation in macrophages via inhibition of
microsomal DGAT1 activity, and this phenomenon was associated with a significant reduction in
cellular oxidative stress.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Increased Susceptibility to diet-induced atherosclerosis
and cholestasis in PON3KO mice
Janet Yu, Yu-Rong Xia, Yi-Shou Shi, XuPing Wang, Aldons J. Lusis, and Diana M. Shih
Division of Cardiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
PON3 is a member of the paraoxonase gene family that includes PON1, PON2, and PON3.
PON3 and PON1 share approximately 60% identity at the amino acid level. Recent studies have
demonstrated that PON3 is present in human and rabbit HDL but not in mouse HDL. Mouse
PON3 appears to be cell-associated and is expressed in a wide range of tissues such as liver,
adipose, macrophage and the artery wall. In vitro studies have shown that PON3 can prevent
LDL oxidation and destroy bacterial quorum sensing molecules. Our previous study also showed
that PON3 transgenic mice were protected against obesity and atherosclerosis. To further
examine the role of PON3 in atherosclerosis, we have generated PON3 knockout (KO) mice
through standard gene targeting techniques and backcrossed them ten times onto the C57BL/6J
genetic background. Female PON3KO and wild-type mice were fed an atherogenic diet
containing 15.75% fat, 1.25% cholesterol, and 0.5 % cholic acid for 16 weeks to induce
atherosclerosis. Unexpectedly, we observed that 32% (8/25) of PON3KO mice died before the
end of the feeding study, whereas only 4% (1/24) of the wild-type female mice died (Mantel-Cox
Logrank test, p = 0.01). We observed that, except for HDL cholesterol, plasma total cholesterol,
free cholesterol, and triglycerides were significantly higher in the PON3KO mice as compared to
the wild-type mice. Plasma total bile acid levels were also significantly elevated in the PON3KO
mice (269 + 62 μM) as compared to those of the WT mice (83 + 9 μM, p = 0.001). In addition,
the PON3KO mice exhibited highly elevated levels of hepatotoxicity markers in circulation,
including ALT, AST, and bilirubin, as compared to the wild-type mice. The hepatic total
cholesterol contents in the PON3KO mice were also significantly elevated as compared to the
wild-type mice. Furthermore, mean atherosclerotic lesion areas at the aortic root region of the
PON3KO mice were 63% larger than those of the WT mice (9090 + 1360 vs. 5570 + 700
μm2/section, p < 0.05). In summary, our data demonstrate that female PON3KO mice are more
susceptible to cholestasis and atherosclerosis as compared to the WT mice. Our study suggests a
role for PON3 in bile acids metabolism and atherosclerosis.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Role of PON2 in atherosclerosis and inflammation
Srinu Reddy
Paraoxonases are a group of enzymes with established properties and enzyme activities that
provide relief from toxic environmental chemicals as well as physiological oxidative stress. My
laboratory is interested in understanding the physiological function and role of paraoxonases
(especially PON2 and PON3) in Atherosclerosis and Inflammation. I will discuss recent studies
that further demonstrate a role for PON2 in the development of atherosclerosis. Briefly, we have
generated PON2-def/ApoE-/- mice. On a chow diet, plasma VLDL/LDL cholesterol levels were
significantly elevated from ApoE-/- mice; however, plasma VLDL/LDL cholesterol
concentrations were significantly reduced (p<0.01) in the PON2-def/ApoE-/- mice when placed
on a high fat western diet. HDL and LDL isolated from PON2-def/apoE-/- mice were more
inflammatory as measured by their ability to prevent or promote monocyte chemotactic activity,
respectively, when compared to corresponding lipoproteins from apoE-/- mice independent of
the diet. Atherosclerotic lesions were significantly larger (p<0.001) in PON2-def/apoE-/- mice
on both standard chow and high fat western diet, when compared to apoE-/- mice on the
corresponding diet. Enhanced inflammatory properties of LDL, attenuated anti-atherogenic
capacity of high-density lipoprotein, and a heightened state of oxidative stress in PON2-deficient
macrophages appear to be the main mechanisms behind the larger atherosclerotic lesions in
PON2-deficient mice.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Oppositional regulation of paraoxonase-2 expression by endoplasmic
reticulum stress and disturbance of calcium-homeostasis
Sven Horke*, Ines Witte, Petra Wilgenbus, Sebastian Altenhöfer, Maximilian Krüger, Huige Li,
Ulrich Förstermann
From the Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany. *Send
correspondence to: [email protected]
Background- Paraoxonase-2 (PON2) is a ubiquitously expressed anti-oxidative protein.
Substantial amounts of PON2 were found in the endoplasmic reticulum (ER) and its expression
was induced by the ER stress pathway “unfolded protein response” (UPR). Moreover, PON2
reduced cellular susceptibility to UPR-induced caspase-3 activation. Using PON2 as a model for
a UPR-induced gene, we examined the connection(s) between ER stress-inducing mechanisms
and gene expression.
Methods and Results- ER stress was induced in endothelial cells by various compounds; gene
reporter analyses revealed transcriptional activation of PON2 expression irrespective of the ER
stress causing mechanism. We further show that UPR activation caused by compounds
interfering with protein modification (tunicamycin or dithiotreitol) increased PON2 mRNA and
protein levels. In contrast, UPR induction by disturbance of Ca2+-homeostasis (A23187 or
thapsigargin) resulted in PON2 mRNA degradation, which was mediated by the 5’-untranslated
region. In addition, A23187 and thapsigargin induced PON2 protein degradation by an unrelated
calpain-dependent mechanism. Both PON2 mRNA and protein degradation resulted from Ca2+deregulation rather than UPR activation, because PON2 degradation, but not UPR activation,
was inhibitable by an intracellular Ca2+-chelator. Functionally, PON2 protein levels correlated
inversely with caspase-3 activity; PON2 overexpression protected largely against tunicamycinand dithiotreitol-, but very moderately against thapsigargin-induced caspase-3 activation.
Conclusions- It appears that the primary effect of ER stress is an upregulation of PON2
transcription. However, secondary mechanisms (e.g. those leading to enhanced cytosolic Ca2+)
can obliterate the primary effect and could cause PON2 degradation. Thus, the degree of
cytoprotection achieved against ER stress depends on the net PON2 protein level.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
IMMUNOHISTOCHEMICAL ANALYSIS OF PARAOXONASES-1, 2, AND
IN HUMAN ATHEROMA PLAQUES
Judit Marsillach1, Bharti Mackness2, Michael Mackness2, Francesc Riu1, Raul Beltrán1, Jorge
Joven1, Jordi Camps1
1
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Institut d’Investigacions Sanitàries Pere Virgili,
Universitat Rovira i Virgili, Reus, Spain. 2 Division of Cardiovascular and Endocrine Sciences, University of
Manchester, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, UK
Background/Aims: The paraoxonase (PON) multigene family comprises three members PON1,
PON2 and PON3. Paraoxonases protect LDL from oxidation, and play an anti-inflammatory and
anti-atherogenic role. We analysed the immunohistochemical localisation of PONs in excised
human aortas that had different degrees of atherosclerosis.
Methods: The aortas were obtained from necropsies of 15 patients with or without
atherosclerosis identified at post-mortem. PON1, 2, and 3 antibodies were obtained by
inoculating rabbits with peptides derived from specific sequences of mature PONs. Macrophages
were detected by the CD68 antigen; fibroblasts and endothelial cells by the CD34 antigen;
smooth muscle cells by α-smooth muscle actin; monocyte chemoattractant protein-1 (MCP-1) by
a polyclonal antibody. Fibrosis extent was assessed using Masson’s trichrome stain.
Results: In normal aortas, PON1 and PON3 were expressed only in smooth muscle cells of the
media layer. In aortas with atherosclerosis, PON1 and PON3 were still present in muscular cells
of the external area of the media layer where the histological structure was better preserved, but
not in the areas with an altered structure. In these samples, PON1 and PON3 were also expressed
in macrophages, and co-localised with MCP-1 and CD68. We did not observe any PON2
expression in the aortas examined.
Conclusion: The observation that PON1 and PON3 co-localise in macrophages, together with
MCP-1, supports the concept of a local anti-oxidant and anti-inflammatory role for these
proteins. Further studies are needed to confirm the results for PON2.
*Also presented as poster.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P1
PARAOXONASE-1 GENE Q192R POLYMORPHISM IS NOT RELATED
TO THE EXTENT OF ATHEROSCLEROSIS
Ahmet Bayrak1, Ali Deniz2, Tülin Bayrak1, Lale Tokgözoğlu2, Bilge Volkan Salancı3, Ediz
Demirpençe1, Mehmet Ali Kaşifoğlu3
Departments of Biochemistry1 , and Cardiology2 and Pediatric Genetic Unit3 , School of
Medicine, Hacettepe University, Ankara, Turkey
Introduction: Paraoxonase-1 (PON1) is a HDL-associated enzyme. It was first defined as an
organophosphate-hydrolysing enzyme and later it has been shown that PON1 protected LDL
particles from oxidative injury by hydrolysing lipoperoxides. Therefore, PON1 was defined as a
protective factor against atherosclerosis. The aim of this study is to investigate the relation
between PON1-related parameters (serum PON1 activity, serum PON1 concentration, PON1
Q192R polymorphism), and the extent and severity of atherosclerosis.
Method: Blood specimens were collected from 142 individuals who had no coronary artery
lesions angiographically (control group) and 146 individuals who had angiographicallydocumented coronary artery disease at several degrees (patient group). The extent and severity of
arterial lesions were evaluated by Gensini scoring system. PON1 activity was measured in serum
using a spectrophotometric method. PON1 concentration was measured using ELISA. Q192R
polymorphism was evaluated using RFLP after DNA isolation from samples.
Results: Serum PON1 activity was significantly lower in the patient group than controls (126
[42-270] U/mL vs 145 [46-307] U/mL; p<0,05). Serum PON1 concentration was also
significantly lower in the patient group than controls (1,44±0,94 mg/dL vs 2,06±0,84 mg/dL,
p<0,001). In the patient group, there was a negative correlation between PON1 activity, and the
extent and severity of atherosclerosis (r=-0,270; p=0,002). In both groups, there was no
significant difference in the distribution of Q192R polymorphism.
Conclusion: It has been concluded that serum PON1 activity is a more significant factor than
PON1 genotype in determining the extent and severity of atherosclerosis.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P2
Paraoxonase 2 deficiency aggravates atherosclerosis and affects lipid
metabolism in apoE null mice
Bourquard N1,2, Hama SY1, Su F3, Farias-Eisner, R3, and Reddy ST1,2
Department of Medicine1, Molecular and Medical Pharmacology2, and Department of Obstetrics and
Gynecology3, David Geffen School of Medicine, UCLA, Los Angeles, California 90095
Objective. Paraoxonase 2 (PON2) is a widely expressed intracellular enzyme with antioxidant
and anti-inflammatory properties, and has been implicated in the pathogenesis of several
inflammatory diseases including atherosclerosis. PON2 deficiency in C57BL/6J mice fed a high
fat, cholate-containing atherogenic diet accelerates fatty streak formation in the aortic sinus and
significantly affects both the metabolism and the inflammatory properties of lipoproteins relative
to wild-type control mice. To further evaluate the role of PON2 in atherosclerosis and lipoprotein
metabolism, we generated PON2-deficient (PON2-def) mice on an apoE null background
(PON2-def/apoE-/-).
Methods and results. PON2-def/apoE-/- and littermate apoE-/- mice were placed on either
standard chow or high fat western diet for 16 weeks. On a chow diet, plasma VLDL/LDL
cholesterol levels were significantly elevated in the PON2-def mice; however, plasma
VLDL/LDL cholesterol concentrations were significantly reduced in the PON2-def/ApoE-/mice on high fat western diet (1346 vs. 1077 mg/dL, p<0.01). Interestingly, HDL and LDL
isolated from PON2-def/apoE-/- mice were more inflammatory as measured by their ability to
prevent or promote monocyte chemotactic activity, respectively, when compared to
corresponding lipoproteins from apoE-/- mice independent of the diet. Atherosclerotic lesions as
measured by both the aortic root sinus lesion area and en face analysis were significantly larger
in PON2-def/apoE-/- mice on both standard chow and high fat western diet, when compared to
apoE-/- mice on the corresponding diet.
