Lack of p53 protein expression in preneoplastic rat hepatocytes in vitro after exposure to N-acetoxy-acetylaminofluorene, X-rays or a proteasome inhibitor.

Clonal expansion of initiated cells is an important process in carcinogenesis. Loss of functional p53 protein in initiated, preneoplastic cells might be involved in this process because such a loss would favour cell growth at the expense of normal cells upon exposure to genotoxic compounds. We have tested the hypothesis that p53 is not expressed in preneoplastic cells in the rat liver. Hepatocytes were isolated from livers of 10-week-old female rats that contained foci of preneoplastic hepatocytes, generated by 6-7 weekly injections of diethylnitrosamine (0.15 mmol/kg body wt intraperitoneally (i.p.)), starting 24 h after birth. The mixture of phenotypically normal and preneoplastic hepatocytes was exposed to X-rays or N-acetoxy-acetylaminofluorene (NAAAF), both causing DNA damage directly. At 24 and 48 h after exposure the cells were fixed and double stained for glutathione-S-transferase 7-7 (GST7-7), to identify preneoplastic cells, and p53. The percentage of p53-positive cells was much lower in GST7-7 positive (GST7-7+) than in GST7-7 negative (GST7-7-) hepatocytes. Exposure of cells to X-rays or NAAAF induced p53 in GST7-7- cells after 24 h, but GST7-7+ hepatocytes failed to do so. These results suggest that preneoplastic cells do not express p53 or have an attenuated p53 response to genotoxic treatments. This was confirmed when the cells were exposed to a proteasome inhibitor, PSI, which inhibits p53 degradation: a 12-fold increase in p53-positive cells was found after 48 h in GST7-7- hepatocytes, but in GST7-7+ hepatocytes no increase was observed. The percentage of GST7-7+ hepatocytes among surviving cells was increased after exposure to NAAAF, suggesting that these are more resistant to NAAAF than GST7-7- cells. This was not observed with PSI. These results indicate that preneoplastic hepatocytes have a lower p53 protein content and are not able to increase p53 upon inhibition of p53 breakdown or upon induction of DNA damage. Therefore, loss of p53 may favour clonal expansion of preneoplastic hepatocytes in the rat after administration of hepatocarcinogens or X-rays.

Phenobarbital-induced cytoplasmic accumulation of beta1-integrin in rat liver enzyme altered foci; an immunohistological study.

In this immunohistological study we investigated integrin expression in EAF in female rats treated with diethylnitrosamine (DEN) as initiator and phenobarbital (PB) as promotor (DEN-PB treatment) for up to 32 weeks. Using a beta1-integrin antibody, there was an increased cytoplasmic staining and a decreased sinusoidal staining in EAF, as compared to non-EAF areas. The majority of small EAF and all larger EAF exhibited this altered distribution of beta1-integrin. The increased cytoplasmic staining was not found in EAF after a 10 week treatment-free period. In periportal areas in partial hepatectomized control rats a similar increase in cytoplasmic staining was seen. EAF in DEN-initiated and DEN-promoted rats (DEN-DEN treatment) were also studied. This protocol induced rapidly growing EAF. Most lesions did not show the increased cytoplasmic staining. However, after partial hepatectomy of DEN-DEN-treated rats, a cytoplasmic staining was seen in EAF. It is concluded that PB induced a reversible cytoplasmic beta1-integrin expression in many EAF and in all larger EAF. It is suggested that the alteration constitutes part of hepatocyte resistance to toxicological stress and apoptosis in EAF.

Selective toxicity in putative preneoplastic hepatocytes: a comparison of hydroquinone and duroquinone.

In this paper data is presented suggesting selective toxicity towards enzyme altered hepatocytes. Hydroquinone (HQ) treatment 24 or 48 h after diethylnitrosamine (DEN) initiation reduced the number of glutathione S-transferase-P (GST-P)-positive hepatocytes in situ. Furthermore, in experiments on primary cultures of hepatocytes from control rats a synergism in cell killing between DEN and HQ was observed. In another in vitro system the effect of HQ and duroquinone (DQ) on GGT-positive and -negative hepatocytes was investigated. DQ was shown to affect the GGT-positive cells, while HQ mainly affected GGT-negative cells. These results suggest that HQ can reduce the population of enzyme altered foci (EAF) precursor cells by synergistic interactions with DEN, but provide no support for the notion that HQ selectively damage cells in developed EAF. This conclusion is supported by previously published data on effects of HQ on the development of EAF.

