Toxicology Letters ELSEVIER Toxicology Letters 91 (1997) 169-178 Pro-oxidant effects of 6 -aminolevulinic acid (6 -ALA) on Chinese hamster ovary (CHO) cells Rachel Neal a, Ping Yang a, James Fiechtl b, Deniz Yildiz a, Hande Gurer a, Nuran Ercal a,* a Chemistry Department, University of Missouri, 142 Schrenk Hall, Rolla, A40 6.5409, USA b Life Sciences Department, University of Missouri, Rolla, MO, USA Receive’d24 October 1996; received in revised form 17 February 1997; accepted 17 February 1997 Abstract 6-Aminolevulinic Acid (a-ALA) is a heme precursor accumulated in lead poisoning and acute intermittent porphyria. Although no single mechanism for lead toxicity has yet been defined, recent studies suggest at least some of the lead-induced damage may originate from d-ALA-induced oxidative stress. The present study was designed to test the hypothesis that a-ALA accumulation in Chinese hamster ovary (CHO) cells contributes to the cumulative oxidative challenge of lead poisoning as indicated by the oxidative stress parameters glutathione (GSH), glutathione disulfide (GSSG), malondialdehyde equivalents (MDA), and catalase (CAT). It will also examine the possibility that this oxidative challenge can be reversed by treatment with an antioxidant such as N-acetylcysteine (NAC). First in vitro administration of B-ALA to CHO cells was found to have a concentration-dependent inhibitory effect on colony formation and cell survival. NAC administration was shown to alleviate this inhibition in CHO survival. The oxidative status of CIHO cell cultures exposed to increasing concentrations of B-ALA was then examined. Decreases in GSH levels (P < 0.05) were observed in the a-ALA-treated cultures as compared to the controls, while GSSG and MDA levels were significantly increased in d-ALA-treated cells (P < 0.05). CAT activity was not significantly affected. NAC administration concurrent with B-ALA exposure resulted in GSH and GSSG levels similar to the control levels, while :no significant improvement in MDA was observed. These results indicate a state of oxidative stress and suggest that the a-ALA- induced inhibitory effect on CHO colony formation may be due to its pro-oxidant effect. To assess whether this oxidative challenge would induce antioxidant increases during extended exposure to S-ALA, CHO cells were exposed to 5 mM d-ALA for increasing time periods. The GSH and GSSG levels were measured and a rebound effect was observed after 12 h of B-ALA exposure. 0 1997 Elsevier Science Ireland Ltd. Keywords: 6Aminolevulinic acid; Lead-induced damage; Oxidative stress; Chinese hamster ovary cells * Corresponding author. Tel.: + 1 573 3416950; fax: + 1 573 3416033; e-mail: [email protected] 0378-4274/97/$17.000 11997Elsevier Science Ireland Ltd. All rights reserved. PIZSO378-4274(97)03887-3 170 R. Neal et al. /Toxicology 1. Introduction 6 -Aminolevulinic acid (6 -ALA), a component of the heme biosynthesis pathway, is accumulated in lead poisoning and in acute intermittent porphyria [l]. Lead’s inhibitory effect on critical enzymes such as S-ALA dehydratase (6-ALAD), an enzyme in the heme biosynthesis pathway which catalyzes the condensation of two molecules of S-ALA to porphobilinogen , has been well documented (Fig. 1). Lead has been shown to decrease the activity of 6-ALAD, leading to an accumulation of J-ALA [3,4]. Although no single mechanism for lead toxicity has been discovered, recent studies suggest the lead-induced damage may originate in part from 6 -ALA-induced oxidative stress. d-ALA has been shown to have a proclivity for enhancing reactive oxygen intermediates (ROI) and lipid peroxidation [5,6]. The promotion of ROI formation by d-ALA was examined by Monteiro et al. . Spin-trapping experiments were used to support the contention that 6 -ALA-induced oxyhemoglobin autooxidation occurs in conjunction with d-ALA autooxidation. Additionally, Oteiza and Bechara determined a role for d-ALA-induced lipid peroxidation and membrane leakage by using phosphatidyl choline:cardiolipin liposomes as a model system [S]. &-ALA induced the formation of thiobarbituric acid reactive substances (TBARS) in the liposomes as well as stimulating leakage of carboxyfluorescein from the liposome into the media. The formation of TBARS and membrane leakage was also found to be inhibited by the free radical