Conclusion. We conclude that PON2 plays an important role in both the development of
atherosclerosis, as well as in the inflammatory properties of lipoproteins in mice. Our results
suggest that PON2 may be a novel therapeutic target for the treatment of atherosclerosis. This work was supported by the NHLBI Grant 1RO1HL71776
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P3
Hypochlorous acid (HOCl) is a potent inactivator of human proteins
responsible for protection against thrombosis and LDL oxidation: a
comparative study on plasminogen and paraoxonase 1
Alejandro Gugliucci and John Schulze. Glycation, Oxidation and Disease Laboratory, Touro UniversityCalifornia, Vallejo, CA. USA
Background. Myeloperoxidase (MPO), released from degranulated neutrophils, has been
considered an important pathophysiological factor in oxidative stress. Through generation of
hypochlorous acid it plays a key bactericidal role, however, if it goes unchecked, its activity may
lead to inactivation of important proteins. This may occur in the inflammatory microenvironment
of the atheroma plaque. Oxidation of amino acid residues such as tyrosine, leading to the
formation of 3-chlorotyrosine, has been recently shown to inactivate paraoxonase-1 (PON-1) in
HDL. Plasminogen is an important check for thrombus formation, possesses 25 tyrosine residues
and has been previously shown by us to be inactivated by nitration.
Hypothesis. We hypothesized that plasminogen can be also be inactivated by HOCl in a similar
way as PON-1 is, and this inhibition can be counteracted by cysteine.
Methods. Human plasminogen (10 μmol/L) or human HDL prepared by sequential flotation
ultracentrifugation were incubated in PBS containing 2 mmol/L CaCl2, pH 7.4, at 37°C for up to
3 h in the presence or absence of freshly prepared HOCl (0-1000 µmol/L). After extensive
dialysis, spreptokinase-activated plasmin activity was kinetically measured using a synthetic
pNA substrate and PON-1 activity was kinetically measured using paraoxon as a substrate. SDSPAGE gels were run to monitor protein aggregation/fragmentation and Western blots (anti-3
chlorotyrosine antibodies) allowed for monitoring of protein adduct formation.
Results: We confirm inactivation of PON-1 in HDL which achieves over 95% inhibition at 1
mmol/L (IC 120 μmol/L). This correlates with changes in molecular weight and 3-chlorotyrosine
formation. When plasminogen was incubated under the same conditions plasmin activity
(generated by streptokinase) is reduced in a time and concentration dependent fashion (IC 40
μmol/L). Inhibition reaches a plateau of 70 % inhibition at 50-100 μmol/L. Cysteine and taurine
protected both proteins from inactivation in a concentration dependent fashion.
Conclusions: This study addressed the effects of HOCl on 2 key players in the overall process of
atherogenesis. Neutrophils can secrete HOCl at 100 μmol/L. Our data on quick functional
inactivation of plasminogen by chlorination, at IC concentrations 3 times lower than those
needed for inactivation of PON-1, adds a new pathophysiological dimension to our previous
work showing plasminogen as a target for peroxynitrite damage. They suggest that MPO and
HOCl may also be implicated in impaired fibrinolysis as well as in PON-1 inactivation. New
therapeutic approaches in atherosclerosis and diabetes should limit the formation of HOCl,
taurine and thiols may prove beneficial.
Supported by Touro University-California
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P4
Study of Serum Paraoxonase (PON1) activity in Coronary Artery Disease
patients with & without Type 2 Diabetes Mellitus in North Indians
Nidhi Gupta*, Surjit Singh**, Yash Paul Sharma***, K.D. Gill*
Departments of *Biochemistry, **Internal Medicine, ***Cardiology, Postgraduate Institute Of Medical
Education & Research. Chandigarh-160012, India
Background- Paraoxonase (PON1) is an arylesterase associated with high density lipoprotein
cholesterol (HDL-C). By preventing low density lipoprotein cholesterol (LDL-C) from
peroxidation and hydrolysis of lipid peroxides, it is supposed to provide protection against
atherosclerosis & Coronary Artery Disease (CAD).The incidence of CAD is known to be high in
North Indians. Though many factors play a role in it’s pathogenesis, low PON1 activity could be
an independent risk factor.
Methods & Result- We carried out this study to determine PON1 activity in patients with CAD
with and without type II diabetes mellitus and compared it with healthy individuals. A total of
170 patients with angiographically proven CAD (50 with and 120 without type II diabetes) and
100 healthy controls were studied for serum PON1 activity & lipid variables.
Conclusions- On univariate analysis of variance after adjusting for age and sex, no significant
difference could be observed between PON1 activity and age and sex. Significantly lower serum
PON1 activity (P < 0.001) along with lower HDL- C (P < 0.001) levels were observed in CAD
patients as compared to healthy controls. When PON 1 activity was compared between patients
of CAD with & without diabetes, significant difference could be observed (P<0.01). On the basis
of our result, it may be concluded that low serum PON1 activity could be an independent risk
factor for CAD & may provide a therapeutic measure for CAD.
( Along with that we are conducting PON1 coding (Q192 R & L55M) & Promoter ( -108 T/C , 162 A/G & -909 C/G ) polymorphism in CAD patients ( N = 375), CAD patients with T2DM
(N = 375 ) , T2DM patients ( N = 175 ) & Healthy controls individuals ( N = 175 ). These results
will be correlated at the time of presentation.)
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P5
PON1 activity decreasing in stroke patients
Jelena Kotur-Stevuljevic, Aleksandra Stefanovic, Ana Vujovic, Vesna SpasojevicKalimanovska, Zorana Jelic-Ivanovic, Sandra Samardzic, Tatjana Ivanic-Corlomanovic, Slavica
Spasic
Institute for Medical Biochemistry, Faculty of Pharmacy, Belgrade, Serbia
Background: Atherosclerosis and dyslipidemia [elevated low-density lipoprotein cholesterol
(LDL-C) and reduced high-density lipoprotein cholesterol (HDL-C)] are well-known risk factors
for stroke development. Paraoxonase 1 (PON1) is HDL-associated enzyme responsible for its
anti-oxidative properties. The aim of this study was to test PON1 enzyme activity and oxidative
stress status of stroke patients.
Methods: Serum paraoxonase (POase), diazoxonase (DZOase) activities, malodialdehyde
(MDA), superoxide anion (O2.–), advanced oxidation protein products (AOPP), lipid
hydroperoxides (LOOH), superoxide dismutase (SOD) activity and sulphydril (SH) groups
content were measured in fasting samples of 200 stroke patients and 102 healthy subjects
(control group, CG). Lipid status parameters were also measured.
Results: POase activity was significantly lower in stroke patients vs. CG (79±72 U/L vs.
476±361 U/L, P<0.001), such as SOD activity (P<0.001) and SH groups content (P<0.001).
MDA, O2.–, AOPP, LOOH levels were statistically higher in patients comparing to control.
DZOase activity was comparable between the two experimental groups. Moreover, stroke
patients had lower HDL-C than healthy people (0.94±0.32 vs. 1.44±0.35, P<0.001).
Conclusions: Serum PON1 POase activity showed marked decrease in stroke patients along with
decreased HDL-C concentration. Stroke patients were in exacerbated oxidative stress which was
apparent through elevation of MDA, O2.–, AOPP and LOOH levels and also by weakened
antioxidative defence, which could be a reason for significantly diminished POase activity in this
acute condition.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P6
Relationship between Paraoxonase2 C311S polymorphism and Acute
Coronary Syndrome
1
2
1
F. Marchegiani , L. Spazzafumo , F. Olivieri3, M. Cardelli , L. Galeazzi1, F. Lattanzio4, C.
5,6
7
Franceschi , R. Antonicelli .
1
2
Center of Molecular Biology and Genetics, Center of Statistics, 4Scientific Direction, 7Cardiology-CCU Unit,
Italian National Research Centers on Aging (I.N.R.C.A.), Ancona, Italy
3
Faculty of Medicine, Polytechnic University of Marche, Ancona, Italy
5
6
Department of Experimental Pathology and CIG, Interdipartimental Centre “L. Galvani”, University of Bologna,
Bologna, Italy
Objectives: It is well known that oxidative stress plays a role in atherosclerosis and age-related
diseases. Human Paraoxonase gene family (PON1, 2, 3) has been widely investigated for its
antioxidant properties and for its possible involvement in cardiovascular disease. In this casecontrol study we aim to study the relationship between the C311S PON2 polymorphism and the
risk of acute coronary syndrome (ACS).
Methods: We analysed PON2 C311S polymorphism in a total of 459 elderly patients who
underwent an acute coronary syndrome (ACS): 107 patients affected by unstable angina (UA),
215 AMI (acute myocardial infarction) patients affected by STEMI (ST-Elevation) and 137 AMI
patients affected by NSTEMI (No ST-Elevation). For all the patients a follow-up of 12 months
has been performed to evaluate the following outcomes: new cardiovascular events, rehospitalizations, and mortality. In addition, 459 control subjects matched with patients for sex
and age characteristics were recruited. PON2 C311S genotypes were grouped and analyzed as C(SS genotype) and C+ (CS+CC) carriers.
Results: We found that patients carrying CS+CC genotypes (C+ carriers) showed higher levels
of TroponinI (TnI) compared to patients carrying SS genotype, also when they are separately
considered on the basis of the diagnosis of UA, STEMI and NSTEMI. C+ carriers were
significantly overrepresented among patients with an history of type 2 diabetes mellitus and
patients with low levels of HDL cholesterol (<30 mg/dl). PON2 C+ and C- carriers frequency
distributions were not statistically different between patients and control group. Also, these
carriers seem not be involved in the survival at 12 months from the event.
Conclusions: Our study suggests that, although not important for the survival and onset of
acute coronary syndrome, PON2 C311S polymorphism could be important in the pathogenesis
of cardiac ischemic damage. In fact, C+ carrying patients show higher levels of TnI, an
important marker of the extension of myocardial ischemic damage and they are also more
represented among patients with an history of type 2 diabetes mellitus and with low levels of
HDL cholesterol, two typical risk factors for cardiovascular disease.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P7
PON1 ACTIVITIES IN METABOLIC SYNDROME AND
RISK OF CORONARY ARTERY DISEASE
Nicola Martinelli1, Olga Khersonsky2, Dan S. Tawfik2, Patrizia Guarini1, Domenico Girelli1, Oliviero
Olivieri1, Antonella Bassi3, Elisabetta Trabetti4, Leonid Gaidukov2, Simonetta Friso1, Francesca Pizzolo1,
Laura Annarumma1, Renzo Schiavon5, Pier Franco Pignatti4, Roberto Corrocher1.
1
Department of Clinical and Experimental Medicine, University of Verona, Italy
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
3
Institute of Clinical Chemistry, University of Verona , Italy
4
Section of Biology and Genetics, Department of Mother and Child and Biology – Genetics, University of Verona,
Italy
5
Laboratory of Clinical Chemistry, Hospital of Legnago, Verona
2
Background: oxidative stress pathways are thought to play a pivotal role in metabolic syndrome
(MS). Low levels of high-density lipoprotein (HDLs) are one of the most characteristic element in the
cluster of MS. The antioxidant ability of HDLs is largely attributable to serum paraoxonase (PON1).
Methods: MS diagnosis and different PON1 activities (versus classical substrates, i.e. paraoxon
and phenyl acetate, as well as versus novel substrates, i.e. 5-thiobutyl,butyrolactone (TBBL) and 7-Odiethyl phosphoryl,3-cyano,4-methyl,7-hydroxycoumarin (DEPCyMC)) have been analysed in 293
subjects with or without angiographically proven coronary artery disease (CAD).
Results: all the four analysed PON1 activities were lower in MS and there was a decreasing trend
of PON1 activities levels by increasing the number of MS abnormalities. The most significant association
was found for DEPCyMCase activity, which may be considered a surrogate marker of PON1
concentration (20.60±6.05 in MS versus 23.8±5.6 mU/ml in no-MS, P=1.58×10-5; P for linear trend
across the number of MS-abnormalities=3.84×10-6). These associations were independent from HDL and
apolipoprotein A-I concentrations. Moreover, subjects with low HDL levels but without MS had a
DEPCyMCase activity higher than subjects with MS and equally low HDL levels, and similar with that
of subjects with high HDL levels and without MS.
Both low DEPCyMCase activity and MS were independently associated with CAD. Noteworthy,
when compared with no-MS group only MS subjects with concomitant low levels of DEPCyMCase
activity presented an increased risk of CAD (OR 3.54 with 95%CI 1.27-9.92).