Low expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci induced in rat liver by diethylnitrosamine or phenobarbital.

The expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci (EAF) induced by diethylnitrosamine (DEN) and phenobarbital (PB) was examined. p21 expression in the nucleus of hepatocytes in EAF was decreased compared to surrounding tissue. Fifty-eight percent of all GST-P-positive EAF induced by DEN and 79% of the EAF induced by PB were p21-negative. The proportion of p21-negative EAF increased with the size of the foci and more than 90% of the largest EAF were p21-negative. p21 is a mediator of p53 signals leading to block of the cell cycle. In conjunction with previous data indicating that p53 is not induced in GST-P-positive hepatocytes isolated from EAF-bearing rats, the results of this study suggest a role for altered signaling in the G1-S check point in rat hepatocarcinogenesis.

Xenobiotics modulate the p53 response to DNA damage in preneoplastic enzyme-altered foci in rat liver; effects of diethylnitrosamine and phenobarbital.

Enzyme-altered foci (EAF) develop in rat liver in response to carcinogen treatment. Our hypothesis is that EAF adapt to genotoxic stimuli by lowering their expression of p53 and that such decreased p53 expression confers a growth advantage on the hepatocytes present in EAF. After a single neonatal dose of diethylnitrosamine (DEN), rats were treated with either 2 - 12 additional doses of DEN or phenobarbital (PB) for 3 - 14 months. Twenty-four hours prior to sacrifice, all rats also received a challenging dose of DEN. The numbers of p53-positive hepatocytes (demonstrating immunohistological staining in the nucleus) in EAF and surrounding tissue were subsequently determined. In DEN-treated rats, p53 expression was attenuated in EAF compared to surrounding tissue. The longer the period of treatment and the larger the size of the EAF, the fewer the p53-positive hepatocytes/mm2 were observed in these lesions. These data were confirmed by Western blot analysis. PB-treated rats did not demonstrate this effect seen in DEN-treated rats. In this case, the expression of p53 was not related to size of EAF or length of treatment. Many EAF in PB-treated animals contained very large numbers of p53-positive cells. Upon staining for terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (the TUNEL procedure), many apoptotic hepatocytes were also seen in EAF. These data indicate that the p53 response to DNA damage can be modulated by xenobiotics. This can be explained as an adaptive alteration in the p53 response.

In vitro studies on non-genotoxic carcinogens: resistance to DNA synthesis inhibition in GST-P-positive hepatocytes isolated from enzyme-altered foci-bearing rats.

Hepatocytes were isolated from enzyme-altered foci (EAF)-bearing rats. Carcinogen-induced effects on DNA synthesis were studied in primary cultures of isolated cells. The test substance was added at the start and at 24hr. At 48hr the cultures were stained with antibodies against glutathione S-transferase P (GST-P) and DNA synthesis was estimated by thymidine incorporation. It was found that four of nine carcinogens induced a clear selective inhibition of DNA synthesis in GST-P-negative cells. Two of the four inhibiting carcinogens induced p53 in GST-P-negative cells, but did not in GST-P-positive cells. Of four tested non-carcinogens, none induced a selective inhibition of DNA synthesis in GST-P-negative hepatocytes. These findings suggest that this model system is useful when characterizing liver carcinogens and their carcinogenic mechanisms.

Attenuated p53 Expression and Lack of Effect of TGFalpha on Cell Replication in Enzyme Altered Foci.

