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Rodent non-genotoxic hepatocarcinogens such as nafenopin suppress spontaneous and transforming growth factor β1 (TGFβ1)-induced rat hepatocyte apoptosis as well as inducing DNA synthesis. We wished to determine if these two processes are associated. In primary rat hepatocytes, nafenopin suppressed apoptosis from 1.9 to 0.63% but more apoptotic bodies were bromodeoxyuridine (BrdU)-labelled (0.35%) than predicted statistically from a random distribution of apoptosis within the cycling and non-cycling populations (0.10%). In contrast, TGFβ1 induced hepatocyte apoptosis (7.8%) but fewer hepatocytes were BrdU-labelled (0.29%) than predicted (0.82%). Western blot analyses showed that nafenopin and TGFβ1 had opposing effects on cyclin-dependent kinase 4 (CDK4) protein: nafenopin elevated CDK4 compared with controls, whereas TGFβ1 caused a reduction. These data suggest that non-genotoxic hepatocarcinogens suppress apoptosis in the non-cycling population of hepatocytes and elevate CDK4 levels, possibly allowing potentially tumourigenic cells to enter the cell cycle. [ABSTRACT FROM PUBLISHER]

The suppression of apoptosis may contribute to the carcinogenicity of the peroxisome proliferators (PPs), a class of non-genotoxic rodent hepatocarcinogens. Our previous work demonstrated that the PP nafenopin suppressed both spontaneous and transforming growth factor β1 (TGFβ1)-induced hepatocyte apoptosis both in vivo and in vitro. Here, we extend these observations by demonstrating the ability of nafenopin to suppress apoptosis induced by other major candidates for the signalling of cell death in the liver. Treatment of rat or mouse hepatocyte monolayers with TGFβ1 or the DNA damaging drugs etoposide or hydroxyurea induced high levels of apoptosis. Western blot analysis did not support a role for either p53 or p21waf1 in etoposide-induced apoptosis in rat hepatocytes. Treatment of mouse hepatocytes with an agonistic anti-Fas antibody also resulted in an induction of high levels of apoptosis. Preaddition and continued exposure to nafenopin suppressed apoptosis induced by all three stimuli. Overall, our studies demonstrate that the ability of nafenopin to protect hepatocytes from apoptosis is not restricted to species or apoptotic stimulus. It is possible, therefore, that the PPs may suppress apoptosis by acting on diverse signalling pathways. However, it seems more likely that nafenopin suppresses hepatocyte apoptosis elicited by each death stimulus by impinging on a core apoptotic mechanism. [ABSTRACT FROM PUBLISHER]

Non-genotoxic hepatocarcinogenesis may involve suppression of the hepatocyte apoptosis that would normally remove damaged or initiated cells. These protected hepatocytes could then remain as preferential targets for promotion by this class of compounds. Here we demonstrate clearly that the non-genotoxic liver carcinogens and hepatomitogens cyproterone acetate (CPA) and nafenopin, a peroxisome proliferator, both suppress the basal level of rat liver apoptosis in vivo. After 10 days of dosing with CPA (120 mg/kg/day) or nafenopin (25 mg/kg/day) there were 0.005 +/- 0.010 and 0.002 +/- 0.021 apoptotic bodies/100 hepatocytes respectively, compared with 0.031 +/- 0.008 per 100 in controls. Concomitant with this suppression of apoptosis, bromodeoxyuridine (BrdU) labelling indices and mitotic figures rose, confirming a perturbation of both sides of the growth equation between cell death and replication. Withdrawal of CPA or nafenopin resulted in a 100- to 200-fold elevation in apoptosis. This was inhibited by the re-administration of either compound. To investigate if cells protected from apoptosis by non-genotoxic carcinogens are targets for replication, we examined the replicative history of the apoptotic bodies generated upon withdrawal of CPA or nafenopin. Rats were administered BrdU during the hyperplastic phase of compound administration (0-10 days). Livers were examined 5 days after compound withdrawal. With both CPA and nafenopin, apoptotic bodies and S phase were predominantly in the periportal region. However, despite this zonal co-localization, very few (< 10%) of the apoptotic bodies were labelled with BrdU. Overall, our data provide in vivo evidence to support the hypothesis that non-genotoxic hepatocarcinogens such as CPA and the peroxisome proliferators suppress apoptosis. Surprisingly, the majority of the hepatocytes generated during compound-induced hyperplasia were protected from apoptosis during liver regression. These data contribute to our understanding of clonal selection and promotion during non-genotoxic hepatocarcinogenesis.

