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Unlabelled: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells but not in most normal cells and has therefore been proposed as a promising antitumor agent. Recent experiments suggested that isolated primary human hepatocytes but not monkey liver cells are susceptible to certain TRAIL agonists, raising concerns about the use of TRAIL in cancer treatment. Whether TRAIL indeed exerts hepatotoxicity in vivo and how this is influenced by chemotherapeutic drugs or liver disease are completely unknown. Employing different forms of recombinant TRAIL, we found that the cytokine can induce proapoptotic caspase activity in isolated human hepatocytes. However in marked contrast, these different TRAIL preparations induced little or no cytotoxicity when incubated with tissue explants of fresh healthy liver, an experimental model that may more faithfully mimic the in vivo situation. In healthy liver, TRAIL induced apoptosis only when combined with histone deacetylase inhibitors. Strikingly, however, TRAIL alone triggered massive apoptosis accompanied by caspase activation in tissue explants from patients with liver steatosis or hepatitis C viral infection. This enhanced sensitivity of diseased liver was associated with an increased expression of TRAIL receptors and up-regulation of proapoptotic Bcl-2 proteins., Conclusion: These results suggest that clinical trials should be performed with great caution when TRAIL is combined with chemotherapy or administered to patients with inflammatory liver diseases.

One important cell death pathway involves binding of the cell surface receptor Fas, which recruits and activates specific initiator and effector caspases. In this study Balb/c mice were injected with monoclonal antibody to Fas either alone or followed by the tripeptide caspase inhibitor Z-VAD.fmk. At four hours mice were killed along with concurrent controls and tissues processed for histological examination and immunocytochemical staining for cleaved caspase-3. The livers in all animals treated with Fas alone showed massive apoptosis and positive staining for cleaved caspase-3 whereas those treated with Fas and Z-VAD.fmk or controls showed little or no apoptosis or staining for cleaved caspase-3. These features suggest that massive apoptosis may be important in fulminant liver disease.

There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn2+ on transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-beta1 (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 micromol/L Zn2+ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-beta1-treated hepatocytes. Surprisingly, Zn2+ did not inhibit the formation of high-molecular-weight DNA fragments (30-50 kbp to 250-300 kbp). These data provide evidence that Zn2+ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-beta1-induced apoptosis in hepatocytes.

Caspases plays a key role in the execution phase of apoptosis. "Initiator" caspases, such as caspase-8, activate "effector" caspases, such as caspase-3 and -7, which subsequently cleave cellular substrates thereby precipitating the dramatic morphological changes of apoptosis. Following treatment of mice with an agonistic anti-Fas antibody to induce massive hepatocyte apoptosis, we now demonstrate a distinct subcellular localization of the effector caspases-3 and -7. Active caspase-3 is confined primarily to the cytosol, whereas active caspase-7 is associated almost exclusively with the mitochondrial and microsomal fractions. These data suggest that caspases-3 and -7 exert their primary functions in different cellular compartments and offer a possible explanation of the presence of caspase homologs with overlapping substrate specificities. Translocation and activation of caspase-7 to the endoplasmic reticulum correlates with the proteolytic cleavage of the endoplasmic reticular-specific substrate, sterol regulatory element-binding protein 1. Liver damage, induction of apoptosis, activation and translocation of caspase-7, and proteolysis of sterol regulatory element-binding protein 1 are all blocked by the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD. fmk). Our data demonstrate for the first time the differential subcellular compartmentalization of specific effector caspases following the induction of apoptosis in vivo.

Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.

Apoptosis induced in primary hepatocytes by transforming growth factor beta1 and staurosporine produced chromatin condensation, DNA cleavage is detected by in situ end-labelling, field inversion and conventional gel electrophoresis, and cell detachment. These effects were abolished by benzyloxycarbonyl-valinylalanylaspartylfluoromethyl ketone, a cleavage-site-directed inhibitor of interleukin-1beta-converting enzyme-like proteases, and this finding suggests that these enzymes are involved in liver apoptosis.