scavenger, a-tocopherol. Recent studies have implicated the mechanism of d-ALA-induced ROI formation in the pathophysiology of acute intermittent porphyria and plumbism . In situ generation of ROI due to the accumulation of d-ALA in bone marrow and nerve cells has been proposed to trigger the neurological abnormalities typical of porphyrias and plumbism . The present study was designed to investigate the hypothesis that d-ALA accumulation during lead poisoning contributes to the cumulative oxidative challenge, with CHO cells used as an in vitro model system. Colony formation assays were Letters 91 (1997) 169-l 78 performed to determine d-ALA’s inhibitory effect on cell proliferation. The observed inhibition was investigated by measuring indicators of oxidative stress in the presence and absence of d-ALA and without the addition of 1 mM NAC. NAC, a well-known antioxidant and GSH precursor, was included to evaluate the possible therapeutic role of antioxidants on 6 -ALA-induced oxidative stress. A time course study of GSH and GSSG levels in response to b-ALA challenge was included to evaluate a possible induction of an antioxidant response. 2. Materials and methods 2.1. Materials The N-( 1-pyrenyl)-maleimide, 1,1,3,3-tetramethoxypropane, and 2-vinyl pyridine were purGlycine+ Succinyl-CoA ALA-synthetase & b-AminolevulinicAcid (GALA) AL.A-dehydratase(ALAD) J Porphobiiogm 4 Uroporphyrinogen 4 Coproporphyrinogen i ProtoporphyrinIx & Heme Fig. 1. The formation of B-ALA from glycine Co-A and the condensation reaction of h-ALA phobilinogen. and succinyl to form por- R. Neal et al. /Toxicology chased from Aldrich other chemicals were Louis, MO, USA). used in GSH, GSSG (Milwaukee, WI, USA). All purchased from Sigma (St. HPLC grade reagents were and MDA analysis. Letters 91 (1997) 169-I 78 the S-ALA exposed scale vs. the d-ALA scale. 2.5. Oxidative 2.2. Colony formation and counting of colonies The cells were fixed by decanting the media and Carnoy’s fixative adding (3:1, methanol:acetic acid) for 5 min. After the cells were washed, crystal violet was added for 5 min to stain the colonies. The plates were washed with distilled water, allowed to air dry, and the number of colonies were then counted. The plating efficiency (PE) was calculated as follows: PE = Colonies counted/cells seeded x IO0 Results reported from colony formation assays represent at lf:ast three separate experiments performed each time in triplicate. 2.4. Construction A cell ting the counted, times the groups on concentration a logarithmic on a linear stress studies assays CHO cells, an established tumor cell line, were propagated in Ham’s F-12 culture media supplemented with 10% fetal calf serum (FCS) and maintained at 37°C in 5% CO,/95% air. For colony formation assays, exponentially growing cells were collected after trypsinization and centrifuged at 1000 x g for 5 min. The resulting cell pellets were resuspended in fresh media and counted on a hemocytometer. Between 100-2000 cells were plated into small (60 mm) petri dishes and incubated for 4 h to allow cell attachment to the surface. The respective concentration of B-ALA, either with or without 1 mM NAC, was then added to the petri dishes. After 3-4 h of d-ALA or d-ALA plus 1 mM NAC exposure, the media was replaced with fresh media. The cells were incubated for 7-10 days, then fixed and stained (see below). 2.3. Staining 171 of cell survival curve survival curve was constructed by plotsurvival fraction (number of colonies divided by the number of cells seeded, plating efficiency of the control) from Separate cultures of exponentially growing - 5 x lo6 cells/ml were esCHO cells containing tablished. After overnight incubation, the media was replaced with fresh media containing varying concentrations of d-ALA with or without 1 mM NAC. The b-ALA-exposed groups were incubated for 3 h with the &-ALA while the control was incubated in media alone during this time. At the end of the incubation, the media was removed and lactate dehydrogenase (LDH) activity was immediately assayed in the media. The cells were washed, trypsinized, and homogenized for the determination of GSH, GSSG, MDA, and CAT activity. Results for oxidative stress studies are from a minimum of three separate experiments. 