The structures of many dibenz[a,j]acridine (DBAJAC) metabolites formed in vitro in incubations with liver microsomes prepared from 3-methylcholanthrene-pretreated male Wistar rats have previously been determined; they were trans-DBAJAC-3,4-dihydrodiol, trans-DBAJAC-5,6-dihydrodiol, DBAJAC-5,6-oxide, 3-hydroxy-DBAJAC, 4-hydroxy-DBAJAC and several multiply oxidized secondary metabolites. Herein are reported [14-3H]DBAJAC metabolite distributions obtained by h.p.l.c. separation of products produced in incubations with liver and lung microsomes prepared from untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated male Wistar rats. Liver microsomal metabolites were also quantitated in preparations from trans-stilbene oxide-pretreated rats. For all preparations trans-DBAJAC-3,4-dihydrodiol, the candidate proximate carcinogen according to the bay-region theory of carcinogenesis, was the major metabolite (30-40%) while DBAJAC-5,6-oxide and phenols were also quantitatively important. In incubations conducted in the presence of 3,3,3-trichloropropene-1,2-oxide (1.5 mM) formation of dihydrodiol was inhibited by about 85%. DBAJAC-N-oxide was also identified as a minor metabolite (approximately 1%) formed in incubations with phenobarbital-induced and control liver microsomes.

The metabolism of the carcinogenic pentacyclic azaaromatic compound, dibenz[a,j]acridine, has been examined in liver microsomal incubations using preparations from 3-methylcholanthrene-pretreated Wistar rats. Using authentic synthetic standards, u.v. spectroscopy and mass spectrometry, the following were proved to be metabolites: trans-5,6-dihydro-5,6-dihydroxydibenz[a,j]acridine, trans-3,4-dihydro-3,4-dihydroxydibenz[a,j]acridine, dibenz[a,j]acridine-5,6-oxide, 3-hydroxydibenz[a,j]acridine and 4-hydroxydibenz[a,j]acridine. The 3,4-dihydrodiol appeared to be the major metabolite. The secondary metabolites were also examined and evidence is presented for the additional formation of dibenz[a,j]acridine-5,6,8,9-dioxide, tetrols, diol epoxides and phenolic dihydrodiols.

The presence of the proposed proximate carcinogen, trans-3,4-dihydro-3,4-dihydroxy-7-methylbenz[c]acridine (7MBAC-3,4-DHD) among the liver microsomal metabolites of 7-methylbenz[c]acridine (7MBAC) has been demonstrated using gas chromatography mass spectrometry (GCMS) and by co-chromatography with synthetic standards on reverse and normal phase h.p.l.c. 7MBAC-3,4-DHD represented 2.2-3.4% of the total ethyl acetate-extractable metabolites formed from 7MBAC by liver microsomes prepared from untreated and induced rats. About 2.3-2.7% of metabolites formed by lung microsomes was identified as 7MBAC-3,4-DHD. Mutagenicity studies with 7MBAC-3,4-DHD have been carried out in bacterial and mammalian systems using S9 fractions derived from rats pre-treated with Aroclor and guinea pigs pre-treated with 3-methylcholanthrene. Comparative data with other 7MBAC derivatives are also reported. The 7MBAC-3,4-DHD and the analogous dihydro derivative of 7MBAC were the most potent mutagens of those compounds requiring metabolic activation. The data imply that the 3,4-dihydrodiol is metabolised to a bay region diol epoxide as the ultimate carcinogen. In support of this anti-1,2-epoxy-trans-3,4-dihydroxy-7-methyl-1,2,3,4- tetrahydrobenz[c]acridine was a potent mutagen in the Ames and V79 cell systems without activation. The syn-isomer was less active.