Enzyme altered foci (EAF) in rat liver are stress-related precarcinogenic lesions. Control of the cell cycle in EAF hepatocytes is altered, and in vitro data indicate that p53 protein does not accumulate in these cells in response to DNA-damaging agents. In this report we present immunohistological data indicating that this p53 response is also attenuated in EAF in situ. The p53 response was 11 times more frequent in non-EAF than in EAF hepatocytes. The results are compared with previously reported in vitro studies. We also demonstrate that isolated EAF hepatocytes do not respond to TGFalpha and that four different p53-inducing agents, namely DEN, taxol, doxorubicin and araC readily induce p53 in non-EAF, but not in EAF hepatocytes. Taxol induces a similar toxicity in both these cell types. It is suggested that the attenuated response to DNA-damaging agents cannot be explained solely by altered metabolism of xenobiotics. Additional factors, such as an adaptive alteration in the control of p53 expression in EAF hepatocytes, must be involved.

Resistance against ethacrynic acid in glutathione transferase 7-7 (GST-P)-positive hepatocytes isolated from carcinogen-treated rats: the role of cytoskeletal changes and ATP depletion.

Ethacrynic acid (Ea) is a substrate for glutathione transferase 7-7 (GST-P) in rat. Toxic effects of Ea have been related to its metabolism and GSH depletion, but resistance conferred by GSTP1-1 (the human homologue) has also been reported. Hepatocytes from enzyme altered foci (EAF) express GST-P, and a model for selection of resistant EAF cells has been developed using Ea as a toxic agent. In the present study the effects of Ea in this model have been characterized. Hepatocytes from foci-bearing rats were isolated. Isolated cells were exposed to Ea for 1-4 hr in suspension. They were then allowed to attach to collagen-coated plates in a serum-containing medium. Preferentially GST-P-positive cells attached after Ea treatment, thus increasing the number of positive cells per attached cells (GST-P-%). Extracellular GSH, as well as alpha-tocopherol, did not influence the Ea effect. However, the effect of Ea was counteracted by inhibitors of glutathione transferase activity. Taxol, a microtubule stabilizing agent, also counteracted the effect of Ea on GST-P-%. 1,2-Dichloro-4-nitrobenzene (DCNB, 0.4 mM), which is a substrate for other glutathione transferase isoenzymes than GST-P, also increased the GST-P-%. However, the effect of DCNB was not inhibited by taxol. It was also found that Ea induced a drop in ATP levels, but this effect, as well as cell leakage, came later than the loss of attachment. The data suggest that the critical effect of Ea was cytoskeletal changes, and that GST-P conferred resistance by detoxification of Ea.

Nanomolar levels of PAHs in extracts from urban air induce MAPK signaling in HepG2 cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmental mixtures.

Different inhibition of DNA synthesis by transforming growth factor beta and phenobarbital on GST-P-positive and GST-P-negative hepatocytes.

Hepatocyte resistance against inhibition of DNA synthesis by transforming growth factor beta 1 (TGF-beta 1) was studied in vitro. Hepatocytes were isolated from rats that had received diethylnitrosamine (DEN) for 6 weeks. The effect of TGF-beta 1 and phenobarbital (PB) on DNA synthesis in different cell populations was studied using bromodeoxyuridine (BrdU) incorporation and placental glutathione S-transferase (GST-P) as markers. It was found that GST-P-positive cells were resistant to the growth inhibitory effect of TGF-beta 1 and PB, whereas GST-P-negative cells were inhibited. It is concluded that resistance to TGF-beta 1-dependent growth control may develop early during DEN-induced hepatocarcinogenesis.

gamma-Glutamyltranspeptidase-conferred resistance to hydroquinone induced GSH depletion and toxicity in isolated hepatocytes.

Hepatocyte resistance against glutathione (GSH) depleting xenobiotics was studied in an in vitro model. Hepatocytes were isolated from carcinogen treated rats that had received phenobarbital for three weeks. Isolated cells were incubated in GSH containing buffer with hydroquinone, which depleted GSH. Cells were then seeded on collagen coated plates and cultured overnight in complete medium. Attached cells were stained and the proportion of gamma-glutamyltranspeptidase (GGT)-positive cells was counted. It was found that toxicity related to GSH depletion increased the proportion of GGT-positive cells from 10-15% up to 40-60%, indicating that the toxicity mainly affected GGT-negative cells. GSH added to the buffer was essential for this effect. It is concluded that GGT may protect GGT-positive hepatocytes from GSH depletion and toxicity early during liver carcinogenesis.

