378A AASLD ABSTRACTS HEPATOLOGY October 1995

1085 COCAINE-INDUCED HEPATIC INJURY IN RATS IS MEDIATED BY IMPAIRMENT IN MITOCHONDRIAL FUNCTIONS AND DEPRESSION IN ANTIOXIDANT ENZYMES. B. G. Devi. D. L. Schunlev. A.W.K. Chan. Research Institute On Addictions, Buffalo, NY- 14203

Cocaine ~ abuse is associated with extensive morphological and biochemical abnormalitms in liver cells leading to necrosis. Animal studies have shown that the C-induced liver injury is associated with an increase in peroxidative stress, loss of ATP and giutathione (GSH), inareased leakage of aspartate (AST) and alanine aminotransferase (ALT). These changes paralleled the extensive morphological damage to hepatic mitochondria (M). INTENT: Examine the underlying mechanism by which C induces oxidative stress and determine whether M energetics are the target of C toxicity. METHODS: Male Sprague Dawley rats were injected I.P. five times with C over a 3-day period (25 mg/kg). Cytochrome oxidase (CO), AST, MDA (malonaldehyde), D (diene), GSH, ATP, respiration (state 3 & 4) with glutamate and suceinate were assayed. Amioxidant enzymes ¢atelase (CAT), superoxide dismutase (CuZn-SOD and Mn-SOD), GSH peroxidase (GSH Px) and GSH transferase (GST) were quantified. RESULTS: Significant changes include the following: AST level in C group was increased 83%. Cellular and M-MDA levels rose 64% and 21%,~espectively, and ATP levels dropped 35%. Cellular levels of D in C group increased 22%, with a parallel decrease in GSH levels (30%) in cells and M (40%). Exposure to C__ decreased glutamate-stimulated state 3 & 4 respiration by 23% a n d l 9 Suceinate-stimulated state 3 & 4 respiration was decreased to 20%and 25%. Significant decreases in the activity of CO (35%), CAT (19%), GSH Px (29%), and GST (20%) was found in the C group. C exposure significantly increased the levels ofCu Zn-SOD and M-n-SOD by 30~ and 22%, respectively, Preliminary analysis ofGSH Px, GST, and CAT mRNA also agrees with the above findings. CONCLUSION: These observations demonstrate that: 1] acute exposure to C decreases the activities of key antioxidunt enzymes resulting i n elevation peroxidative stress ; 2] In M, the accumulation of MDA is accompanied by significant drop in GSH; 3] The decrease in cellular ATP levels could be directly associated with a reduction in mitochondrial respiration and CO activity; 4] Increased oxidative stress in M may be partly responsible for the observed inhibitory effect of C on energetios and subsequently the development of hepatic injury. (Supported by Tuttle Grant from the Research Foundation for Mental Hygiene Inc.)

1086 ESTROGEN AND ANTIOXIDANTS IN HEXACHLORO- BENZENE-INDUCED HEPATIC PORPHYRIA IN RATS. K.E. Anderson. S. Vadhanavikit and D,E. Goeger, University of Texas Medical Branch, Galveston, TX 77555-1109

Porphyria cutanea tarda (PCT) is due to oxidative inactivation or inhibition of hepatic uroporphyrinogen decarboxylase (UROD) and/or oxidation of its substrate, uroporphyrinogen. Precipitation of the disease requires iron, induction of P450 1A may be involved, and estrogens can contribute by an unknown mechanism. Recent studies suggest that ascorbate can protect against the development of PCT in laboratory animals. We studied the interactions of estrogen and the antioxidants ascorbate and coenzyme Q-10 in the hexachlorobenzene (HCB)-treated rat, a n animal model for human PCT. Male Wistar rats were treated either with standard diet alone, HCB (5g/kg diet) for 15 weeks, diethylstilbestrol (DES, 6 mg/kg body weight i.p. weekly), ascorbate (5 g/kg diet), CoQ-10 (100 mg/kg die0, or combinations of these treatments. Liver porphyrins were increased in all groups treated with H C B . Porphyria was enhanced in all groups treated with DES, compared to appropriate controls. Ascorbate partially prevented porphyrin accumulation with or without DES, whereas CoQ-10 had the opposite effect. UROD was reduced by HCB, but not affected by DES or antioxidants. DES increased malondialdehyde only in rats treated with HCB, and this was not affected by antioxidants. Activity of ethoxyresorufin- O-deethylase (a marker for CYP1A) was markedly induced by HCB and tittle affected by other treatments. DES oxidation was little affected. These results establish clearly that DES can exacerbate PCT in this model. Ascorbate may have a protective role, whereas CoQ at the dose studied does not. We conclude that estrogens exacerbate PCT by an oxidative mechanism that remains to be established, and that ascorbate is at least partially protective.

1087 CALCIUM-INDUCED MITOCHONDRIAL PERMEABILITY TRANSI- TION IN INTACT HEPATOCYTES REVEALED BY LASER SCAN- NING CONFOCAL MICROSCOPY. T Qian. B Herman and JJ. Lemasters. Dept. of Cell Biology, Univ. of North Carolina, Chapel Hill, NC

2+ • . BACKGROUND: In previous work, Ca -dependent toxicity to hepato- cytes was shown to involve uncoupling of mitochondria (Biochem. Biophys. Res. Commun. 167, 600). A possible mechanism of uncoupling is a Ca 2+- induced mitochondrial permeability transition that opens a high conductance pore in the inner mitochondrial membrane, causing depolarization, swelling and uncoupling of oxidative phosphorylation. Accordingly, our AIM was to visualize changes in mitochondrial permeability, polarization and Ca 2+ content

2+. during toxicity with Br-A23187, a Ca Ionophore, using laser scanning confo- cal microscopy.