Conclusions: our results suggest that a low PON1 concentration may be a characteristic of MS
and may modulate the CAD risk associated with MS.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P8
The Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone
induces inflammatory response in the human aortic endothelial cells
Yu-Rong Xia, Brian Kim, Yi-Shou Shi, Aldons J. Lusis, Judith A. Berliner, Srinivasa Reddy,
and Diana M. Shih
Infectious agents such as Chalmydia pneumoniae, Helicobacter pylori, and cytomegalovirus have been
associated with cardiovascular disease. It is postulated that chronic infections induce inflammation and
promote atherogenesis. Pseudomonas aeruginosa is an opportunistic pathogen that significantly
contributes to morbidity and mortality in patients with cystic fibrosis. It is also a leading source of
opportunistic nosocomial (hospital acquired) and a significant cause of community acquired infections. A
recent study has shown that rats with chronic lung infection of Pseudomonas aeruginosa exhibited
increased atherosclerosis as compared to the uninfected controls, suggesting a relationship between P.
aeruginosa infection and atherosclerosis. P. aeruginosa utilizes quorum sensing molecules such as N-3oxododecanoyl homoserine lactone (3O-C12-HSL) to regulate genes involved in virulence and biofilm
formation. There is growing evidence that 3O-C12-HSL also exerts biological effects on host cells, such
as macrophages and epithelial cells. Recent reports also demonstrated that all of the three paraoxonase
gene family members, PON1, PON2, and PON3, can hydrolyze and inactivate 3O-C12-HSL, suggesting a
novel role of PON proteins in host defense. In this study we utilized global gene expression profiling and
quantitative-RT-PCR techniques to examine the effects of 3O-C12-HSL in altering gene expression of
primary human aortic endothelial cells (HAEC). We observed that HAEC treated with 25 μM 3OC12HSL for 5 hours showed increased expression of the adhesion molecules such as ICAM-1 and E-selectin
by 2.5 and 5.2 fold, respectively, as compared to the vehicle-treated cells, whereas 50 μM 3OC12-HSL
treatment for 5 hours exhibited increased expression of ICAM-1 and E-selectin by 5.4 and 27 fold,
respectively, as compared to the controls. In addition to ICAM-1 and E-selectin, both of which are NF-κβ
target genes, 16 additional NF-κβ target genes are up-regulated by the treatment of 50 μM 3OC12-HSL
for 5 hours. These include COX2 (1.9 fold), HO-1 (2.3 fold), interleukin-1β (4.5 fold), and interleukin-8
(2.5 fold). Genes involved in unfolded protein response (UPR), such as XBP-1, and ATF3, are also
induced by 50 μM 3OC12-HSL treatment for 5 hours, by 1.6 fold and 2.5 fold, respectively. In addition,
genes involved in apoptosis, such as caspase 6, is also induced by 50 μM, but not 25 μM, 3OC12-HSL
treatment for 5 hours. In summary, our data suggest that 3OC12-HSL promotes the expression of genes
involved in leukocyte-endothelial cell interaction, inflammation, UPR, and apoptosis in HAEC.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P9
Paroxonase activity in high density lipoproteins (HDL):
guardian angel of cell membrane?
Tiziana Bacchetti, Simona Masciangelo, Gianna Ferretti
Istituto di Biochimica, Università Politecnica delle Marche
High density lipoproteins (HDL) are inversely correlated with the risk of atherosclerosis and
coronary heart disease (1). The protective effect exerted by HDL has been related to their role in
cholesterol reverse transport (from peripheral tissue to the liver) and to their ability to inhibit
lipid peroxidation, an atherogenic modification of low density lipoproteins (LDL) (1-3). Several
lines of evidences suggest that the protective effect of HDL is at least partially related to the
enzyme paraoxonase (PON1), a calcium dependent esterase associated with HDL surface (4).
Studies in vitro have demonstrated that PON1 is able to hydrolyze oxidized fatty acids in
oxidized lipoproteins and/or biological membranes (5). Furthermore it has been demonstrated
that PON1 hydrolyzes homocysteine thiolactone (HTL), a toxic metabolite of homocysteine (6).
Therefore it has been hypothesized that PON1 may have two independent antiatherogenic roles:
a) by preventing the accumulation of oxidized lipids from oxidized lipoproteins (LDL and HDL)
and thereby by inhibiting the atherogenic and inflammatory response induced by lipid
peroxidation products; b) by detoxifying HTL involved in homocysteinilation of plasma proteins
and lipoproteins (6). A lower PON1 activity has been observed in patients affected by diseases
associated with oxidative damage and with a higher risk for coronary artery disease such as in
insulin-dependent and non-insulin dependent diabetes, obesity, hypercholesterolemia, renal
failure (7-9). The lower PON1 activity is associated with higher levels of lipid peroxides
associated with HDL or LDL isolated from plasma of the patients (7-9). Several studies have
demonstrated that oxidatively-modified HDL (Ox-HDL), show structural and functional
alterations (3), thereby we may expect that HDL with lower PON1 activity protect less
efficiently circulating cells against oxidative damage. To further investigate the role exerted by
HDL and PON1 against oxidative damage of biological membranes, we incubated oxidativelymodified erythrocyte membranes (OX-membranes) with HDL isolated from normolipemic
subjects or from patients affected by of PON activity. A decrease of lipid hydroperoxides has
been demonstrated in OX-membranes incubated with HDL and HDL of subjects with higher
PON1 activity protect more efficiently erythrocytes against oxidative damage and we confirm
the significant relationship between PON1 activity and the antioxidant role of HDL. Bowry et al
(10) have demonstrated that HDL are the main carriers of lipid hydroperoxides from oxmembranes to liver. Recently, Rosenblat et al (11) reported that HDL-associated PON1 could
play a regulatory role also in HDL binding to cell membrane and in HDL-mediated cholesterol
efflux in macrophages. Studies are in progress to better investigate the interactions between HDL
and biological membranes and the molecular mechanism of HDL –mediated efflux of lipid
peroxides in normal and pathological conditions.
References
1. Tall AR Cholesterol efflux pathways and other potential mechanisms involved in the atheroprotective effect of high density lipoproteins. J Intern Med 2008 ;263(3):256-73
2. Negre-Salvayre A, Dousset N, Ferretti G, Bacchetti T, Curatola G, Salvayre R. Antioxidant
and cytoprotective properties of high-density lipoproteins in vascular cells. Free Radic Biol
Med. 2006;41:1031-40.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
3. Ferretti G, Bacchetti T, Salvayre AN, Salvayre R, Dousset N, Curatola G. Structural
modifications of HDL and functional consequences. Atherosclerosis. 2006;184:1-7
4. Aviram M, Rosenblat M.Paraoxonases and cardiovascular diseases: pharmacological and
nutritional influences. Curr Opin Lipidol. 2005;16(4):393-9.
5. Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M.
Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the
biological activity of minimally oxidized low density lipoprotein. J Clin Invest.
1995;96(6):2882-91.
6. Jakubowski H.Protein homocysteinylation: possible mechanism underlying pathological
consequences of elevated homocysteine levels. FASEB J. 1999 ;13(15):2277-83.
7. Ferretti G, Bacchetti T, Masciangelo S, Nanetti L, Mazzanti L, Silvestrini M, Bartolini M,
Provinciali L. Lipid peroxidation in stroke patients Clin Chem Lab Med. 2008;46(1):113-7
8. Ferretti G, Bacchetti T, Masciangelo S, Pallotta G. Lipid peroxidation in hemodialysis
patients: Effect of vitamin C supplementation Clin Biochem. 2007;41(6):381-6.
9. Ferretti G, Bacchetti T, Moroni C, Savino S, Liuzzi A, Balzola F, Bicchiega V, Paraoxonase
activity in high density lipoproteins: a comparison between healthy and obese females. J
Clin Endocrinol Metab, 2005;90(3): 1728-33
10. Bowry VW, Stanley KK, Stocker R. High density lipoprotein is the major carrier of lipid
hydroperoxides in human blood plasma from fasting donors. Proc Natl Acad Sci U S A.
1992; 89(21):10316-20.
11. Rosenblat M, Karry R, Aviram M Paraoxonase 1 (PON1) is a more potent antioxidant and
stimulant of macrophage cholesterol efflux, when present in HDL than in lipoproteindeficient serum: relevance to diabetes. Atherosclerosis. 2006;187(1):74-81.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P10
Structural analysis of rePON1 mutants and complexes
Moshe Ben-David1, Israel Silman2, Dan S. Tawfik3 and Joel L. Sussman1
Departments of 1Structural Biology, 2Neurobiology and 3Biological Chemistry, The Weizmann
Institute of Science, Rehovot 76100, Israel
As its name implies, paraoxonase 1 (PON1) has the ability to hydrolyze toxic organophosphates
(OPs). It is thus considered to be a promising catalytic bioscavenger for nerve agents. Directed
evolution studies on PON1 produced a recombinant variant (rePON1-G2E6) that was overexpressed in soluble form in E. coli, thus permitting its crystallization, and the subsequent
determination of its 3D structure [1]. The crystal structure has shed considerable light on its
specificity and mechanism, in particular with respect to its lactonase activity [2]. However, the
residues that mediate its action as an OP hydrolase have yet to be unequivocally identified.
Recently evolved mutants of rePON1, in particular mutants bearing the H115W and V346A
mutations, display enhanced activity towards various OPs [3]. Consequently, their crystal
structure may provide insights as to how these mutations influence activity. So far, we have
obtained needle crystals of the H115W mutant that are too small for structure determination;
optimization of crystallization conditions is underway. In addition, we are attempting cocrystallization of rePON1 with various inhibitors, such as 2-hydroxyquinoline. The 3D structure
of such complexes will also permit mapping of the active site. This, in turn, will pave the way to
the rational design of PON1 mutants capable of rapid hydrolysis of a variety of toxic OPs.
References:
1. Harel, M., Aharoni, A., Gaidukov, L., Brumshtein, B., Khersonsky, O., Meged, R., Dvir, H.,
Ravelli, R.B.G., McCarthy, A., Toker, L., Silman, I., Sussman, J.L. & Tawfik, D.S. (2004)
"Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotc
enzymes." Nature Struct. Mol. Biol. 11:412-419.
2. Khersonsky O, Tawfik DS. (2005) "Structure-reactivity studies of serum paraoxonase PON1
suggest that its native activity is lactonase." Biochemistry 44:6371-6382.
3. Amitai G, Gaidukov L, Adani R, Yishay S, Yacov G, Kushnir M, Teitlboim S, Lindenbaum
M, Bel P, Khersonsky O, Tawfik DS, Meshulam H. (2006) "Enhanced stereoselective hydrolysis
of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase."
FEBS J. 273:1906-1919.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P11
Distribution of the PON1, PON2 and PON3 immunohistochemical
expressions in the tissues of normal mice
Judit Marsillach1, Bharti Mackness2, Michael Mackness2, Francesc Riu1, Raul Beltrán1, Jorge
Joven1, Jordi Camps1
1
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Institut d’Investigacions Sanitàries Pere Virgili,
Universitat Rovira i Virgili, Reus, Spain; 2University of Manchester, Cardiovascular Research Group, School of
Clinical & Laboratory Sciences, Manchester Royal Infirmary, UK.
Background/Aims: In contrast to the information provided by gene expression studies, there is a
paucity of information on the histological distribution of the PON family of proteins in different
tissues of humans or experimental animals. In this communication, we describe the
immunohistochemical localisation of the PON proteins in the normal mouse.
Methods: The study was performed in 13 tissues from 2 normal mice, male and female, from the
C57BL/6J strain. Antibodies were obtained by inoculating rabbits with peptides derived from
specific sequences of mature PONs. Immunohistochemical analyses were performed by using
ABC-peroxidase as a chromogenic system.
Results: PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous
glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and
the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal
cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes,
chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous
tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of
PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland,
nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium.
Apolipoprotein A-I did not co-localise with PONs, suggesting local synthesis.
Conclusion: This study provides with an experimental model to investigate the role played by
these enzymes as antioxidants and their relationship with the development of a variety of
diseases.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P12
Evaluation of a semi-automated method for the measurement of
the serum PON1 lactonase activity. Influence of genotypes and
application to the study of patients with liver disease
Judit Marsillach,1 Gerard Aragonès,1 Raul Beltrán,1 Joan Caballeria, 2 Joan C. Pedro-Botet,3
Carlos Morcillo,4 Arcadi Navarro,4 Jorge Joven,1 and Jordi Camps1
1
Centre de Recerca Biomèdica, Hospital Universitari de Sant Joan, Institut d’Investigacions Sanitàries Pere Virgili,
Reus, Spain. 2 Liver Unit, Hospital Clínic Universitari, Institut d’Investigacions Biomèdiques Agustí Pi i Sunyer,
Barcelona, Spain. 3 Department of Internal Medicine, Hospital del Mar, Barcelona, Spain.4 Centro Nacional de
Genotipado, Universitat Pompeu Fabra, Barcelona, Spain.
Background/Aims: We previously suggested that PON1 measurement may improve the
evaluation of liver function. However, the standard method uses a highly toxic substrate
(paraoxon) and is strongly influenced by PON1 genetic polymorphisms. The aims of the present
study were to (a) evaluate the analytical performance of a new lactonase activity PON1 assay;
(b) investigate the influence of genetic variability in a population-based study; (c) investigate the
efficacy of the lactonase assay in the assessment of liver damage.
Methods: We studied a sample of 633 Caucasian individuals and 369 patients with chronic liver
disease. Serum lactonase activity was determined by the hydrolysis of TBBL. Paraoxonase-1, 2,
and 3 gene polymorphisms were analyzed by the Iplex Gold MassArrayTM method.