2.6. Glutathione and glutathione determinations by HPLC disuljide 2.6.1. GSH determination A new method of GSH determination was developed in this laboratory to analyze y-glutamyl cycle intermediates . Cell pellets were resuspended in serine-borate buffer (100 mM TrisHCl, 10 mM borate, 5 mM serine, 1 mM diethylenetriaminepentacetic acid (DETAPAC), pH 7.0). The cell suspension was homogenized on ice for 2 min with 5-s intervals of homogenization and rest, and derivatized with N-(lpyrenyl)-maleimide (NPM). This compound reacts with free sulfhydryl groups to form fluorescent derivatives (Fig. 2). Each sample was first diluted with distilled water to make a volume of 250 ~1 NPM (750 ~1, 1 mM in acetonitrile) was then added; the resulting solution was mixed and incubated at room temperature for 5 min. One ~1 of 2 N HCl was added to stop the reaction. After filtration through a 0.2 pm acrodisc, the derivatized samples were injected in a reverse phase onto a 3 pm C,, column HPLC system. R. Neal et al. /Toxicology 172 Letters 91 (1997) 169-178 SR -_15 0 0 / i> / I +Ii-SR 0 A l NPM THIOL Fig. 2. Reaction of thiol with N-(I-pyrenyl)-maleimide 2.62. GSSG determination The determination of GSSG was accomplished by adding 44 ~1 of water to 40 ~1 of the sample. To the diluted sample, 16 ~1 of 6.25% 2vinylpyridine in absolute ethanol was added, and the mixture was allowed to incubate at room temperature for 60 min. After 95 ,ul of a 2 mg/ml solution of NADPH and 5 ~1 of a 2 U/ml solution of glutathione reductase were added, the solution was subsequently mixed and an aliquot of 100 ~1 was immediately withdrawn. To this aliquot, 150 ~1 of HPLC grade water and 750 ~1 of 1.0 mM NPM were immediately added to perform the GSH derivatization, as mentioned above. 2.6.3. HPLC system The HPLC system (Shimadzu) comprised a model LC-1OA pump, a Rheodyne injection valve with a 20-~1 injection filling loop, and a model RF535 fluorescence spectrophotometer operating at an excitation wavelength of 330 nm and an emission wavelength of 375 nm. The HPLC column was 100 x 4.6 mm and packed with 3 ,um particles of C,, packing material. The mobile phase was 35% water and 65% acetonitrile containing 1 ml/l acetic acid and 1 ml/l o-phosphoric acid. The NPM derivatives were eluted from the column isocratically at a flow rate of 0.5 ml/min. Quantitation of the peaks from the HPLC system was performed with a Chromatopac, model CR601 (Shimadzu). THIOL-NPM DERIVATIVE to produce fluorescent thiol-NPM derivative. 2.7. Lipid peroxidation determinations by HPLC The cell pellets were homogenized in serine borate buffer. To 0.250 ml of homogenate, 0.650 ml of 5% trichloroacetic acid (TCA) and 0.100 ml of 500 ppm butylated hydroxytoluene (BHT) in methanol were added. The sample was then heated in a boiling water bath for 30 min. After cooling on ice, the sample was centrifuged. The supernatant was mixed 1: 1 with saturated thiobarbituric acid (TBA) [lO,l 11. The sample was once again heated in a boiling water bath for 30 min. After cooling on ice, 0.50 ml of the sample was extracted with 1.00 ml of n-butanol and centrifuged to facilitate the separation of the two phases. The resulting organic layer was first filtered through a 0.45 pm acrodisc and then injected onto a reverse phase 250 x 4.6 mm 3 pm C,, column. The mobile phase for this system was composed of 30% acetonitrile and 0.6% tetrahydrofuran in 5 mM phosphate buffer. The reaction complexes were eluted from the column isocratitally at a flow rate of 0.75 ml/min. 2.8. Catalase activity assays CAT activity was determined spectrophotometrically by the method of Beers and Sizer, and was expressed in U/mg protein as described by Aebi [12,13]. This method measures the exponential disappearance of H,O, (10 mM) at 240 nm in the presence of cellular homogenates. The equation which was used to fit the exponential decay of H,O, is as follows: R. Neal et al. /Toxicology Letters 91 (1997) 169-l 78 113 A,, = Aini e - kt 3.2. where k is the rate constant, which is dependent on the catalase activity. Table 1 displays the results of the GSH and GSSG measurements after culture exposure to various concentrations of S-ALA. GSH levels in CHO cells treated with 3 mM and 5 mM d-ALA were significantly lower than those levels observed in control cultures (P < 0.05). GSSG levels were found to be significantly elevated in CHO cultures treated with &-ALA when compared to those levels in control cultures (P < 0.05). Additionally, the ratios of GSH/GSSG were significantly decreased in the CHO cultures exposed to J-ALA when compared to those ratios observed in the control cultures (P < 0.05). Simultaneous exposure to 1 mM NAC and S-ALA returned the GSH levels to control levels and significantly decreased the levels of GSSG. 2.9. Lactate dehydrogenase activity The lactate dehydrogenase (LDH) activity assay was performed according to the method of Hassoun et al.  with a minor modification. The activity of LDH in 100 ~1 of media was determined by direct calculation based on the decrease in absorbance . 