DNA fragmentation in isolated rat liver nuclei is a Mg(2+)-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into > or = 700, 200-300 and 30-50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/Mg(2+)-dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N-ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg(2+)-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30-50, 200-300, > or = 700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.

Internucleosomal DNA fragmentation is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. In this study we demonstrate that DNA is initially cleaved into > or = 700, 200-250, and 30-50 kilobase pair (kbp) fragments via a Mg(2+)-dependent, multistep process which can be potentiated by Ca2+. The subsequent internucleosomal cleavage requires both Ca2+ and Mg2+. Furthermore, we show that the heavy metals Cd2+ and Hg2+, dichloroisocoumarin (a general serine protease inhibitor), and N-ethyl maleimide (NEM, a specific thiol reagent) are potent inhibitors of both the Mg(2+)- and Ca2+/Mg(2+)-stimulated DNA fragmentation. In contrast, two other serine protease inhibitors, N-alpha-tosyl-L-lysine chloromethylketone and N-tosyl-L-phenylalanine chloromethylketone are weak and ineffective, respectively, as inhibitors of DNA cleavage. Increasing inhibition of DNA cleavage is accompanied by a shift in the size of the cleaved DNA fragments, which increases from mono- + oligo-nucleosomes-->30-50 kbp-->200-300 kbp--> > or = 700 kbp-->intact DNA. Dithiothreitol, a dithiol, blocks NEM and dichloroisocoumarin inhibition, and since Cd2+ and Hg2+ are also potent--SH blocking agents it is proposed that a critical thiol is involved in the cleavage of DNA into both large kbp fragments and oligonucleosomal-sized fragments.

Internucleosomal cleavage of DNA has often been regarded as the biochemical hallmark of apoptosis. We now demonstrate in isolated rat liver nuclei that DNA is initially cleaved into > or = 700, 200-250 kbp and 30-50 kbp fragments via a multi-step process, which is activated by Mg2+ and Mg2+(+)Ca2+ but not by Ca2+ alone. The subsequent internucleosomal cleavage requires both cations. These findings demonstrate that a key event in the apoptotic process is the fragmentation of DNA into large kbp fragments by either a Mg(2+)-dependent process (which can be potentiated by Ca2+) and/or by a Ca2+/Mg2+ activated endonuclease(s).

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.

We have investigated the influence of the elevation of pulmonary glutathione (GSH) levels on the toxicity of the rodenticide alpha-naphthylthiourea (ANTU) to rat lung. Administration of phorone (diisopropylidene acetone; 200 mg/kg i.p.) caused an initial depletion of both pulmonary and hepatic GSH followed after 48 hr by a marked elevation in both tissues, due most probably to a compensatory rebound synthesis. In control rats, ANTU produced a dose-dependent lethality, hydrothorax and loss of ability of lung tissue to accumulate adenosine and spermidine (markers of endothelial and epithelial cell function, respectively). These effects were prevented or markedly ameliorated when ANTU was given 48 hr after pretreatment with phorone. The mechanism of the protection by phorone pretreatment against ANTU-induced pulmonary toxicity is unclear. It may be due, in part, to elevated GSH levels in pulmonary endothelial cells and, in addition, to increased detoxification of ANTU in the liver, resulting in a decreased availability to the lung.

Quinones may be toxic by a number of mechanisms, including arylation and oxidative stress caused by redox cycling. Using isolated hepatocytes, we have studied the cytotoxicity of four quinones, with differing abilities to arylate cellular nucleophiles and redox cycle, in relation to their effects on cellular pyridine nucleotides. High concentrations of menadione (redox cycles and arylates), 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) 2,3-dimethoxy-1,4-naphthoquinone (a pure redox cycler) and p-benzoquinone (a pure arylator) caused an initial decrease in NAD+ and loss of viability, which was not prevented by 3-aminobenzamide, an inhibitor of poly(ADP-ribose)polymerase. In contrast, 3-aminobenzamide inhibited the loss of NAD+ and viability caused by dimethyl sulphate so implicating poly(ADP-ribose)polymerase in its toxicity but not that of the quinones. Non-toxic concentrations of menadione, 2,3-dimethoxy-1,4-naphthoquinone and 2-hydroxy-1,4-naphthoquinone all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD+ was accompanied by a small, transient increase in NADP+ and followed by a larger, prolonged increase in NADPH and total NADP+ + NADPH. Nucleotide changes were not observed with non-toxic concentrations of p-benzoquinone. Our findings suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides, in an attempt to combat the effects of oxidative stress.