Immunohistochemical detection of induced expression of wild-type p53 tumor suppressor protein in the livers of rats treated with diethylnitrosamine.

Paraffin sections of formalin-perfused rat livers were stained immunohistochemically for p53. In livers from untreated rats, no p53 expression was observed. p53 expression was induced in a response to treatment with diethylnitrosamine 24 h prior to sacrifice. Staining for p53 was localized in the nucleus of perivenous hepatocytes. In serial sections p53-immunopositive areas were found to co-localize with increased expression of TUNEL-positive cells. Without formalin perfusion, the staining for p53 was uneven and often barely detectable. Perfusion with saline prior to formalin resulted in a rapid decrease in the detectability of p53, indicating rapid degradation of this protein under these conditions. We conclude that rapid fixation by formalin perfusion increases the detectability of p53 by immunohistochemical staining. This provides a convenient procedure for studying the response of wild-type p53 in rodent liver. This procedure is also suitable for in situ investigations on the degradation of p53 protein stabilized by DNA damage.

Fas-mediated apoptosis is attenuated in preneoplastic GST-P-positive hepatocytes isolated from diethylnitrosamine-treated rats.

Previous reports have indicated that apoptosis is selectively decreased in enzyme-altered foci (EAF) in the livers of rats treated with a carcinogen. Here we have investigated the effects of an anti-Fas antibody (anti-Fas Ab) on EAF cells in vitro. Hepatocytes were isolated from rats treated repeatedly with diethylnitrosamine (DEN), whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Subsequently, primary cultures of GST-P-positive and GST-P-negative hepatocytes were established and exposed to anti-Fas Ab. Anti-Fas Ab (4 microg/ml) preferentially induced apoptosis in GST-P-negative cells. Furthermore, GST-P-positive cells were shown to be resistant to p53-mediated apoptosis. We conclude that EAF hepatocytes are resistant to Fas-mediated apoptosis in vitro. This lack of response may explain the selective decrease in apoptosis in EAF.

GST-P-positive hepatocytes isolated from rats bearing enzyme-altered foci show no signs of p53 protein induction and replicate even when their DNA contains strand breaks.

Hepatocytes were isolated from rats with enzyme-altered foci (EAF) in their livers and were studied in primary cultures. Cultures were treated with two doses of 0.6 mM diethylnitrosamine (DEN) at 1.5 and 24 h. At 48 h the cultures were double stained with antibodies against glutathione S-transferase P (GST-P) and p53 protein. Ten percent of the GST-P-immunonegative cells were p53-immunopositive. Thymidine incorporation was blocked in these cells. Both p53 expression and the block in thymidine incorporation could be eliminated by p53 antisense oligonucleotides. Less than 1% of the GST-P-positive cells in the same cultures were p53-immunopositive. Thymidine incorporation was less affected than in GST-P-negative cells. DNA strand breaks were also monitored by an immunological technique. Twenty-three percent of the GST-P-negative cells and 7% of the GST-P-positive cells were positive for this marker. Seven percent of the GST-P-positive cells with DNA strand breaks incorporated thymidine. Virtually none of the GST-P-negative cells with DNA strand breaks demonstrated thymidine incorporation. We suggest that GST-P-positive cells lack functional p53 protein and that this permits cells with damaged DNA to replicate.

Wild-type p53 expression in liver tissue and in enzyme-altered foci: an in vivo investigation on diethylnitrosamine-treated rats.