2. IO. Protein determination The Bradford method was used to determine the protein content of the cell samples using concentrated Coomassie Blue (Bio-Rad) and optical density determinaf.ons at 595 nm . A standard curve using bovine serum albumin was constructed. The homogenized cell pellets were subjected to appropriate dilutions before protein determination was performed. 2. Il. Statistical analysis Tabulated values represent means f SD. of at least three separate experiments. One-way analysis of variance (ANOVA) and the Student-NewmanKeuls multiple comparison test were used to analyze data from experimental and control groups. P-values < 0.05 were considered significant. 3.3. GSH and GSSG GSHIGSSG time course Fig. 4 shows the GSH/GSSG ratio in CHO cells exposed to 5 mM d-ALA over a 12-h period compared to the GSH/GSSG levels in control CHO cells (media alone). The GSH/GSSG ratio of 6-ALA-exposed CHO cells decreases between 3 and 6 h of exposure. By 12 h, the GSH/GSSG level of J-ALA-exposed cells had begun to increase. 3. Results 3.1. Survival curve Fig. 3 represents a survival curve, generated by plotting the survival fractions of d-ALA-treated cultures against increasing 6 -ALA concentrations. J-ALA inhibited CHO colony formation in a concentration-dependent manner. As shown in Fig, 3, 1 mM NAC incubated concurrent with 1, 3 and 5 mM concentrations of S-ALA results in significantly increased cell survival. I 0.1 0 -AlA+NAC .-..-- Am I 1 I I , 1 2 3 4 5 delta-ALA concentrations (IIIM) Fig. 3. Survival curve of CHO and without 1 mM NAC. cells exposed to d-ALA, with 174 R. Neal et al. /Toxicology Table 1 Effect of 1, 3 and 5 mM J-ALA, Control 1 mM 3 mM 5 mM 1 mM I mM 3 mM 5 mM a-ALA a-ALA h-ALA NAC B-ALA+NAC 6-ALA+NAC S-ALA+NAC with or without GSH (nmol/mg 72.98 76.47 65.35 60.49 85.71 90.50 77.69 69.64 k k k + k * k f Letters 91 (1997) 169-178 1 mM NAC supplementation, protein) 0.95 1.24 1.27* 4.84* 3.22 1.15** 4.03** 0.29** All values represent mean k S.D. for three separate experiments. *P<O.O5 compared to control. **P<O.O5 NAC treated groups compared to corresponding d-ALA 3.4. Ca talase Table 2 contains the results from CAT activity assays. CAT activity had not significantly decreased in CHO cultures exposed to S-ALA when compared to the control cultures. NAC (1 mM) treatment during exposure to S-ALA resulted in no significant improvement in CAT activity. 3.5. Malondialdehyde equivalents (MDA) Table 2 shows the results of MDA levels for control and S-ALA- exposed CHO cells. MDA levels of &-ALA-exposed CHO cells were significantly higher than those observed for the control CHO cells (P < 0.05). NAC had no observed beneficial role. 3.6. Lactate dehydrogenase (LDH) activity Table 2 also shows the results of the LDH activity assay. As shown, no significant differences were seen between control, d-ALA, and NACtreated cells. 4. Discussion Lead has wide-ranging effects on a number of human organ systems. It has been implicated in cases of anemia and immunosuppression [17- 191. The mechanism by which lead causes its deleteri- GSSG 5.33 7.64 13.85 15.45 7.50 8.82 8.81 9.83 (nmol/mg * + + k k * + f 1.38 2.10 1.27* 3.82* 1.84 1.45 1.97** 1.23** on CHO protein) cell GSH and GSSG levels GSH/GSSG 13.70 10.01 4.72 3.92 11.43 10.26 8.82 7.08 + 0.69 f 0.59* k 1.OO* k 1.27* * 1.75 & 0.79 k 2.05** _+0.24** groups. ous effects has yet to be elucidated; however, part of lead’s effect may be due to the accumulation of d-ALA. The accumulation of d-ALA originates from lead’s inhibition of ALAD, an enzyme in the heme biosynthesis pathway which catalyzes the condensation of two molecules of b-ALA to porphobilinogen [2-41. ALAD is sensitive to oxygen, and requires a high exogenous thiol concentration