Results: Lactonase assay was linear up to 10 U/L. Detection limit was 0.12 U/L. Inter-assay
imprecision was ≤ 17.7%. There were no interferences by jaundice, hemolysis, hyperlipemia, or
paraproteinemia. Median and 95% lactonase activity in our population were 5.99 (3.29-13.61)
U/L. Lactonase activity showed a lower influence by PON’s genetic polymorphisms than
paraoxonase activity. Both measurements showed a similar efficiency in testing for liver
dysfunction.
Conclusion: We reported a reliable assay for the measurement of serum lactonase activity, based
in a non-toxic substrate, with lower influence by genetic variability and that may add a
significant contribution to the evaluation of liver impairment.
Acknowledgments: We thank Drs. Dan Tawfik, Olga Khersonsky and Leonid Gaidukov from the
Weizmann Institute of Science, Rehovot, Israel, for the generous gift of the TBBL reagent.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P13
Purification and Characterization of Functional Human Paraoxonase 1
Expressed in Insect Larvae
Tamara C. Otto1, Elena Kovaleva2, Zhi Liu2, George Buchman2, Marita Tolosa2, Robert
Balcerzak2, David E. Lenz1 and Douglas M. Cerasoli1
1
US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD;
2
Chesapeake-PERL, Inc., Savage, MD
Human paraoxonase 1 (HuPON1) is notoriously difficult to either purify from plasma or
functionally express in high yield from recombinant sources. This may be because HuPON1
requires a cofactor for functionality (such as HDL association) or specific glycosylation not
provided by many hosts. Here, we describe the characterization of functional HuPON1
expressed and purified from cabbage loopers (Trichoplusia ni larvae) infected with an orally
active form of baculovirus. This baculovirus encodes the complete amino acid sequence of
HuPON1 with a C-terminal 6X-His tag. After clarification and filtration, samples of
homogenized insect larvae were bound to an IMAC column. Eluate was passed over G-25 and
anion exchange columns before analysis. The resulting samples yielded only three bands of
approximately 39, 41, and 43 kDa observed by both Coomassie and anti-HuPON1 western
analyses. MALDI-TOF confirmed the identity of each of the three bands as HuPON1, with
greater than 95% confidence. While the molecular basis for the three isoforms of HuPON1
remains unclear, deglycosylation experiments using PNGase F suggest that they result from
differential glycosylation of the enzyme. Purified samples of this recombinant HuPON1
hydrolyzed paraoxon and the organophosphorus nerve agents GD, VX and VR with kinetic
properties comparable to those of HuPON1 expressed in 293T cells.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P14
Paraoxonase 2 (PON2) localizes to and modulates
function of mitochondria in response to oxidative stress
Devarajan A1, Reddy ST2
Dept. of Medicine1, Molecular and Medical Pharmacology2, David Geffen School of Medicine, UCLA,
Los Angeles, CA 90095
Objective: Dysfunction of mitochondrial electron transport chain (ETC) complexes, play an
important role in the development of many inflammatory diseases including cardiovascular
diseases (1). Reduced activities of NADH-Ubiquinone oxidoreductase (Complex I) and
Ubiquinol cytochrome c oxidoreductase (complex III), result in the increased production of
reactive oxygen species (ROS). Although cytochrome oxidase (Complex IV) itself is not a
source of ROS, inhibition of complex IV facilitates ROS production via complexes I and/or III.
PON2 is a membrane-associated protein and its deficiency results in increased ROS
accumulation and atherosclerosis in mice (2). The mechanism by which PON2 contributes to
ROS production is not known. In the present study, we sought to determine i) whether PON2 is
localized to the mitochondrial respiratory complexes, and ii) whether PON2 expression
influences mitochondrial function.
Methods and Results. For localization studies, submitochondrial particles from the livers of
C57BL/6J wild type mice were isolated and mitochondrial respiratory complexes were
immunoprecipitated using immunocapture antibodies. PON2 was detected predominantly in the
inner mitochondrial membranes and was found associated with complex III. To study the
functional significance of PON2 in the respiratory complex, we compared the activities of
mitochondrial ETC complexes from the livers of wild type and PON2-def mice fed either a chow
or an atherogenic diet. On a chow diet, the activities of complex I+III and complex IV were not
significantly different in PON2-def mice compared to wild type mice (although lower in PON2def mice). Interestingly, on an atherogenic diet, complex I+III activity was ~50% lower (p<0.01)
in PON2-def mice when compared to wild type mice. Complex IV was also significantly lower
in PON2-def mice compared to wild type mice (395±26 vs. 330±40 cytochrome c
oxidized/min/mg/protein, p<0.05). Moreover, mitochondria from PON2-def mice on an
atherogenic diet had significantly elevated oxidative stress as measured by TBARS (p<0.05),
when compared to wild type mice. To determine whether overexpression of PON2 alleviates
mitochondrial oxidative stress, HeLa Tet-On-PON2 cells (PON2 gene under the control of a
tetracycline inducible promoter) and control HeLa Tet-On cells were either treated with
doxycycline (+) or left untreated (-) for 48 h and further incubated with hydrogen peroxide (300
mM) for 1 hour. Mitochondrial membrane potential was analyzed using the lipophilic cationic
dye 5,5’ 6,6’–tetrachloro 1-1’, 3-3’- tetrathylbenzimidazolyl carbocyanine iodide (JC1) by flow
cytometry. Interestingly, 45% of PON2 overexpressing cells had intact membrane potential in
contrast to only 14% in control cells, suggesting that PON2 overexpressing cells are able to
protect mitochondria from hydrogen peroxide-induced dysfunction.
Conclusion. PON2 is predominantly localized to the inner membranes of mitochondria, and
associated with complex III. PON2 deficiency decreases the activities of ETC complexes
resulting in increased oxidative stress under atherogenic conditions. Finally, overexpression of
PON2 in HeLa cells protects mitochondria from hydrogen peroxide-induced oxidative damage.
Our results suggest that PON2 plays an important role in mitochondrial function.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P15
Characterization and sequencing of a physiological partner of HPON1: the
Human Phosphate Binding Protein (HPBP)
Mikael Elias1, Hélène Diemer2, Frédérique Renault3, Daniel Rochu3, Carlos Contreras-Martel4,
Christine Schaeffer2, Alain Van Dorsselaer2, Eric Chabriere1
1
Architecture et Fonction des Macromolécules Biologiques, CNRS-Université de la Méditerranée, 13288 Marseille,
France
2
Laboratoire de Spectrométrie de Masse Bioorganique, Institut Pluridisciplinaire Hubert Curien, UMR 7178 (CNRSULP) ECPM, 25 rue Becquerel F67087-Strasbourg-Cedex 2, France
3
Unité d’Enzymologie, Département de Toxicologie, Centre de Recherches du Service de Santé des Armées, 38702
La Tronche, France
4
Laboratoire de Cristallogenèse et Cristallographie des Protéines, Institut de Biologie Structurale JP EBEL, 38027
Grenoble, France
E-mail: [email protected]
HPON1 environment has been shown to be of crucial importance for the modulation of its catalytic
activities. Thus, the study of the serendipitously discovered Human Phosphate Binding Protein (HPBP),
that is associated with HPON1 in vivo, is of particular interest to understand the HPON1 physiological
role(s). The fact that HPBP modulates HPON1 activities suggests that the presence of HPBP must be
taken into account in HPON1 dosage. In that regard monoclonal antibodies against HPBP have been
obtained. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in
eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal
residues, are involved in different human pathologies such as rheumatoid arthritis [1], kidney stones [2]
and atherosclerosis [3], and are systematically absent from eukaryotic genomes databases. Consequently,
HPBP amino acids sequence had to be first assigned from the electronic density map at 1.9Å resolution.
Then, an original approach combining X-ray crystallography and mass spectrometry has allowed to obtain
the complete and a priori exact sequence of HPBP, a 38kDa protein [4]. The complete sequence obtained
by this study will be useful to study further the interaction between HPON1 and HPBP, thus increasing
the global understanding of HPBP and HPON1 physiological involvement(s).
[1] Mehta A, Lu X, Block T, Willis A, Dwek R, Tennant B, Blumberg B., Arthritis and rheumatism, 2001, 44, 486487.
[2] Morales R, Berna A, Carpentier P, Contreras-Martel C, Renault F, Nicodeme M, Chesne-Seck ML, Bernier F,
Dupuy J, Schaeffer C, Diemer H, Van-Dorsselaer A, Fontecilla-Camps JC, Masson P, Rochu D, Chabriere E.,
Structure, 2006, 14, 601-609.
[3] Kumar V, Yu S, Farell G, Toback FG, Lieske JC., American journal of physiology, 2004, 287, 373-383.
[4] Elias M, Diemer H, Renault F, Rochu D, Contreras-Martel C, Schaeffer C, Van Dorsselaer A, Chabriere E.,
Proteins, 2007, in press.
Keywords: HPON1 partner, ab initio protein sequencing, DING protein, missing genes, antiHPBP antibodies.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P16
Phylogeny-based directed Evolution of PON2 and PON3 for increased
bacterial expression
Adrian Hugenmatter, Olga Khersonsky, and Dan S. Tawfik
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Serum paraoxonases (PONs) are enzymes with key roles in organophosphate detoxification,
prevention of atherosclerosis, and bacterial defense. However, the inability to express PONs in
bacteria constrained their detailed biochemical characterization and engineering. Directed
evolution of PON1 by family shuffling resulted in a recombinant protein, expressing in soluble
and active form in E. coli (1) and opened the way to 3D structure determination (2) and in depth
biochemical characterization (3,4,5).
Our current strategy to increase bacterial expression of PON3 includes DNA shuffling of three
mammalian PON3 genes (family shuffling), followed by introducing ‘back to consensus’
mutations. These are mutations towards evolutionary conserved residues, which are likely to
contribute to protein stability. As a result, recombinant PON3 enzymes with properties identical
to natural PON3, yet with functional expression levels of >10mg/l, were obtained. Specific
activities of these variants were similar to that of human PON3 (6), and as is case of PON1 (7),
binding to HDL particles, was found to stimulate the lactonase activity. Structural determination
and further biochemical characterization of the recombinant PON3 are currently in progress.
In the case of PON2, a phylogeny-based approach was pursued. We reconstructed the
phylogenetic tree of the PON2 family, and inferred the ancestor sequences. The common
ancestor gene of the PON2 group was synthesized, cloned, and was found to functionally
express at a moderate yield. The ancestor PON2 showed activity with all previously identified
substrates of human PON2, and an enhanced TBBL activity compared to its progenitors.
Deepened phylogenetic analysis for refined library design is currently under investigation, with
the aim of obtaining a stable, bacterially-expressed PON2 variant whose enzymatic parameters
are the same as native mammalian PON2s.
The structural and functional characterization of PONs is of great medical interest, and our
results considerably widen our understanding of these interesting enzymes. Moreover, we show
that directed evolution in combination with phylogenetic sequence information can lead to stable
proteins of so far intractable proteins.
References
1. Aharoni, A., Gaidukov, L., Yagur, S., Toker, L., Silman, I., and Tawfik, D. S. (2004) Proc Natl Acad Sci U S A
101, 482-7.
2. Harel, M., Aharoni, A., Gaidukov, L., Brumshtein, B., Khersonsky, O., Meged, R., Dvir, H., Ravelli, R. B.,
McCarthy, A., Toker, L., Silman, I., Sussman, J. L., and Tawfik, D. S. (2004) Nat Struct Mol Biol 11, 412-9.
3. Khersonsky, O., and Tawfik, D. S. (2005) Biochemistry 44, 6371-82.
4. Khersonsky, O., and Tawfik, D. S. (2006) J Biol Chem 281, 7649-56.
5. Rosenblat, M., Gaidukov, L., Khersonsky, O., Vaya, J., Oren, R., Tawfik, D. S., and Aviram, M. (2006) J Biol
Chem 281, 7657-65.
6. Draganov, D. I., Teiber, J. F., Speelman, A., Osawa, Y., Sunahara, R., and La Du, B. N. (2005) J Lipid Res 46,
1239-47.