Previous reports have documented an attenuated p53 response to DNA damage in hepatocytes isolated from enzyme-altered foci (EAF). Here, we have studied this p53 response in vivo in rats with EAF. These animals received repeated doses of diethylnitrosamine (DEN) for 6 weeks and a challenging dose 24 h before death. Liver sections were then analysed using an immunohistological procedure for p53, or a double-staining procedure for p53 and glutathione-S-transferase pi (GST-P). In control rats or rats with EAF not given the challenging dose of DEN, there was no p53 staining. In control rats, only given the challenging dose of DEN, there was a centrilobular p53 nuclear staining that co-localized with TUNEL staining. In an experiment involving four rats with EAF 389 +/- 39 hepatocytes/mm2 of non-EAF tissue stained positively for p53, while the corresponding value for EAF tissue was 27.6 +/- 7.5. Thus, p53-positive cells were 14.6-fold more frequent in non-EAF than in EAF tissue. In many EAF no p53-positive cells were seen at all and 83% of the EAF demonstrated <20% of the number of p53-positive cells seen in non-EAF tissue. Very few EAF had as high a proportion of p53-positive cells as did the average non-EAF tissue. EAF >0.06 mm2 had significantly fewer p53-positive cells than smaller EAF. The ratio of p53 expression in non-EAF tissue and large EAF was 32.6. In a control experiment, four EAF-bearing rats were used as donors to prepare primary cultures of hepatocytes. After 24 h of exposure to DEN, many of the cultured cells became p53-positive. Among GST-P-negative hepatocytes, 12.8% were p53-positive, whereas only 0.25% of the GST-P-positive hepatocytes were p53-positive. Literature data suggest that the altered xenobiotic metabolism in EAF may give rise to a 3-4-fold difference in DNA damage between non-EAF and EAF tissues. It is concluded that GST-P-positive EAF hepatocytes have an attenuated p53 response to DNA damage. This attenuated response may facilitate clonal expansion of EAF under stress induced by DNA-damaging chemicals.

p53 expression and TGF-alpha-induced replication of hepatocytes isolated from rats exposed to the carcinogen diethylnitrosamine.

Previous reports indicate that the expression of transforming growth factor alpha (TGF-alpha) is increased in enzyme-altered foci (EAF) arising in livers of rats treated with a carcinogen. Here we have investigated the effects of TGF-alpha on EAF cells in vitro. Hepatocytes were isolated from rats that had received repeated treatment with diethylnitrosamine (DEN) and whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Primary cultures of GST-P-positive and GST-P-negative hepatocytes were exposed to TGF-alpha. TGF-alpha (20-40 ng/ml) increased DNA replication in the GST-P-negative, but not in the GST-P-positive cells. Furthermore, it was shown that this effect on GSTP-negative cells could be blocked by p53 antisense oligonucleotides. We conclude that EAF hepatocytes do not respond to TGF-alpha in vitro. This lack of response may reflect the attenuated expression of p53 in these cells. These data corroborate previous findings that, in response to DNA damage, many EAF hepatocytes do not accumulate p53.

gamma-Glutamyltranspeptidase-positive rat hepatocytes are protected from GSH depletion, oxidative stress and reversible alterations of collagen receptors.

The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.

Peroxisome proliferation and resistance to hydrogen peroxide in rat hepatocytes: is development of resistance an adaptation to cytotoxicity?

In this work the resistance of peroxisome-proliferated hepatocytes to hydrogen peroxide (H2O2) has been studied. The question has been raised as to whether this resistance is a response to cytotoxicity. In an initial series of experiments, hepatocytes were isolated from rats that had been treated with nafenopin (NAF-hepatocytes). Isolated cells were exposed to a H2O2-generating system or to H2O2 in pulses. The ability to attach to collagen was used as a toxicological endpoint. Loss of attachment was found to be correlated to glutathione (GSH) depletion, and NAF-hepatocytes were more resistant to GSH depletion and to loss of attachment induced by H2O2 than were control hepatocytes. NAF-hepatocytes were not resistant to hydroquinone or to adriamycin. It was also indicated that this resistance was related to an altered metabolism of H2O2, less dependent on GSH. In a second series of experiments, hepatocytes from altered hepatic foci-bearing rats, treated with nafenopin or di(2-ethylhexyl)phthalate (DEHP), were used. This model was used in an attempt to monitor the development of resistance in different subpopulations of hepatocytes. It was found that the majority of hepatocytes developed resistance towards H2O2, and that, for example, foci marker-positive hepatocytes were as resistant as marker-negative cells. In control experiments with this model, it was found that marker-positive cells were more resistant towards diethyl maleate (DEM) or phorone than were marker-negative cells. In addition to demonstrating the validity of the model, these control experiments indicate an increased steady-state level of H2O2 in cells from peroxisome proliferator-treated rats. Other control experiments suggested that a low GSH-peroxidase activity protected from, rather than aggravated, the effect of peroxisome proliferation on marker-negative and GSH-depleted cells. It is concluded that H2O2 metabolism may affect the function of collagen receptors, but that a shift in H2O2 metabolism, so that it becomes less dependent on GSH, conferred resistance to this effect. The apparent non-focal induction of resistance to peroxisome proliferators, as opposed to the focal induction of resistance induced by most liver carcinogens, may explain the lack of development of gamma-glutamyltranspeptidase-positive foci in peroxisome proliferator-treated rats.