for full catalytic activity due to the oxidative susceptibility of the enzyme’s 32 reactive thiol groups composing many possible active sites. Lead is known to bind to ALAD through one of these active sites causing dramatically lowered enzyme activity resulting in J-ALA accumulation . At a pH range of 7.0-8.0, J-ALA enolizes and this enol undergoes autooxidation resulting in the formation of the superoxide and the hydroxyl radical. d-ALA has also been shown to undergo iron-catalyzed oxidation with ROI generation and to induce Ca2+ release from mitochondria through oxidative damage to the inner mitochondrial membrane . To investigate b-ALA’s role in lead-induced oxidative stress, first colony formation assays were performed in the presence of increasing concentrations of d-ALA. The results of these assays, as shown in Fig. 3, indicated that J-ALA acts in a concentration-dependent manner to inhibit CHO cell colony formation. This was evidenced by a decrease in survival fraction as d-ALA concentration increases. The administration of 1 mM NAC results in increased survival fraction at all R. Neal et al. /Toxicology Letters 91 (1997) 169-l 78 175 -CONTROL 20- --+- 5 mM G-ALA /--4 _H----- time (hours) Fig. 4. The effect of 5 mM J-ALA on the GSH/GSSG ratio of CHO cells exposed to d-ALA for 12 h S-ALA concentrations studied. Since d-ALA has been shown to have a proclivity for enhancing ROI, the observed inhibition is believed to be the result of 6-ALA-induced oxidative stress . The degree of oxidative challenge can be quantified by measuring GSH, GSSG, lipid peroxidation byproducts (MDA), and the activities of antioxidant enzymes such. as catalase, superoxide dismutase, and glutathione peroxidase. Together these proteins combine to mediate the intracellular response to oxidative stress . GSH functions both as a direct scavenger of ROI and as a cofactor in their metabolic detoxification [21-231. As a result of oxidative stress, GSH is oxidized rapidly to GSSG; consequently, a decrease in GSH and an increase in GSSG (i.e. a decreasing ratio of GSH to GSSG) is suggestive of oxidative stress [24,25]. Results from the study illustrated a decrease in the GSH/GSSG ratio after d-ALA indicating that d-ALA was inducing exposure, oxidative stress in CHO cells. A time course experiment was performed to evaluate the impact of prolonged d-ALA exposure on the GSH/GSSG ratio in CHO cells. In Fig. 4, even at 3 h, d-ALA is shown to induce oxidative stress as the GSH/ GSSG ratio falls. This trend continues until 12 h when an increase in the GSH/GSSG ratio of the d-ALA-treated cells was observed, indicating a possible initiation of antioxidant defense. Catalase is a heme-containing antioxidant enzyme that converts H,O, to 0, and water . Its activity has been shown to change upon oxidative challenge in vitro. The activities of antioxidant enzymes are dependent upon the nature of the oxidant and the duration of challenge. In the present study, no increases in CAT activity were observed in the d-ALA concentrations studied. It is possible that these concentrations were simply not significant to cause an observed change in CAT activity. Oxidative stress is also shown by an increase in lipid peroxidation byproducts, such as MDA [27,28]. MDA is a degradation product of the highly unstable lipid peroxides that are generated from the interaction of pro-oxidants such as the hydroxyl radical with membrane lipids. It is routinely measured to show the degree of lipid peroxidation due to oxidative stress [29,30]. Lead does not directly undergo an oxidation-reduction cycle that produces ROI. Therefore, the effect of lead on lipid peroxidation in our previous study may have been due to an indirect mechanism involving the inhibition of enzymes such as ALAD . Such inhibition could result in increased substrates capable of undergoing a redox cycle that would produce ROI [25,32]. To assess this hypothesis, CHO cells were exposed to increasing concentrations of d-ALA. We have shown that MDA levels increased in CHO cell cultures ex- R. Neal et al. /Toxicology 176 Table 2 Effect of 1, 3 and 5 mM d-ALA, hamster ovary (CHO) cells Group Control 1 mM 1 mM 3 mM 5 mM 1 mM 3 mM 5 mM both with and without CAT activity NAC b-ALA b-ALA b-ALA B-ALAfNAC O-ALA+NAC 6-ALA+NAC All values represent *PiO.O5 compared 0.041 0.039 0.028 0.034 0.036 