Activation of poly(ADP-ribose)polymerase by DNA damaging agents causes a depletion of intracellular NAD+ and subsequent lowering of ATP pools, which if extensive may lead to cell death. We have studied the cytotoxicity to isolated hepatocytes of dimethyl sulphate, a direct-acting carcinogen and mutagen, hydrogen peroxide, generated by glucose/glucose oxidase, and menadione (2-methyl-1,4-naphthoquinone) in relation to their effects on intracellular NAD+ and ATP levels. Both dimethyl sulphate and glucose/glucose oxidase caused a depletion of NAD+, which was apparently due to an activation of poly(ADP-ribose)polymerase as it was prevented by inhibitors of the polymerase, i.e. 3-aminobenzamide and nicotinamide. This protection of intracellular NAD+ was accompanied by a prevention of the cytotoxicity of both dimethyl sulphate and glucose/glucose oxidase, while it did not alter the decrease in intracellular ATP they induced. This apparent dissociation of effects on ATP from NAD+ does not support the suggestion that activation of poly(ADP-ribose)polymerase leads to a decrease in cellular ATP as a consequence of NAD+ depletion. Menadione also caused a depletion of NAD+ which preceded cytotoxicity, but in contrast to dimethyl sulphate and H2O2 this depletion did not involve poly(ADP-ribose)polymerase as it was not prevented by inhibitors of the enzyme. Our results also indicate that the cytotoxicity of menadione is not mediated by H2O2 alone. Marked depletion of intracellular NAD+ prior to toxicity and a protection against toxicity associated with maintenance of NAD+ suggest a possible role for the maintenance of intracellular NAD+ in cellular integrity.

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.

The possible mechanisms of naphthoquinone-induced toxicity to isolated hepatocytes were investigated using three structurally-related naphthoquinones, 1,4-naphthoquinone (1,4-NQ), 2-methyl-1,4-naphthoquinone (2-Me-1,4-NQ) and 2,3-dimethyl-1, 4-naphthoquinone (2,3-diMe-1,4-NQ). 1,4-NQ was more toxic than 2-Me-1,4-NQ whereas 2,3-diMe-1,4-NQ did not cause cell death at the solubility-limited concentrations used. All three naphthoquinones extensively depleted intracellular glutathione (GSH). However, the depletion of GSH induced by 1,4-NQ and 2-Me-1,4-NQ prior to cell death was more rapid and extensive than that induced by the nontoxic 2,3-diMe-1,4-NQ. Further studies demonstrated that 2,3-diMe-1,4-NQ was cytotoxic in the presence of dicoumarol, a compound which also potentiates the cytotoxicity of 1,4-NQ and 2-Me-1,4-NQ. To investigate the differential cytotoxicity of these three naphthoquinones, their relative capacities to redox cycle and to bind covalently to cellular nucleophiles were assessed. Redox cycling was investigated using rat liver microsomes where the order of potency for quinone-stimulated redox cycling was 1,4-NQ approximately 2-Me-1,4-NQ much greater than 2,3-diMe-1,4-NQ as indicated by nonstoichiometric amounts of NADPH oxidation and O2 consumption. NADPH-cytochrome P-450 reductase was implicated as the enzyme primarily responsible for naphthoquinone-stimulated redox cycling. The reactivity of the naphthoquinones with glutathione and, by implication, with other cellular nucleophiles was 1,4-NQ greater than 2-Me-1,4-NQ much greater than greater than 2,3-diMe-1,4-NQ. Overall, these studies indicate that 2,3-diMe-1,4-NQ is not cytotoxic (except in the presence of dicoumarol) and this lack of toxicity may be related either to its lesser capacity to redox cycle and/or its inability to react directly with cellular nucleophiles.