Isolation of glutathione S-transferase P-positive hepatocytes from carcinogen treated rats by use of ethacrynic acid as selecting agent.

Rat liver responds to carcinogen treatment with growth of glutathione S-transferase P (GST-P)-positive enzyme-altered foci. In this paper a method is described where GST-P-positive hepatocytes are isolated from carcinogen-treated rats. The method utilizes ethacrynic acid, which is a good substrate for GST-P, and which induces toxicity mainly in GST-P-negative cells. The toxicity results in a loss of attachment to collagen. The method gives a 70% pure population of GST-P-positive cells attached to collagen-coated plates. Use of additional markers supports the conclusion that the GST-P-positive cells were derived from foci. Isolated GST-P-positive hepatocytes spread out and formed primary cultures of normal appearance. It was also shown that they synthesized DNA and did not respond to transforming growth factor beta 1. It is concluded that isolated GST-P-positive hepatocytes can be used for studies on alterations in enzyme-altered foci that cannot be done with in situ immunohistochemistry or in situ hybridization.

The role of GSH depletion and toxicity in hydroquinone-induced development of enzyme-altered foci.

Hydroquinone (HQ) may activate oxygen via redox cycles in biological systems and may also deplete glutathione (GSH). Both these reactions are potentially harmful, and we have studied their possible involvement in hydroquinone-induced development of gamma-glutamyltranspeptidase (GGT)-positive enzyme-altered foci in rat liver. The effect of HQ was compared to the effect of duroquinone, catechol, resorcinol and phenol. The dose was 100 mg/kg per day and the test substances were administered for 7-12 weeks in these foci experiments. HQ gave an increased number of foci and increased the foci volume, while none of the other compounds had any significant effect on these parameters. HQ, duroquinone and resorcinol were also tested at a higher dose level (200 mg/kg per day), but this dose gave a lower number of foci than the 100-mg dose. HQ, duroquinone and catechol induced single-strand breaks in hepatic DNA. Single doses of HQ (200 mg/kg) increased malondialdehyde excretion in urine, indicating in vivo lipid peroxidation. Duroquinone, phenol and resorcinol were negative with respect to malondialdehyde excretion. Catechol could not be properly tested as the 200-mg dose killed several animals. HQ and catechol induced hepatic ornithine decarboxylase activity. This effect was correlated to GSH depletion. An in vitro model for toxicity studies with hepatocytes from carcinogen-treated rats was also used. In this model HQ could be shown to be selectively toxic to GGT-negative cells in the presence of extracellular GSH. The toxicity was preceded by a rapid depletion of GSH. Catechol also depleted GSH and could be shown to be selectively toxic, but higher concentrations than those used for HQ had to be used. Duroquinone, phenol and resorcinol were not selectively toxic to GGT-negative cells. As duroquinone can be regarded as a more potent inducer of redox cycles than HQ, it can be concluded that the foci data provide no evidence for an involvement of redox cycles in HQ induced development of enzyme-altered foci. They suggest that GSH depletion may act to develop enzyme-altered foci, and the in vitro data indicate a mechanism by which GSH depletion and toxicity may induce this effect.