7. Gaidukov, L., and Tawfik, Dan S. (2005) Biochemistry 44, 11843-54.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P17
Comparison of the Effects of Amino Acid Substitutions on the Stereoselective
Hydrolysis of Soman by Human and Bacterially-Expressed PON1
Dustin S. Morrow, Christina K. Harsch1, Thomas J. Magliery1,2, Douglas M. Cerasoli, and David E. Lenz
US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, APG, MD 210105400
Departments of Chemistry1 and Biochemistry2, The Ohio State University, 100 W. 18th Avenue,
Columbus, OH 43210-1185
Human paraoxonase 1 (HuPON1) has been shown to hydrolyze the organophosphorus
nerve agent soman (GD) with modest stereoselectivity for the two less toxic (C±P+)
stereoisomers. We have examined the stereoselectivity of five mutants of HuPON1 expressed in
mammalian tissue culture cells, as well the bacterially expressible G2E6 chimeric PON1 and
three mutants thereof. All of the PON1 enzymes catalyzed the hydrolysis of each GD
stereoisomer, with the exception of the H115W mutant of HuPON1. H115W HuPON1 had no
detectable activity for GD, but was capable of recognizing GD as a competitive inhibitor. In
contrast, the H115W mutant of G2E6 did catalyze the hydrolysis of all four isomers of GD but at
a slower relative rate than the unmutated G2E6. With the exception of H115W HuPON1, all
mutants of HuPON1 and G2E6 displayed the same relative preference for the C±P+
stereoisomers of GD. These results are supportive of previous findings indicating that
substitution of the amino acid at position 115 of PON1 leads to different interactions with V-type
nerve agents. Taken together, the results suggest that the identity of the amino acid residue at
115 can effect binding of nerve agent substrates to PON1. Furthermore, they indicate that the
effects on enzymatic activity caused by amino acid substitutions of the G2E6 variant of PON1
may not predict the effects of the same substitution on HuPON1.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P18
Structure activity relationship studies towards the understanding
of active site residues in Paraoxonase-1: interplay of
experimental and computational studies
Siva Muthukrishnan, Vivekanand S. Shete, Toby T. Sanan, Lauren M. Porter, Thomas J. Magliery*,
Christopher M. Hadad*
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210
[email protected], [email protected], FAX: (614) 292-1685
Paraoxonase-1 (PON1) is a human enzyme capable of hydrolyzing organophophorous
(OP) nerve agents with moderate activity. In order develop a more viable prophylactic bioscavenger, it is necessary to develop mutants of PON-1 with several fold improvement in
activity. However, the mechanism of hydrolysis of PON1 is poorly understood and no crystal
structure with substrate or inhibitor bound is available. In order to identify the key residues
involved in the binding and catalysis of the substrates to a chimeric, recombinant form of PON-1
(crePON-1), we employed both experimental and computational structure activity relationship
studies. We designed de novo and synthesized a library of 50 OP compounds (see Figure 1) and
screened them against crePON-1 and identified 8 inhibitors and 10 substrates. We performed
extensive molecular dynamics simulations on a computational model of the protein and docked a
library of 120 substrates to the MD relaxed protein. Key residues involved in the binding were
identified as L69, K70, Y71, P72, G73, H285, I291, and F292 (see Figure 2). Relevant
experimental and computational data will be presented.
O
P O
O
O
R
SAR27
O
P O
O
O
R
O
P O
O
O
R
O
P O
O
O
R
SAR54
SAR55
O
P O
O
O
R
4
SAR56
O
P O
O
O
R
6
4
SAR58
O
P O
O
O
R
SAR124
SAR125
SAR57
O
P O
O
O
R
O
P O
O
O
R
SAR122
SAR123
6
SAR60
SAR59
O
P O
O
O
R
O
P O
O
O
R
O
P O
O
O
R
5
O
P O
O
O
R
SAR126
6
O
P O
O
O
R
SAR127
R=
NO2
Figure 1. Several representative substrates and inhibitors of crePON-1
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
Figure 2. Enzyme model after 20 ns of MD simulations (water box not shown for clarity). The
residues in green, blue, red and purple appear to be the most important in substrate binding.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P19
Recombinant Human Paraoxonase 1 (PON1) Engineered in an E. coli
Expression System
Stephanie M. Suzuki1,2, Richard C. Stevens1, Rebecca J. Richter1, Toby B. Cole1, Sarah S. Park1,
Tamara C. Otto3, Douglas M. Cerasoli3, David E. Lenz3 and Clement E. Furlong1.
1
Depts. of Medicine - Division of Medical Genetics, and Genome Sciences, 2Molecular and Cellular
Biology Program, University of Washington, Seattle, WA, USA 98195
3
Research Division, US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Rd.,
APG, MD 21010
Purification of active recombinant human PON1 (rHuPON1) in E. coli provides several
advantages over eukaryotic expression systems. Recombinant PON1 derived from E. coli lacks
glycosylation and avoids this source of immunogenicity of protein derived from non-human
eukaryotic sources. Bacterial expression of active PON1 also provides a system in which PON1
can be engineered for catalytic efficiencies sufficient to protect against or treat specific OP
exposures. Human PON1 has been shown in mouse models to protect against OP exposures
including chlorpyrifos oxon (CPO) and diazoxon (DZO). Improved catalytic efficiency is
required for use in protecting against paraoxon (PO) or nerve agent exposure. We have
developed a protocol for engineering and expressing active, untagged rHuPON1 in E. coli.
Purification to near homogeneity of naturally occurring PON1 variants with arginine or
glutamine at position 192 as well an engineered variant with lysine at position 192 has been
accomplished using a series of column chromatography methods involving both anion exchange
and hydrophobic interaction chromatography. The rHuPON1s have been characterized for their
ability to hydrolyze CPO, DZO, PO and phenyl acetate. The purified rHuPON1 is non-toxic
when injected into PON1-/- mice, has a half-life similar to purified human plasma PON1 and
protects against exposure to three times the LD-50 of DZO. Each of the PON1192 variants was
also capable of catalyzing the hydrolysis of the nerve agents VX and VR. Preliminary data
suggests that the order of catalytic efficiency of hydrolysis was Q192 > R192 > K192.
Supported by: NIH ES09883, ES04696, ES07033, ES09601/EPA: RD-83170901. TCO, DMC
and DEL supported by NIH U54 Center Grant 5 U54 NS058183. TCO is a senior NRC fellow.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P20
Characterization of the PON1 active site using modeling simulation, in
relation to PON1 lactonase activity
Hagai Tavori†,‡ Soliman Khatib†, Michael Aviram‡, Jacob Vaya†*,
† Laboratory of Natural Medicinal Compounds, MIGAL - Galilee Technology Center, P.O. Box 831, Kiryat
Shmona 11016, and Tel Hai College, Israel., ‡ Rappaport Family Institute for Research in the Medical Science,
Rambam Medical Center, Haifa 31096, Israel
The precise biological role of PON1 however has remained elusive and a recent interest has focused on the
possible protective role of PON1 in vascular disease as a result of its inhibitory effects on lipoprotein oxidation.
PON1 catalyzes the hydrolysis of various substrates such as organophosphates, esters and certain lactones.
According to recent finding lactonase activity is considered as the primary activity of PON1 in vivo.
The main objective of the present study is to build a prediction model that will help identifying PON1
endogenous/exogenous potential substrates by using two types of modeling methodologies: a) an
independent analysis of parameters related to PON1 known substrate and b) docking of various substrates
into enzyme three-dimensional crystal structure, and measuring the free and docking energies of the
interactions. On using substrates most stable conformation and upon calculating their molecular moment
dipole, a negative correlation (R2=0.71, P<0.0001) between the published rates for PON1 hydrolysis of
over 20 different lactones, to substrate dipole moment on the Y-axis. Previous publication , predicted
PON1 active site base on the elucidation of PON1 crystallographic structure, and suggested the
involvement of one Ca+2 ion (out of the two present in PON1) and also that of Asn168, Asn270, Asn224,
Asp269 and Glu53. Using AutoDock4 software, performing superposition analysis, we were able to
allocate the possible PON 1 active site and to partially approve the authors’ assumptions. We suggest that
indeed Asn168, Asn224, and to some extent, His115, form hydrogen bonds with the substrate and thus
have a major role in the enzyme active site function, while Asp269, Asn 270, and Glu53 seem more likely
to contribute only to the active site structure. Finally, we have found also a strong correlation between the
calculated docking energy of the substrate during their interaction with the enzyme active site and PON1
known rate of lactones hydrolysis (R2= 0.73, P<0.0001), and to the number of carbons constructing the
adjacent chain of the γ, or δ lactones (R2= 0.85, P<0.0001).
In conclusion, our results may contribute to the effort of identifying exogenous and endogenous natural
substrates for PON1.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P21
Paraoxonases hydrolyze the iodoeicosanoid delta-iodolactone
and attenuate its toxicity in cell cultures
John F. Teiber1, Junhui Xiao1, Gerald L. Kramer1, Dragomir I. Draganov2 , Robert W. Haley1
1
Department of Internal Medicine, Division of Epidemiology, The University of Texas Southwestern Medical
Center, Dallas, TX 75390, USA
2
Department of Metabolism, WIL Research Laboratories, LLC, Ashland, OH 44 805, USA
Delta-iodolactone (δ-IL) is formed in the thyroid as a result of the oxidation of iodide by
thyroperoxidase in the presence of arachidonic acid. In vivo δ-IL inhibits thyroid enlargement
induced by goitrogens. In vitro, both molecular iodine and δ-IL exhibit potent antiproliferative
activity in thyroid cells and it is postulated that δ-IL mediates this activity of iodine. Due to the
structural similarity of δ-IL to known paraoxonase (PON) polyunsaturated fatty acid lactone
substrates, we tested the ability PONs to hydrolyze this lactone. All three recombinant PONs
hydrolyzed δ-IL with rates of hydrolysis in the order PON3>PON1>PON2. δ-IL has two chiral
centers giving rise to four stereoisomers. Only half of the racemic δ-IL preparation we have
synthesized was hydrolyzed by the PONs even after extended treatment indicating that only
certain δ-IL stereoisomers were substrates for the enzymes. We demonstrate that δ-IL decreases
the viability of several different human cell lines in a dose dependent manner. Compared to
control HEK 293T cells, cells transiently transfected with human PON1 or PON3 exhibited
higher δ-IL lactonase activities and were also protected from δ-IL toxicity. These results suggest
that the PONs may regulate the biological activity of δ-IL and iodine in vivo.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P22
Role of Paraoxonase 2 in acute gram-negative microbial infection
Bourquard N1,2, Reddy ST1
Department of Medicine1 and Molecular and Medical Pharmacology2, David Geffen School of Medicine,
UCLA, Los Angeles, California 90095
Objective. Paraoxonase 2 (PON2) is one member of a multigene family that has been implicated in a
number of inflammatory diseases. The paraoxonases share a lipo-lactonase activity and were recently
shown to hydrolyze 3OC12-homoserine lactone (HSL), the major quorum sensing autoinducer of the
opportunistic gram-negative pathogen, Pseudomonas aeruginosa. We previously reported that
inactivation of 3OC12-HSL is impaired in PON2-deficient (PON2-def) murine airway epithelial cell
lysates suggesting that PON2-def mice may be more sensitive to P. aeruginosa infection. Conversely, we
have also reported that PON2-def macrophage are hyper-responsive to LPS as measured by increased
ROS production and cytokine expression suggesting that PON2-def mice may be less susceptible to an
acute microbial infection. The objective of this study is to determine the role of PON2 in an acute
infection model of P. aeruginosa in vivo.
Methods and results. PON2-def and littermate wild type male mice were intraperitoneally challenged
with 1x107 cfu P. aeruginosa (PA01 strain) and survival was monitored for 72 hours. PON1 and PON3
mRNA levels were not significantly different between the two groups of mice. Interestingly, PON2-def
mice were more resistant to P. aeruginosa infection when compared to control mice. To determine the
mechanism, we next sought to assess the level of bacterial clearance and inflammation in control and
PON2-def mice following acute systemic PA01 infection. Six week old PON2-def and littermate wild
type male mice were intraperitoneally injected with 5.2x106 cfu of PA01 and were sacrificed 3 hours
post-infection. Liver, spleen and lungs isolated from PON2-def mice had significantly lower bacterial
burden than control mice (p<0.05). Moreover, tissue expression of IL-6 mRNA was 5-fold less (p<0.01)
and KC mRNA was 4.7-fold lower (p<0.01) in PON2-def mice compared to controls reflecting a reduced
state of tissue inflammation corresponding to the reduced bacterial burden.
Conclusions. Our results demonstrate that PON2-def male mice are resistant to an acute PA01 systemic
infection when compared to littermate wild type mice. Heightened macrophage activation as
characterized by increased ROS production and cytokine expression in PON2-def mice in response to
LPS, the major virulence factor in an acute gram-negative infection model, may explain the lack of
susceptibility observed in PON2-def mice.