The mechanisms of toxicity to isolated rat hepatocytes of two structurally related naphthoquinones have been studied. Both 5-OH-1,4-naphthoquinone (5-OH-1,4-NQ; juglone) and 2-OH-1,4-naphthoquinone (2-OH-1,4-NQ; lawsone) caused a concentration-dependent cytotoxicity to hepatocytes which was preceded by a depletion of intracellular glutathione. 5-OH-1,4-NQ caused a depletion of intracellular glutathione when incubated either at 4 degrees C or 37 degrees C whereas 2-OH-1,4-NQ caused a depletion of intracellular glutathione when the hepatocytes were incubated at 37 degrees C but not at 4 degrees C. 5-OH-1,4-NQ but not 2-OH-1,4-NQ reacted with glutathione in buffered solution. These results suggested that the depletion of intracellular glutathione by 2-OH-1,4-NQ is enzyme mediated whereas in the case of 5-OH-1,4-NQ the direct chemical reaction with gluathione may be largely responsible for the depletion. A critical role for depletion of protein thiols in menadione-induced cytotoxicity has been proposed. In agreement with earlier work, menadione caused a decrease in protein sulphydryls prior to cell death, however, at cytotoxic concentrations of both 2-OH-1,4-NQ and 5-OH-1,4-NQ this decrease only accompanied rather than preceeded cell death. The mechanism of toxicity of 5-OH-1,4-NQ is similar to that of other naphthoquinones and involves formation of its corresponding naphthosemiquinone, active oxygen species and redox cycling as it stimulated a disproportionate increase in both microsomal NADPH oxidation and oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)

Quinones may be toxic by a number of mechanisms, including oxidative stress caused by redox cycling and arylation. This study has compared the cytotoxicity of four quinones, with differing abilities to arylate cellular nucleophiles and redox cycle, in relation to their effects on cellular pyridine nucleotides and ATP levels in rat hepatocytes. Non-toxic concentrations (50 microM) of menadione (redox cycles and arylates), 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) and 2,3-dimethoxy-1,4-naphthoquinone (a pure redox cycler) all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD+ was accompanied by a small, transient increase in NADP+ and followed by a larger, prolonged increased in NADPH and total NADP+ + NADPH. At toxic concentrations (200 microM), the quinones caused an extensive depletion of NAD(H), an increase in levels of NADP+ and an initial rise in total NADP+ + NADPH, prior to a decrease in ATP levels and cell death. Nucleotide changes were not observed with non-toxic (20 microM) or toxic (100 microM) concentrations of p-benzoquinone (a pure arylator) and ATP loss accompanied or followed cell death. A novel mechanism for the activation of 2-hydroxy-1,4-naphthoquinone has been implicated. Our findings also suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides, possibly mediated by an NAD+ reduction, in an attempt to combat the effects of oxidative stress.

Benzo[alpha]pyrene is metabolised by isolated viable hepatocytes from both untreated and 3-methylcholanthrene pretreated rats to reactive metabolites which covalently bind to DNA. The DNA from the hepatocytes was isolated, purified and enzymically hydrolysed to deoxyribonucleosides. The hydrocarbon-deoxyribonucleoside products after initial separation, on small columns of Sephadex LH-20, from unhydrolysed DNA, oligonucleotides and free bases, were resolved by high pressure liquid chromatography (HPLC). The qualitative nature of the adducts found in both control and pretreated cells was virtually identical; however pretreatment with 3-methylcholanthrene resulted in a quantitatively higher level of binding. The major hydrocarbon-deoxyribonucleoside adduct, found in hepatocytes co-chromatographed with that obtained following reaction of the diol-epoxide, (+/-) 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene with DNA. Small amounts of other adducts were also present including a more polar product which co-chromatographed with the major hydrocarbon-deoxyribonucleoside adduct formed following microsomal activation of 9-hydroxybenzo[alpha]-pyrene and subsequent binding to DNA. In contrast to the results with hepatocytes, when microsomes were used to metabolically activate benzo[alpha]-pyrene, the major DNA bound-product co-chromatographed with the more polar adduct formed upon further metabolism of 9-hydroxybenzo[alpha]pyrene. These results illustrate that great caution must be exercised in the extrapolation of results obtained from short-term mutagenesis test systems, utilising microsomes, to in vivo carcinogenicity studies.