METHODS: Overnight cultured rat hepatocytes were loaded with Rhod- 2, calcein, Rhodamine 123, and propidium iodide to measure free Ca 2+, mite- chondrial membrane permeability, mitochondrial membrane potential and loss of cell viability, respectively. To increase cellular Ca 2+, hepatocytes were ex- posed to 10 p_M Br-A23187 in Krcbs-Ringer-HEPES buffer (KRH) containing 0.1 or 0.5 mM CaC12. Fluorescent images were collected with Bio-Rad MRC- 600 and Zeiss LSM 410 laser scanning confocal microscopes.

RESULTS: In hepatocytes incubated in KRH containing 0.1 mM Ca 2+, Br-A23187 produced an increase of mitochondrial Rhod-2 fluorescence, indi- cating increased mitochondrial free Ca 2+. Rhodamine 123 and calcein images remained unchanged. When extracellular Ca ~+ was changed to 0.5 mM, mito- chondrial Ca 2+ increased further. Concomitantly, Rhodamine 123 fluorescence was lost from mitochondria, and calce!n redistributed from the cytosol into the mitochondrial matrix, indicating the onset of the mitochondrial permeability transition. Hepatocytes then lost viability after 10-15 minutes. Pretreatment with 1 pM cyclosporin A and 5 p.M trifluoperazine, pharmacological inhibitors of the permeability transition, blocked these chunges and delayed cell killing.

CONCLUSION: The mechanism of the Ca2+-dependent toxicity by Br- A23187 involves mitochondrial depolarization and uncoupling due to onset of the mitochondrial permeability transition.

1088 I N H I B I T I O N O F I R O N T O X I C I T Y I N H U M A N H E P A T O C Y T E C U L T U R E S B Y T H E P Y O V E R D I N S P a A and Pf. G LesanaL N Hubert. N Chenoufi. O ~ . G ~ 1 • Pasdeinup. M A Abdullah~ and P Brissot. INSERM I349 and Cllnique des Maladies du Foie, CHRU Pontchailloe, Rennes ; 1 - La~ratoire de Biologic Celtulaire, Facult~ de Pharmacie, Rc~ws ; 2 - Laboratoire de Chimie Mic~bie~e~UniveTsit~ Louis Pastet~, Strasbomg, Frmce.

In the trenUnent of iron overload sitoadoes, desferrioxamin¢ B is until now the only useful drug. However, desferriox~nine B is ineffective by the oral mute and must be given by subeutane~ms i~usien. The~fo~, there is a need for the development of an oral active chelating agent to Ire.at iron overloaded patients. The present work was undertaken to evaluate two candidate compounds, the bacterial pyoverdins PaA and Pf, isolated respectively from Pseudomonas aerugiansa and fluarescens. These substances are chromopeptides possessing three bidentate groups which bind iron (HI) giving very stable complexes CK A ffi 1032). Human hepatocyte cultures were used ; they were maintained in the control cundifiun, in the presence of iron citrate alune (50 or 100 I.tM), PaA or Pf alune (50 or 100 ttM), iron citrate (50 er 100 IxM) plus PaA er Pf (50 er 100 ~d), Extracellular LDIL AST and ALT, expressed in per cent of the total activity of the culture and MDA production, were measured as indexes of cytotoxicity. In order to demonsWate that these chelators were able to decrease iron uptake or increase iron release from the hepatecytes, labelled cells were obtained by maintaining the cultures in the presence of 1 ttM 55Fe ferric chloride plus 50 ttM iron citrate. Evaluation of iron twdcity : In tbe conlxol cultures, the exWacellular enzyme/total enzyme ratios were 9, 23 and 9% for LDH, AST and ALT respectively ; in the presence of 100 ttM of iron, the same ratios increased to 27, 45 and 28%. MDA concentration was also increased by a factor 23 and 34 respectively with 50 or 100 ~ of iron. Protective effect of the chelators : The addition of 50 er 100 ~ PaA or Pf to the culture medium did not exhibit any toxic effect ; the enzyme leakages were ~ and a maximal effect o f 20% was observed with 100 ttM. We observed also that 50 and 100 ~M PaA or Pf were effective to inhibit the increase in the enzyme releases resulting from the addition of 50 or 100 pM iron to the culture medium. Effect of the chelators on 55Fe uptake : The bepatocytes were maintained in the presence of 1 ;uM 55Fe plus 50 gM iron citrate without cbelator or with 50 or 100 I.tM PaA or Pf. In the presence of tbe chelators iron uptake was decreased by 95%. Effect of the chelators on 55Fe release : The cultures were treated for 1 day with 1 p.M 55Fe plus 50 ~ iron cllrate ; then the bepatocytes were maintained 1 day more with either ccmrol medium er medium supplemented with 50 or 100 ttM PaA or Pf. The addition of 50 or 100 IxM PaA resulted in 34 and 47% decrease in intracellulas iron level. In the presence of 50 or 100 ttM Pf this decrease was 41 and 63% ~ t i v e l y . Conclusion : The pyovardins PaA and Pf are effective in the protection of human bepatocytes against the toxic effect of iron by decreasing iron uptake by the cells. However, P£ appears more effective than PaA on iron release by the human hepatocytes.