This work was supported by the NHLBI Grant 1RO1HL71776 and predoctoral AHA fellowship 0715008Y
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P23
PON1 in Murine Cecum Ligation and Puncture (CLP) Model of Sepsis
Dragomir I. Draganov1,2, Jean Nemzek1, John F. Teiber1,3, Charles L. Bisgaier1,4, Daniel
Remeck1, Bert N. La DU1
1
University of Michigan, Ann Arbor, MI 48105, USA; 2WIL Research Laboratories, LLC, Ashland, OH 44 805,
USA; 3The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; 4Esperion Therapeutics,
Division of Pfizer Global Research and Development, Ann Arbor, MI 48108, USA
In the murine CLP model of sepsis, the slow leakage of intestinal flora in the abdominal cavity
produces gradual disease development that closely mimics human sepsis. The severity and
progression of the sepsis in this model is proportional to the size of the needle used: 25 gauge
(G) induces mild or absent systemic inflammation (less than 10% lethality) whereas 21 G
induces sepsis in about 90% of the animals with ~50% lethality. CLP (21 G) in Balb-c mice
caused decrease in serum PON1 activity by 50% over 2 days after the surgery followed by a
slow recovery to 85% from the basal level over 1 week after the CLP; PON1 activity in the nonsurvivors was significantly lower than in the survivors prior and after the CLP, thus we
hypothesized that low or absent serum PON1 will increase sensitivity of mice to CLP-induced
sepsis. There were no significant differences in 21-day survival of PON1 knockout mice on
Balb-c background compared to wild type (wt) after 25 G-CLP (15/18 PON1-KO vs. 10/11 wt)
or 21 G-CLP (12/24 PON1-KO vs. 5/11 wt). PON1-KO mice compared to wt had significantly
lower white blood cell counts and lost more weight after CLP. We attempted to improve
survival of wt Balb-c mice after 21G-CLP by increasing serum PON1 levels by sc injections of
purified human PON1 or rabbit HDL (total of 6 injections every 12 hours post-CLP). There
were no significant differences in 21-day survival (10/24 hPON1-treated vs. 9/21 vehicle and
8/18 rabit HDL vs. 9/18 vehicle). In conclusion, serum PON1 levels were decreased after CLP,
however, supplementation with exogenous PON1 did not improve survival in this murine model
of sepsis.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P24
INVESTIGATION OF PARAOXONASE-1 STATUS IN AUTISM
SPECTRUM DISORDERS
Maria Dronca, Sergiu P. Paşca, Eleonora Dronca, Bogdan Nemeş, Tamás Kaucsár, Emõke
Endreffy, Ileana Benga, Rodica Cornean, Felicia Iftene.
Faculty of Medicine, Iuliu Haţieganu University of Medicine and Pharmacy,
Cluj-Napoca, Romania
ABSTRACT
Background: Autism spectrum disorders (ASD) comprise a complex and heterogeneous group
of conditions of unknown etiology, characterized by significant disturbances in social,
communicative, and behavioral functioning. Recent evidence pointed to a possible implication of
the HDL-associated esterase/lactonase paraoxonase-1 (PON1) in autism. We aimed at
investigating the PON1 status in children with autism spectrum disorders.
Methods: We evaluated PON1 bioavailability (i.e., arylesterase activity) and catalytic activity
(i.e., paraoxonase activity) in plasma using spectrophotometric methods and the two common
polymorphisms in the PON1 coding region (Q192R, L55M) by employing Light Cycler RTPCR, in 50 children with ASD as compared to healthy controls.
Results: Both PON1 activities were lower in autistic children, despite no association with less
active variants of the PON1 gene. The distribution of the PON1 phenotype was similar between
patients and controls.
Conclusions: PON1 arylesterase and PON1 paraoxonase activities are decreased in patients with
ASD, despite no association with the Q192R and L55M polymorphisms in the PON1 gene.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P25
ASSOCIATION BETWEEN HUMAN PARAOXONASE 1 ACTIVITY AND
INTIMA-MEDIA THICKNESS IN SUBJECTS UNDER 55 YEARS OF AGE
WITH CAROTID ARTERY DISEASE
Harangi M, Fulop P, Seres I, Magyar MT, Csipo I, Sipka S, Valikovics A, Csiba L, Bereczki
D, Paragh G.
1st Department of Medicine, University of Debrecen, Debrecen, Hungary.
[email protected]
BACKGROUND: Human serum paraoxonase (PON1) protects lipoproteins against oxidation by
hydrolyzing lipid peroxides in oxidized low-density lipoprotein (oxLDL); therefore, it may
protect against atherosclerosis. PON1 activity and polymorphisms have been inconsistently
associated with carotid artery disease. The goal of this study was to clarify the role of PON1
activity and phenotype on carotid artery disease and its correlation with some inflammatory and
immune markers in subjects under 55 years with early-onset carotid atherosclerosis.
METHODS: Sixty patients with occlusive carotid artery disease and 30 healthy controls were
enrolled. Intima-media thickness (IMT) was measured by high-resolution ultrasound of both
common carotid arteries. Anti-oxLDL antibody levels were determined by ELISA.
RESULTS: In the whole study population we found a negative correlation between PON1
activity and IMT (r = -0.27, p = 0.011), and between salt-stimulated PON1 activity and IMT (r =
-0.24, p = 0.02). Both PON1 activity and salt-stimulated PON1 activity negatively correlated
with anti-oxLDL levels (r = -0.28, p = 0.008; r = -0.26, p = 0.01). PON1 activity was lower in
patients compared to controls; however, the difference was not significant.PON1 phenotype
distribution of patients and controls did not differ significantly.
CONCLUSION: The importance of PON1 activity as a predictive risk factor for early-onset
occlusive carotid artery disease should be assessed in future studies.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P26
Reduced paraoxonase 1 activity in sleep apnea-hypopnea patients:
effects of 6 month treatment with continuous positive airway pressure
treatment. A pilot study
Alejandro Gugliucci1, Kazuhiko Kotani 2, Kokoro Tsuzaki,3 Naoki Sakane3 , Ichiro Komada4,
John Schulze,1 and Satoshi Kimura5.
1: Glycation, Oxidation and Disease Laboratory, Touro University-California, Vallejo, CA. USA. 2: Tottori
University School of Medicine, Japan. 3: Clinical Research Institute for Endocrine and Metabolic Disease, National
Hospital Organization, Kyoto Medical Center, Japan. 4: Department of Otorhinolaryngology, Shiga Hospital of
Social Insurance, Japan. 5: Department of Central Clinical Laboratory, Showa University Northern Yokohama
Hospital, Yokohama City, Japan.
Background. Obstructive sleep apnea-hypopnea (OSAH) is associated with an increased rate of
cardiovascular morbidity, which has been suggested to be partly related to increases in oxidative
stress and a state of inflammatory cell activation. Recent studies have demonstrated a greater
degree of oxidative stress, increased biomarkers of cardiovascular risk, and dysfunctional HDL
in subjects with OSAH. Paraoxonase 1 is an esterase carried by HDL particles with
antiatherogenic functions, mainly through protection of LDL from oxidation as well as through
its homocysteine-thiolactonase activity. It has been shown is to lose activity under strong
oxidative insults.
Hypothesis. We hypothesized that PON-1 activity is reduced in OSAH patients as compared to
control subjects and that this change could be ameliorated by treatment.
Methods. Ten patients (5 male, 5 female) with OSAH (more than 15 events of apneahypopnea/hour) and 20 age-matched controls were studied. The patients were studied at
diagnosis and after 6 months of continuous positive airway pressure treatment (CPAP). PON-1
activity was determined from the initial velocity of p-nitrophenol production at 37C and recorded
at 405 nm in a VERSAmax (Tunable) Microplate Reader. Arylesterase activity was determined
from the initial velocity of phenylacetate hydrolysis at 37C and recorded at 270 nm in a
Beckman DU spectrophotometer. Other parameters were measured using standard clinical
laboratory methods.
Results: PON-1 activity (measured as arylesterase) is 29% lower in OSAH patients than in
control subjects (p < 0.001). After treatment both paraoxonase and arylesterase levels increased
significantly (50% and 53% respectively) in females but not in males. HDL-cholesterol
increased only 9% (NS). The increase in arylesterase or paraoxonase does not correlate with
changes in triglycerides but correlates with the change in HDL-cholesterol (r = 0.75).
Conclusions: Our data show that PON-1 activity is consistently lower in OSAH patients.
Interestingly, the CPAP treatment (which minimizes the cycles of hypoxia/reoxygenation) seems
to have a positive effect on PON-1 activity at least in females, that cannot be fully explained by
the changes in HDL–cholesterol. This pilot study should foster more research at PON-1 changes
in OSAH as another aspect of dysfunctional HDL. The higher cardiovascular risk in OSAH
patients may be, in part, linked to impaired PON-1 activity and the current treatment may be
efficacious in restoring it.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P27
Human PON1 During Development: Effects of Age and
Genetic Variability in a Latino Birth Cohort
Karen Huen1, Kim Harley1, Sherri Rose1, Asa Bradman1, Clement Furlong2, Brenda Eskenazi1,
Nina Holland1
1
University of California, Berkeley, School of Public Health 2 University of Washington, Department of
Genome Sciences and Medicine
Studies in both animals and humans report significantly lower serum and liver PON1 activity at
birth in comparison to adults. Therefore, young infants may be more susceptible to OP pesticide
exposures and oxidative stress. Few studies have characterized the age dynamics of PON1 levels
and activities in young children. PON1 provides a distinctive example of a gene containing a
common missense SNP (Q192R) that dramatically affects functional activity. PON1 activity is
easily quantified by measuring rates of hydrolysis of various substrates including paraoxon and
phenyl acetate. Previously, we showed that haplotype analysis of PON1 polymorphisms did not
provide an advantage to analysis of individual genotypes when examining the effects of PON1
genotypes on PON1 functional activity in Latino mothers and children. Here, we (1) genotyped
several PON1 polymorphisms including the Q192R coding SNP and the C-108T promoter SNP,
and measured PON1 enzyme activity and PON1 levels in n=172 children at birth, 24 months,
and 60 months of age and (2) re-sequenced the PON1 gene in N=30 Latino subjects in order to
identify additional PON1 SNPs, insertions, and deletions in a minority population. Hierarchical
linear models were used to examine how PON1 levels and activity change over time from birth
through 60 months of age and to determine if PON1 polymorphisms affect this age-related
change. Both age and PON1 Q192R genotype were associated with PON1 levels and PON1
activity although no significant interaction between the two variables was identified. Contrary to
previous reports that by 24 months children reach a plateau comparable with adults, on average,
PON1 levels, measured by arylesterase activity, increased from birth through 39 months
(p<0.005) while PON1 activity towards the substrate paraoxon, continued to increase from birth
through 47 months (p<0.005) . Further, children with the PON1 192 RR genotype have slightly
lower PON1 levels than children with fewer R alleles (p=0.003) after adjusting for age. In
contrast, mean PON1 activity was highest in children with the PON1 192 RR genotype after
adjusting for age (p<0.005). Thus, early childhood represents a critical period of vulnerability to
OP exposures and oxidative stress which is further influenced by PON1 genotypes.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P28
The influence of oxidative stress to paraoxonase activity in stroke patients
Jelena Joksić, Ivana Srećković, Vera Markovic, Tamara Gojkovic, Jelena Kotur-Stevuljevic,
Vesna Spasojevic-Kalimanovska
Institute for Medical Biochemistry, Faculty of Pharmacy, Belgrade, Serbia
Introduction: Many experimental researches indicate that the generation of free radicals plays
an important role in the pathogenesis of ischemic brain injury. Paraoxonase, enzyme associated
to HDL, exerts an anti-oxidative and a protective role against oxidative damage of lipoproteins
and cells, and therefore in atherosclerosis.
The aim of the study: Our research has shown that the level of paraoxonase activity was
significantly decreased in stroke patients (SP) compared to control, therefore the aim of this
investigation was to establish weather the decrease of paraoxonase activity in SP could be related
to blocking of enzyme’s SH-groups. The degree of oxidative stress was determined by measuring
the final product of lipid-peroxidation malondialdehid (MDA) and also product of oxidative
protein damage, reactive protein carbonyls (RPC).
Material and methods: The research focused on 35 healthy individuals (control group, CG) and
45 SP, (24 survivors and 21 with lethal outcome). Paraoxonase activity was determined using
paraoxon as a substrate. SH-groups and MDA were determined using spectrofotometry methods,
both in plasma and HDL-lipoprotein fractions, isolated using precipitation method. SH-groups
concentrations were measured using method by Ellman. MDA level was determined as
thiobarbituric acid reactive substances (TBARS). The level of RPC was determined by reaction
with dinitrophenylhydrazine (DNPH), also using spectofotometry methods.
Results: Paraoxonase activity is significantly lower in SP comparing to CG (402.87±221.01 U/L
CG vs. 72.96±61.76U/L SP, p<0.001). Concentration of SH-groups is significantly decreased
both in plasma and HDL-fraction, in patients compared to controls, but not between two groups
of patients. In patients with lethal outcome, concentration of MDA in HDL-fraction is
significantly increased compared to survivors, and controls, respectively (P=0.037 and P=0.027
respectively). Negative correlation (ρ=–0.424, p=0.024) between plasmatic SH-groups
concentrations and MDA in HDL-fraction was observed. The level of RPC is significantly
increased (P<0.05) in SP compared to CG, but not among patients.
Conclusion: SP have an increased oxidative protein damage which leads to oxidative stress.
Decreased paraoxonase activity could be a consequence of decreased concentrations of SHgroups in HDL-fraction, whereas increased concentration of MDA in HDL-fraction represents
significant prognostic parameter. Therefore, recomendation will be introducing additional
therapy based on antioxidants.