Polycyclic aromatic hydrocarbons are metabolically activated by microsomal enzymes to reactive metabolites which covalently bind to DNA. The qualitative and quantitative nature of the hydrocarbon-deoxyribonucleoside adducts formed are markedly dependent on the balance of the oxidative and conjugating enzymes present in the activation system. Thus, utilising rat liver microsomes, to metabolically activate benzo(a)pyrene, the major hydrocarbon-deoxyribonucleoside adduct formed is due to metabolic activation of 9-hydroxybenzo(a)pyrene. In striking contrast to this when isolated rat hepatocytes are used to metabolically activate [3H]-benzo(a)pyrene, 9-hydroxybenzo(a)pyrene is conjugated primarily with UDPglucuronic acid and the major hydrocarbon-deoxyribonucleoside adduct formed is due to further metabolism of 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene. Thus the balance and nature of conjugating enzymes present in a tissue will, by determining the nature and amounts of adducts formed, also modulate the biological susceptibility of a particular tissue or cell. In this regard it may be of particular interest that whereas in isolated rat hepatocytes and short-term organ cultures of rodent lung and trachea conjugation with UDPglucuronic acid is quantitatively the major route of conjugation, with short-term organ culture of human lung, sulphate ester conjugation of phenolic substrates appears to be a major route of metabolism. Thus in vivo or in microsome or cell mediated mutagenesis assays of polycyclic aromatic hydrocarbons the susceptibility of a particular cell or tissue will be dependent in part on the relative activities of the oxidative and conjugating enzymes.

A patient underwent a routine percutaneous liver biopsy and subsequently developed gastrointestinal hemorrhage, biliary colic, and bilirubinuria suggesting the presence of hemobilia. After a negative arteriogram endoscopic cholangiography was used to confirm the diagnosis.

The mechanism(s) of toxicity of 1-naphthol and two of its possible metabolites, 1,2- and 1,4-naphthoquinone, to freshly isolated rat hepatocytes has been studied. 1-Naphthol and both naphthoquinones exhibited a dose-dependent toxicity to hepatocytes. [1-14C]-1-Naphthol was metabolised by hepatocytes predominantly to its glucuronic acid and sulphate ester conjugates, but small amounts of covalently bound products were also formed. Blebbing on the surface of the hepatocytes was observed following exposure to 1-naphthol and the naphthoquinones, together with a dose-dependent decrease in intracellular glutathione (GSH), which preceded the onset of cytotoxicity. The toxicity of 1-naphthol and the naphthoquinones was potentiated by dicoumarol, an inhibitor of DT-diaphorase (NAD(P)H:quinone oxidoreductase). This enhanced toxicity was accompanied by a greater amount of surface blebbing, an increased depletion of intracellular GSH, particularly in the case of 1-naphthol and 1,4-naphthoquinone, and a decreased metabolism of 1-naphthol to its conjugates with variable effects on the amount of covalently bound products formed. These results support the suggestion that the toxicity of 1-naphthol may be mediated by the formation of 1,2-naphthoquinone and/or 1,4-naphthoquinone, which may then be metabolised by one electron reduction to naphthosemiquinone radicals. These, in turn, may covalently bind to important cellular macromolecules or enter a redox cycle with molecular oxygen thereby generating active oxygen species. Both of these processes appear to play a role in producing the cytotoxic effects of 1-naphthol.

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.