0.030 0.032 0.029 (Ujmg Letters 91 (1997) 169-l 78 I mM NAC supplementation, protein) * 0.007 * 0.008 + 0.006 f 0.004 f 0.009 f 0.006 & 0.008 f 0.002 MDA (nmol/lOO 12.3 f 0.7 11.9+2.3 29.2 + 2.2* 25.5 + 5.5* 26.1 + 2.0* 33.2 k 3.0 25.1 & 3.3 28.1 * 1.5 on CAT, mg protein) MDA, and LDH in Chinese LDH (U/ml) 40.25 40.01 39.36 38.60 37.78 40.60 39.12 37.78 + * k + * + f & 2.62 3.14 2.72 2.31 1.81 2.07 3.56 6.66 mean + S.D. to control. posed to increasing concentrations of d-ALA, once again indicating that S-ALA can induce oxidative stress in CHO cells. Lipid peroxidation is known to change membrane fragility, motility and permeability [33-351. LDH is a cytoplasmic enzyme and its leakage from injured cells into the culture medium has been shown to be useful as an indicator of cellular membrane damage. In the present study, we measured LDH activity in a culture medium in order to assess the membrane damage induced by 6ALA-derived ROI. No differences in LDH activity were noted regardless of whether incubation time or d-ALA concentration was the independent variable. These findings suggest two possibilities. The concentrations in this study of d-ALAderived ROI does not change the native membrane permeability of the CHO cells as HermesLima et al. proposed for mitochondrial inner membrane . Another possibility is that B-ALA may simply inhibit LDH activity. When the results of the GSH, GSSG, and MDA in 6-ALA-exposed CHO cells are examined, the hypothesis that d-ALA is at least partially responsible for the ROI-induced oxidative stress present in lead poisoning is supported. One possible remedy for this oxidative stress would be replenishment of the cellular GSH pool. However, because GSH does not readily cross the outer cell membrane, direct GSH supplementation is not insuccessful . To circumvent this limitation, tracellular L-cysteine pools may be elevated by NAC administration. NAC is readily deacetylated to yield L-cysteine, which is the rate limiting component of GSH synthesis. NAC is also a wellknown antioxidant with a proven high toxicity threshold in vivo and a well-documented history of clinical application . The antioxidant effects of NAC supplementation were evidenced by the increase in GSH/GSSG ratios (Table 1) and increased cell survival (Fig. 3). These findings support a protective role of NAC in J-ALA exposed cultures and suggest that NAC might have a therapeutic effect on diseases such as lead poisoning where d-ALA accumulation occurs. Our previous study showed that lead-exposed CHO cells undergo increased oxidative stress . The present study was undertaken to examine d-ALA’s contribution to lead-induced oxidative stress. Study results based on data obtained through the measurement of the various oxidative stress parameters indicate that d-ALA does play a role in the induction of oxidative stress in CHO cells. This study, in conjunction with our previous study , suggests that oxidative stress in lead poisoning is caused by lead both directly and indirectly by the accumulation of S-ALA. This leads to the conclusion that antioxidants such as NAC should be employed in cases of lead poisoning to help restore oxidative balance. R. Neal et al. /Toxicology Acknowledgements The authors are thankful to Dr. D. Spitz and Dr. P. Lutz for their comments on the manuscript. Special thanks also to A. Gambill, J.T. Cochran and Dr. Serdar Oztezcan for their technical help. Dr. Ercal was supported by lR15ES08016-01 from the NIEHS, NIH and the contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views 01‘the NIEHS or NIH. H. Gurer was supported by the Turkish Scientific and Technical Research Council. References [l] Hindmarsh, J.T. (1986)The porphyrias: recent advances. Clin. Chem. 32, 1;!55-1263.  Haeger-Aronsen, B., Abdulla, M. and Fristedt, B.I. (1971) Effect of lead on delta aminolevulinic acid dehydratase activity in red blood cells. Arch. Environ. Health 23, 440-445.  Ribarov, S.R. and Bochev, P.G. (1982) Lead-hemoglobin interaction as a possible source of reactive oxygen species a chemiluminescent study. Arch. Biochem. Biophys. 213, 288-292.  Gibbs, P.N.B., Gore, M.G. and Jordan, P.M. (1991) Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolevulinic dehydratase. Biochem. J. 225, 573-580.  Hermes-Lima, M.. Valle, G.R.V.. Vercesi. 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