Key words: stroke, oxidative stress, SH-groups, MDA, RPC
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P29
HIV-1 Infection Increases Human Paraxonase-2 Activity In Vivo
Jinyun Yuan1, Menghua Zhang1, Stella Evans1, Rosita Moya-Castro1,
Noam Bourquard2, Srinivasa T. Reddy2 and Prasad S. Koka1
1
Torrey Pines Institute for Molecular Studies, San Diego, CA
and
2
UCLA Division of Cardiology, Los Angeles, CA
Paraoxanase-2 (PON2) is a member of the cardioprotective enzyme family. Whereas PON1 and
PON3 are HDL associated and found in the serum, PON2 is tissue associated but not with HDL.
PON2 is found in various human and mouse organs. We have found that PON2 enzyme activity
is increased by up to 2.2-fold in the hematopoietic CD34+CD4+ cell line, TF-1, upon HIV-1
infection in vitro. We have also found that PON2 enzyme activity is increased by up to 4.1-fold
in the fetal thymocytes, upon HIV-1 infection in vivo. However, the PON2 activity in the coexisting human CD34+ pluripotent progenitor stem cells that were not susceptible to the virus
infection in the conjoint human fetal hematopoietic organ was unchanged. Increase in PON2
activity is associated with a correlation in its expression. HIV infection generally causes increase
in the oxidative stress and in levels of pro-inflammatory cytokines triggering activation of the
mitogen-activated protein kinase (MAPK) pathway. We have found that the MAPK and STAT5
phosphorylation is decreased in the virus infected TF-1 cells compared to mock infected cells.
We are investigating the likelihood of HIV infection increasing the PON2 activity via MAPK or
STAT5 signaling events. Thus we observe multiple levels of protection from PON2 including a
possible innate immunity against the virus infection.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P30
PON1 ACTIVITY IN A BRAZILIAN POPULATION GROUP OF
HEALTHY AND DISLIPIDEMIC ADULTS AND INFLUENCE
OF DIET AND LIFESTYLE
J.S. Lima, P.C.B.Alexandre, L.S. Leão, L. G. Costa, M. V. Almeida, D. L. Fontoura, L.L Fonseca
LACAT, Instituto Biomédico, Universidade Federal do Estado do Rio de Janeiro, UNIRIO, Rio de
Janeiro, Brazil.
Paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme capable to protect
low density lipoprotein (LDL) from oxidative modifications. Recent studies also describe that
PON1 activity is modulated by dietary, lifestyle and environmental factors. The purpose of this
work was to describe PON1 activity in a group of Brazilian subjects, to compare PON1 activities
between groups of subjects with different lipid profiles, and to investigate whether diet and
lifestyle might affect this activity. We studied groups of healthy and dislipidemic subjects
comprising a total cohort of 94 adult volunteers, age range of 18-60. Variables studied included
basal and salt stimulated PON1 activity, arylesterase activity, PON1 ratio (PON1 salt
stimulated/arylesterase); serum total cholesterol, HDL, LDL, VLDL and triglycerides. Other
variables such as energy consumption and nutrients intakes were assessed from a validated food
consumption frequency questionnaire. Lifestyle variables such as smoking and alcohol
consumption status were also analyzed. Correlation analysis, T test and ANOVA were used for
statistical analysis. We have observed significant associations between higher activity levels of
PON1 and higher levels of LDL and lower HDL levels. We stratified groups setting
recommended dietary intakes as cut off points. Using this approach, no associations were
observed between PON1 activity and nutrient intakes. When groups were dichotomized using the
first quartile of PON1 activity distribution as a cut-off, there were significant associations
between higher levels of PON1 and higher intakes of total fat and cholesterol. Our results show
that PON1 activity may be an important biomarker to assess risk of diseases associated with
individual lipid status and that diet may affect PON1 activity. These results also points out the
need of more studies concerning paraoxonase polymorphism in Brazil, since little is known
about PON1 gene polymorphism in the multiethnic Brazilian population.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P31
The potential role of Paraoxonase-2 in
oxidative stress induced death
Mohammad Parsanejad1, Yi Zhang1, Maxime W.C. Rousseaux1, Noam Bourquard2, Hossein Aleyasin1,
Isabella Irrcher1, Steve Callaghan1, Ruth S. Slack1, Daniel Figeys3, Srinivasa T. Reddy2, David S.Park1
1
Ottawa Health Research Institute, Neuroscience Group, University of Ottawa, Ottawa, K1H 8M5, ON, Canada
Atherosclerosis Research Unit, Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles,
Calif 90095, USA.
3
Ottawa Institute of Systems Biology (OISB), University of Ottawa, Ottawa, K1H 8M5, ON, Canada.
2
Loss-of-function mutations in DJ-1 gene can cause early-onset autosomal recessive Parkinson's
disease. Importantly, loss of DJ-1 also sensitizes to insults in models of PD and stroke. We have
previously reported that dopaminergic neurons lacking DJ-1 are sensitive to oxidative stress
induced by hydrogen peroxide, neurotoxin MPTP, or focal ischemia. However, the
mechanism(s) by which DJ-1 regulates neuronal survival in these death paradigms remains
largely unknown. Interestingly, we recently identified, in an interaction screen, Pon2 as a
potential DJ-1 interactor. This was later confirmed through further immunoprecipitation
interaction analyses. The Paraoxonases, a multigene family that comprises three related genes
Pon1, Pon2 and Pon3 with structural homology, play major protective roles in antioxidant
defense systems. In this study we propose Paraoxonase-2 (Pon2) may be a target of DJ-1 in these
Parkinson’s disease models. We show that Pon2 overexpression, like DJ-1, can protect
embryonic cortical neurons against MPTP active metabolite MPP+. Importantly, oxidative stress
induces an increased Pon2 lactonase activity in both cortical neurons and murine embryonic
fibroblasts (MEF), while DJ-1 deficient cells display a decrease Pon2 activity compared to wild
type cells especially after oxidative insult. Taken together our data suggest a physical link
between DJ-1 and Paraoxonase-2 and suggest that DJ-1 may mediate neuronal survival in
response to oxidative stress through enhancing Pon2 activity. This work was supported, in part
by grants from Pakinsons Society Canada, Parkinsons Research Consortium, Canadian Institute
of Health Research and Heart and Stroke Foundation of Ontario.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P32
Paraoxonase 1 (PON1) Status as a Risk Factor for Disease or Exposure
RJ Richter1, LG Costa2,5, GP Jarvik1, CP Zabetian3,6, H Checkoway2,4, A Samii3,6, A Griffith7,
JW Roberts8, D Yearout3,6, CE Furlong1
1
Departments of Medicine - Division of Medical Genetics, Genome Sciences, 2Environmental and
Occupational Health Sciences, 3Neurology, and 4Epidemiology University of Washington, Seattle, WA
98195; 5Dept. of Human Anatomy, Pharmacology and Forensic Science, University of Parma, Italy; 6VA
Puget Sound Health Care System, Seattle, WA; 7 Evergreen Hospital Medical Center, Kirkland, WA;
8
Virginia Mason Medical Center, Seattle, WA
Human paraoxonase 1 (PON1) has broad substrate specificity and has been shown to protect
against exposure to some organophosphorus insecticides via its ability to hydrolyze toxic
metabolites of some organophosphorothioate insecticides. PON1 status has been shown to be
important in protecting against vascular disease via the role of PON1 in metabolizing oxidized
lipids. More recently, all three PONs (1,2 &3) have been shown to inactivate the quorum
sensing factor N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) of Pseudomonas.
Many studies have been carried out that examined only DNA single nucleotide polymorphisms
as possible risk factors for disease or exposures. For all of the known functions of PON1, the
level of PON1 is important and in some cases, also the Q192R polymorphism. A simple twosubstrate assay/analysis, plotting rates of diazoxon hydrolysis vs. paraoxon hydrolysis, provides
both PON1 levels and functional Q192R phenotype/genotype. We have developed a new, nontoxic substrate assay that provides PON1 status without use of toxic substrates. However, one of
the most exciting recent findings has been the observation that a modification of the two
substrate assay developed by La Du and co-workers reveals differences between male
Parkinson’s disease (PD) patients and male control subjects. PD patients have higher rates of
phenyl acetate hydrolysis relative to their rates of paraoxon hydrolysis. The most obvious
explanation for this observation is that the HDL environment is subtly different in PD patients,
perhaps reflecting differences in oxidative stress status. Supported by: NIH ES04696, ES09883,
K08NS044138; VA Merit Award
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P33
POSSİBLE İNFLUENCES OF BMI, MENAPAUSE PERİOD ON THE
ANTİOXİDANT EFFECTS OF RALOXİFEN TREATMENT İN
POSTMENOPAUSAL OSTEOPOROSİS
Authors: Eser Y. Sözmen1, Yasemin Akçay1, Sibel Eyigör2, Muammer Karadenis3, Yeşim
Kirazlı2
1
Dept of Biochemistry Faculty of Medicine, 2Dept. Of Physical Medicine and Rehabilitation,
3
Dept. of Endocrinology and Metabolism, Faculty of Medicine, Ege University, Izmir/ Republic
of Turkiye
Osteoporosis is a skeletal disease characterized by the loss of the normal density of bone
due to the lack of estrogen. Estrogen normally inhibits some proinflammatory cytokines IL-1,
IL-6, IL-7, M-CSF and TNF-α which may be involved in bone resorption and also decrease
oxidative stress by suppression of reactive oxygen species. Raloxifen, a selective oestrogen
modulator which is used for the treatment of osteoporosis for its estrogen agonist effects on bone
and lipids.
The aim of this study was to investigate the antioxidant effects of raloxifen treatment and
also the relationship between its antioxidant (by measuring antioxidant enzymes and total
antioxidant activity) effects and BMI & postmenopausal period in women with osteoporosis.
Fourteen women with postmenopausal osteoporosis, which was diagnosed according to WHO
criteria, were treated by Raloxifen hydrochloride 60 mg (EVİSTA) for 6 months.
Osteocalcin, serum dien, TEAC levels was significantly decreased after the treatment
(p=0,039). There was an unsignificant increase in eCAT, arilesterase enzyme activities and
FRAP levels with the treatment. When antioxidant activities compared to BMI levels (cut off
limit was 30kg/m2), PON1 activity significantly (31,47±28 vs 45,2±40 in BMI<30 and 18,6±7,4
vs 26,8±10,7 in BMI>30) and TEAC, FRAP, e-CAT activities insignificantly increased in both
groups. When it has been evaluated according to postmenopausal period, serum TEAC, FRAP
and e-CAT and especially PON1 activity (36,1±32U/L vs 52 ±46U/L) increased significantly
after treatment provided that it started to use in the first 10 years of menaupose. These results are
similar with the studies showing that raloxifen treatment decreases inflamatory cytokines and
treatment is beneficial in postmenopausal osteoporosis.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P34
RED WINE CONSUMPTION IS MORE EFFICIENT ON ANTIOXIDANT
SYSTEM IN PATIENTS WITH METABOLIC SYNDROME
Authors: Eser Y. Sözmen1, Muammer Karadenis2, Hatice K. Yıldırım3, Candeger Yılmaz2
1
Dept of Biochemistry Faculty of Medicine, 2Dept. of Endocrinology and Metabolism, Faculty of
Medicine, 3Dept. of Food technology, Faculty of Food Engineering
Ege University, Izmir/ Republic of Turkiye
Epidemiological studies showed that moderate red wine consumption can reduce the risk of
cardiovascular disease due to phenolic compounds which are efficient scavengers of free radicals
and breakers of lipid peroxidative chain reactions. Phenols also have antioxidant activity, antiinflamatuary effect and may protect low-density lipoproteins (LDL) against oxidative
modification. Previously, it has been determined that red wine consumption prevents lipid
peroxidation in healthy subjects as well as diabetic patients. The aim of this study was to
investigate the effects of red wine consumption on LDL oxidation, total antioxidant activity and
antioxidant enzymes (catalase and superoxide dismutase in erythrocytes and
paraoxonase/arylesterase in serum) in patients with metabolic syndrome (independent risk factor
for cardiovascular disease) which is diagnosed with hyperlipidemia, high BMI, high glucose
concentration. Healthy subjects (n=14) and patients with metabolic syndrome (n=10) drank 200
ml of red wine each day during 6 weeks. Blood samples were taken basally and after 6 weeks.
Cabernet Sauvignon wine caused a significant increase in arylesterase activity (p=0.073) and
Total antioxidant capacity (p=0.043) in patients with metabolic syndrome compared to the
baseline levels, it led to an insignificant decrease in serum oxidation (basal (p=0.063) and
Copper-stimulated (p=0.089) diene levels). Our data indicated that red wine consumtion is more
efficient on prevention of lipid peroxidation and protection of antioxidant capacity in patients
with metabolic syndrome rather than healthy controls.
Key words: Red wine, antioxidant enzymes, serum oxidation, metabolic syndrome.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P35
Paraoxonase-1 Attenuates Hyperoxia-induced Lethality in Drosophila
David A. Stoltz1, Peter J. Taft1, Michael Rector2, Joseph Zabner1
1
Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine; 2 Department of Biomedical
Engineering; University of Iowa, Iowa City, IA
Hyperoxia shortens lifespan in a number of organisms, including Drosophila, and aging has been
associated with increased oxidative stress. In addition, hyperoxia has been linked to a number of
pulmonary disorders including bronchopulmonary dysplasia, acute respiratory distress
syndrome/diffuse alveolar damage, and bleomyin toxicity. Paraoxonases (PONs) consist of three
family members and have been demonstrated to be important in cardiovascular disease due to
their protective effects on lipid oxidation. We hypothesized that expression of PON1 in
Drosophila would protect flies from hyperoxia-induced lethality. The UAS/Gal4 system and
daughterless promoter were used to express PON1 ubiquitously in Drosophila. 1-3 day-old male
flies, wildtype or PON1, were placed in an exposure box with continuous 100% oxygen flow.
Fly survival and physical activity was assessed daily. The presence of PON1 in transgenic flies
was confirmed with both immunoblotting and an assay for arylesterase activity. Hyperoxia
exposure shortened fly survival in both groups from about 60-80 days to less than 10 days.
However, hyperoxia-induced lethality was attenuated in the PON1-expressing flies (50% killing
was observed at approximately 7.5 days in wildtype flies compared to 8.5 days in PON1 flies).
Hyperoxia produced a time-dependent decrease in fly physical activity, measured in a climbing
assay, which was attenuated in the PON1 flies. In summary, we found that PON1 attenuates
hyperoxia-induced lethality in Drosophila. These data suggest that PONs may represent a
component of the protective response to oxidative stress.
David A. Stoltz is a Parker B. Francis Fellow.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P36
Comparison of PON1 –C108T and Q192R genotypes and
Phenotype in three Mexican mestizo populations
Quintanilla-Vega B.1, Pérez-Herrera N.1, Rojas-García E.2, May-Pech C1,3, González-Horta
MC.4, Carballo-Carballo F.4, Torres-Zalba R.4, Sánchez-Ramírez BE.4, Erosa de la Vega G.4,
Arévalo-Gallegos S.4
1
Sección Externa de Toxicología, CINVESTAV-IPN, Mexico City, Mexico. 2Dirección de
Fortalecimiento de la Investigación, Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico.
3
Facultad de Química, Universidad Autónoma de Yucatán, Yucatán, Mérida, Mexico. 4Facultad
de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chihuahua, Mexico.
Epidemiological studies have related a wide variation in serum PON1 activity among subjects,
and this variability has been attributed to PON1 polymorphisms. Several studies have showed the
role of PON1 genotype or phenotype on the susceptibility to organophosphorous (OP) toxicity
and to develop cardiovascular heart disease (CAD). OP poisoning and CAD are important public
health problems in Mexico. We evaluated PON1 –C108T and Q192R genotypes and phenotype
on three populations from Mexico: agricultural workers exposed to pesticides from northern
Mexico (n=51), farmers exposed to OP pesticide from southern Mexico (n=90), and non OP
exposed men from the central part of the country (n=214). Participants provided a blood sample;
PON1 –C108T and Q192R polymorphisms were evaluated by real-time PCR or RFLP and serum
PON1 phenotype by using phenylacetate and paraoxon as substrates. PON1 192R allele
frequencies were 0.49 (central), 0.48 (northern) and 0.56 (southern) with no differences among
them. While, PON1 -108T allele frequencies were 0.28, 0.23 and 0.32 for central, northern and
southern Mexico, respectively. PON1 –C108T genotype frequencies were different (p=0.052)
between northern and southern Mexico. Paraoxonase and arylesterase activities as a function of
PON1 Q192R genotypes were observed in all populations. Interestingly, the genotype-phenotype
association according to –C108T polymorphism was observed in individuals from central
Mexico, but not in farmers from northern or southern Mexico. The southern population showed
the highest percent of subjects with -108TT and 192RR genotypes that confer susceptibility to
CAD or toxicity to certain OP insecticides. Study supported by CONACYT (Grants 44643 and
C01-134).
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P37
Paraoxonase (PON1) Modulates the Toxicity of OP Mixtures
TB Cole1,2, K Jansen1, W-F Li3, CE Furlong2, LG Costa1,4
Depts. of 1Environmental and Occupational Health Sciences, 2Medicine (Div. of Medical Genetics) & Genome
Sciences, University of Washington, Seattle, WA, USA; Dept. of 3Environmental Health and Occupational
Medicine, National Health Research Institutes, Zhunan Town, Taiwan; 4Dept. of Human Anatomy, Pharmacology
and Forensic Science, University of Parma, Italy4.
Most chemical exposures involve complex mixtures. The role of paraoxonase 1 (PON1) and the
Q192R polymorphism in detoxication of individual organophosphorous (OP) compounds has
been well established. The extent to which PON1 protects against a given OP is determined by
its catalytic efficiency. We used a humanized transgenic mouse model of the Q192R
polymorphism to demonstrate that PON1 modulates the toxicity of OP mixtures by altering the
activity of another detoxication enzyme, carboxylesterase (CaE). Chlorpyrifos oxon (CPO),
diazoxon (DZO), and paraoxon (PO) are potent inhibitors of CaE, both in vitro and in vivo. We
hypothesized that exposure of mice to these OPs would increase their sensitivity to the CaE
substrate, malaoxon (MO), and that the degree of effect would vary among PON1192 genotypes if
the OP was a physiologically significant PON1 substrate. When wild-type mice were exposed
dermally to CPO, DZO, or PO and then, 4-hours later, to different doses of MO, the toxicity of
MO was increased compared to mice that received MO alone. This potentiation was attributed to
CaE inhibition. The potentiation of MO toxicity by CPO and DZO was higher in PON1 knockout
mice, which have greatly reduced capacity to detoxify CPO or DZO. Potentiation by CPO was
higher in PON1Q192 mice than in PON1R192 mice due to the decreased ability of the PON1Q192
alloform to detoxify CPO. Potentiation by DZO was similar in the PON1Q192 and PON1R192
mice, due to their equivalent efficiencies at detoxifying DZO. PO exposure resulted in
equivalent potentiation of MO toxicity among all four genotypes. These results indicate that
PON1 status can have a major influence on the ability of CaE to detoxicate OP compounds or
pyrethroid insecticides and metabolize drugs such as Ritalin when an individual is confronted
with mixed exposures. Supported by NIEHS ES04696, ES09883, ES09601/EPA: RD-83170901.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P38
Mouse serum paraoxonase-1 and ES-1 carboxylesterase display
distinct fatty acid lactonase activity
Philip W. Connelly1, Clive M. Picardo1, Philip M. Potter2, Xing Chen1, Graham F. Maguire1, Dominic S. Ng1,3. 1
Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Canada. 2St. Jude
Children's Research Hospital, Memphis, TN.
We observed fatty acid lactonase activity in serum from Pon1-/- mice that was resistant to
inactivation by EDTA, sensitive to the serine esterase inhibitor phenyl methyl sulfonyl fluoride,
and co-eluted with carboxylesterase activity by size-exclusion chromatography. Analysis of
serum from the Es1e mouse strain, which is deficient in carboxylesterase ES-1, proved that this
activity was due to ES-1. PON1 activity predominated at early time points (30 seconds),
whereas both PON1 and ES-1 contributed equally at later time points (15 minutes). Thus ES-1
can function as a fatty acid lactonase at neutral pH. Further, a dual lactonase activity is a
characteristic of mouse serum. The fatty acid lactonase activity of ES-1 provides an alternative
pathway for the metabolism of fatty acid lactones in mouse serum. This alternative pathway
may, at least in part, ameliorate the consequences of PON1 deficiency in mice.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P39
Optimal Liposomal Composition Determination and Physicochemical
Characterization of Stealth Liposomes (SL) Encapsulating
Organophosphorous Hydrolase (OPH)
M. Budai 1,3, P. Chapela1, J. Grimsley2, B. N. Novikov2, P. Gróf3, I. Klebovich3, A. Zimmer4,
M. Szilasi5, M. E. Wales2, J. R. Wild2, I. Petrikovics1
1
Sam Houston State University, Dept. of Chemistry & Forensic Science, Huntsville, TX-7341; 2Dept. of
Biochemistry & Biophysics, Texas A&M University, College Station, TX-77843; 3Semmelweis University,
Dept. of Pharmaceutics, Budapest, Hungary, H-1092, 4Karl-Franzens University, Department of
Pharmaceutics, Graz, Austria, A-8010
5
Debrecen University, Health Center, Department of Pulmonology, Debrecen, Hungary, H-4010
The toxicity of organophosphorous (OP) compounds is attributed to the inhibition of
acetylcholinesterase. The current treatment for OP exposure in the USA is the combination of
atropine and pralidoxime (2-PAM). Previous research has demonstrated an enhanced antidotal
efficiency when encapsulated OPH was combined with the current treatment. The enzyme must
be encapsulated in a stealth carrier system to protect OPH from in vivo degradation. Minimizing
the immunologic reactions is crucial for making this approach effective. This study focused on
the liposomal encapsulations. The optimal composition of liposomes for the encapsulation of
OPH was determined by evaluating the encapsulation efficiency using a spectrophotometric
assay. The molar ratios of dipalmitoylphosphatidylcholine (DPPC),
palmitoyloleoylphosphatidylcholine (POPC), and cholesterol were varied to determine the effect
on encapsulation. Physicochemical characterization of the liposomal OPH delivery system is
essential in order to get information on their in vitro stability and in vivo fate. Osmolarity, pH,
viscosity and encapsulation efficiency of the SL preparation and the surface potential of the
liposomes were determined. The membrane rigidity and the impact of OPH enzyme on it was
studied by electron-paramagnetic resonance (EPR) spectroscopy using spin probes. The in vitro
stability of the preparation, the vesicle size distribution and its alteration during a 3-week storage
period was followed by dynamic light scattering measurements.
These studies were supported in part by research funds from the National Institute of Health (Grant #: 5
U01 NS058035-02).
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
P40
Role of some PON1 genetic polymorphisms on semen quality
and sperm DNA integrity in farmers from southern
Mexico exposed to organophosphorous pesticides
Pérez-Herrera N.1, Polanco-Minaya H.2, Salazar-Arredondo E.3, Solís-Heredia M.J.1, HernándezOchoa I.1, Rojas-García E.4, Alvarado-Mejía J.5, Borja-Aburto V.H.6, Quintanilla-Vega B.1
1
Sección Externa de Toxicología, CINVESTAV-IPN, Mexico City, Mexico. 2Facultad de Química,
Universidad Autónoma de Yucatán, Mérida, Yucatán, Mexico. 3FES-Iztacala, Universidad Nacional
Autónoma de México, Mexico City, Mexico 4Dirección de Fortalecimiento de la Investigación,
Universidad Autónoma de Nayarit, Tepic, Nayarit, Mexico. 5Facultad de Medicina, Universidad
Autónoma de Yucatán, Mérida, Yucatán, Mexico. 6Coordinación Nacional de Salud en el Trabajo,
Instituto Mexicano del Seguro Social, Mexico City, Mexico.
Human paraoxonase (PON1) participates in organophosphorous pesticides (OP) deactivation and
may have a role on their susceptibility, due to PON1 polymorphism. We evaluated the role of
PON1 polymorphisms at promoting or coding regions: -A162G, -C108T, L55M and Q192R on
the susceptibility to OP toxicity on semen quality and DNA integrity in farmers from southern
Mexico chronically exposed to pesticides, mostly OP. Agricultural workers (n=54) from 18-55
years old provided semen and blood samples; semen quality was evaluated according to WHO,
and sperm DNA damage by in situ-nick translation (NT-positive cells), PON1 polymorphisms at
regions -A162G, L55M and Q192R by real-time PCR and -C108T by RFLP. We constructed an
OP exposure index during 3 months before sampling from an structure questionnaire as a
reflection of the exposure at one spermatogenic cycle. PON1 frecuencies in this population with
Mayan ascendancy were 0.85, 0.67, 0.20 and 0.54 for -162G, -108T, 55M and 192R alleles,
respectively. A dose-effect association was observed between sperm morphology, viability and
motility and NT-positive cells and OP exposure index only in homozygote 192RR farmers.
Sperm morphology and viability were related to OP exposure in farmers with -162GG genotype;
and sperm viability was associated with OP exposure only in individuals featuring -108CT
genotype. PON1L55M polymorphism was not associated with these effects. Our results suggest
that PON1, mostly Q192R polymorphism, participates in modulating the effects on semen
quality and sperm DNA integrity in occupationally OP exposed men. Study support by
CONACYT (Grant CO1-134) given to BQV.
3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
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3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
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3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
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3rd INTERNATIONAL CONFERENCE ON PARAOXONASES
September 7-10, 2008
Los Angeles, California
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