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Sequential treatment of partially hepatectomized male Wistar rats with diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) induces the emergence of diploid hepatocyte populations. These carcinogen-induced hepatocytes are thought to include the precursor cells of liver carcinomas that arise later in this treatment protocol. The growth of the diploid hepatocytes is promoted by AAF and it has been suggested that the action of the arylamine may be receptor-mediated. AAF has been shown to bind specifically to the aryl hydrocarbon (Ah) receptor and the so-called 4S polycyclic aromatic hydrocarbon (PAH) binding protein. The present study addresses the question of whether the concentrations of the two binding proteins differ in diploid and polyploid hepatocytes from DEN/AAF-treated rats. Hepatocytes from carcinogen-treated rats were isolated and diploid, and tetraploid hepatocytes separated by means of centrifugal elutriation. Whereas Ah receptor concentrations in diploid hepatocytes were insignificantly lower (21.8 +/- 5.9 versus 29.2 +/- 6.6 fmol/mg cytosolic protein; n = 4; P = 0.1), levels of the 4S PAH binding protein in diploid hepatocytes were twice as high as in tetraploid hepatocytes (252.3 +/- 93.6 versus 124.0 +/- 18.5 fmol/mg cytosolic protein; n = 4; P = 0.04). We conclude from our results that the differences in growth control in polyploid and carcinogen-induced diploid hepatocytes are not associated with changes in the levels of the Ah receptor. The role of the 4S PAH binding protein in the process of hepatocarcinogenesis remains to be established.

5L cells, dedifferentiated descendents of the rat hepatoma line H4IIEC3, constitute one of the rare continuous lines which are sensitive to the toxic effects of 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). In the present study we investigated the nature of TCDD toxicity in these cells. The following results were obtained: (1) Exposure to 0.1 n m TCDD for 48 hr inhibits the proliferation of 5L cells by more than 50%, as determined by the increase in the number of cells and the amount of DNA per culture. (2) TCDD doubles the amount of protein and the uptake of neutral red per cell during the 48-hr exposure period. (3) TCDD restores neither constitutive levels of tyrosine aminotransferase, a marker of liver-specific functions, nor its inducibility by dexamethasone. (4) The effects of TCDD are reversible when TCDD-containing growth media are replaced by TCDD-free medium. (5) 5L cells grown at 2% of fetal bovine serum are considerably more sensitive to TCDD than those grown at 10% serum. The results indicate that TCDD inhibits the proliferation of 5L cells without retarding the rate of growth or overtly changing the status of differentiation. The dioxin possibly causes unbalanced cell growth by interfering with the action of hormones or factors contained in the growth medium.

The aromatic amides 2-acetylaminofluorene (2-AAF), its isomer 4-acetylaminofluorene, 2-acetylaminophenanthrene (AAP), trans-4-acetylaminostilbene (AAS) and the corresponding amines differ in their carcinogenic effects in the rat. Only 2-AAF and 2-aminofluorene (2-AF) are tumor promoters in rat liver. We have determined the competitive binding affinities of these compounds to the rat hepatic cytosolic aromatic hydrocarbon (Ah) receptor, which has been associated with tumor promotion. Binding affinity was determined by displacement of labelled 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. The rank order of affinities and the apparent inhibitor constants measured with the sucrose density gradient centrifugation technique were: AAF (2.3 microM) greater than AAP (2.7 microM) greater than 2-AF (7.0 microM) greater than trans-4-aminostilbene (7.7 microM) greater than 2-aminophen-anthrene (10.4 microM). 4-Acetylaminofluorene, 4-aminofluorene and AAS did not displace TCDD from the receptor protein. A number of other fluorene derivatives did not bind, except 2-nitrofluorene (apparent inhibitor constant (1.4 microM). The in vivo biological potency of the aromatic amines was monitored by measuring the induction of ethoxy-resorufin-O-deethylase (EROD) and aromatic hydrocarbon hydroxylase activities. In vitro binding to the Ah receptor correlates roughly with the induction of EROD activity. The effect of 2-AAF was dose dependent, supporting a receptor-mediated mechanism. The correlation between Ah receptor affinity and tumor-promoting properties of the tested aromatic amines is only limited, but the results are not at variance with the concept of a receptor-mediated process in 2-AAF tumor promotion.

Dioxins are potent mammalian carcinogens and toxins affecting liver, skin, and immune and reproductive systems. The intracellular Ah receptor, a ligand-dependent transcription factor of the basic region/helix-loop-helix/Per-Ahr/Arnt-Sim homology domain (bHLH-PAS) protein family, mediates responses to dioxins. Target genes of the Ah receptor that mediate dioxin toxicity and carcinogenicity are, however, mostly unknown. We used 5L rat hepatoma cells to identify dioxin-inducible genes by suppression subtractive hybridization. Eleven cDNA fragments were identified that represented novel sequences or genes for which induction by dioxins had not been known. N-myristoyltransferase 2 (NMT2) is one of the later dioxin-inducible genes. Induction of NMT2 was confirmed in livers of mice in vivo. NMT2 induction was a direct consequence of Ah receptor activation in 5L cells. [(3)H]myristic acid incorporation into 5L cell proteins was inducible by dioxins, indicating that protein myristoylation is a regulated rather than a housekeeping function and that NMT activity is limiting in noninduced 5L cells. Here we show for the first time that expression of NMT2 and induced protein myristoyltransferase activity are direct responses to carcinogen exposure. Because inappropriate protein NH(2)-terminal myristoylation appears to play a role in carcinogenesis, induction of NMT2 may play a central role in dioxin carcinogenicity.

Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause similar teratogenic effects (neural tube defects) in human and mice. In this study a new molecular bioassay is presented using following endpoints: differentiation of F9 teratocarcinoma cells, altered cell morphology, induction of possible targeted genes, and the induction of viral RSV-promoter. The induction of a transiently transfected viral (RSV) promoter driven luciferase gene by VPA was used to screen a set of VPA-derivatives. Structure-activity investigations showed: the longer the aliphatic side chain the more the induction of the RSV-reporter gene. The specific induction was stereoseletive. The teratogenic enantiomer S-4-yn-VPA (2-propyl-4-pentynoic acid) induced the RSV-driven reporter gene while the non teratogenic R-4-yn-VPA does not. Heptyl-4-yn-VPA was the most potent teratogen in vitro and in vivo. Non teratogenic VPA-derivatives like R-4-yn-VPA and 2-en-VPA (2-propyl-2-pentenoic acid) were ineffective in this system. Thus, the teratogenic effect of VPA and VPA-derivatives in the mouse correlated with the specific induction of the viral RSV-promoter controlled reporter in F9-cells. Acid compounds such as fatty acids are known to interact with peroxisome proliferator-activated receptors (PPARs). To test structure-activity relationships by VPA or its derivatives we used CHO cells stably expressing hybrid proteins of the ligand-binding domain of either of the PPARs. The teratogen VPA and the teratogenic derivatives of VPA activated the PPAR-delta construct in a very specific structure- and stereoselective way which correlated well with the activities in the reporter gene assay (bioassay) and those in vivo. No such correlation was found with respect to activation of PPAR-alpha or PPAR-gamma. These structure-activity relationships indicate that PPAR-delta may be a potential mediator of VPA-induced differentiation of F9 cells and may possibly be involved in the mechanism of teratogenicity of VPA in vivo. Furthermore two bioassays were designed with clearly defined endoints, amenable to automation and screening of great number of compounds. The test system allows to replace animal experiments in the preclinical development of new antiepileptics drugs with reduced teratogenic risk. Supported by BgVV-ZEBET (Berlin).

Hypolipidaemic fatty acid derivatives and polyunsaturated fatty acids decrease concentrations of plasma triacylglycerol by mechanisms that are not fully understood. Because poor susceptibility to beta- and/or omega-oxidation is apparently a determinant of the peroxisome proliferating and hypolipidaemic capacity of fatty acids and derivatives, the relative importance of activation of the peroxisome-proliferator-activated receptor alpha (PPARalpha), fatty acid oxidation and triacylglycerol synthesis were examined. We have compared the effects of differentially beta-oxidizable fatty acids on these parameters in primary cultures of rat hepatocytes. Tetradecylthioacetic acid (TTA), 2-methyleicosapentaenoic acid and 3-thia-octadecatetraenoic acid, which are non-beta-oxidizable fatty acid derivatives, were potent activators of a glucocorticoid receptor (GR)-PPARalpha chimaera. This activation was paradoxically reflected in an substantially increased oxidation of [1-(14)C]palmitic acid and/or oleic acid. The incorporation of [1-(14)C]palmitic acid and/or oleic acid into cell-associated and secreted triacylglycerol was decreased by 15-20% and 30% respectively with these non-beta-oxidizable fatty acid derivatives. The CoA ester of TTA inhibited the esterification of 1, 2-diacylglycerol in rat liver microsomes. Both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) activated GR-PPARalpha. EPA increased the oxidation of [1-(14)C]palmitic acid but DHA had no effect. The CoA ester of EPA inhibited the esterification of 1, 2-diacylglycerol, whereas DHA-CoA had no effect. The ratio between synthesized triacylglycerol and diacylglycerol was lower in hepatocytes cultured with EPA in the medium compared with DHA or oleic acid, indicating a decreased conversion of diacylglycerol to triacylglycerol. Indeed, the incorporation of [1-(14)C]oleic acid into secreted triacylglycerol was decreased by 20% in the presence of EPA. In conclusion, a decreased availability of fatty acids for triacylglycerol synthesis by increased mitochondrial beta-oxidation and decreased triacylglycerol formation caused by inhibition of diacylglycerol acyltransferase might explain the hypolipidaemic effect of TTA and EPA.

The induction of ornithine decarboxylase (ODC) in rat liver by the carcinogen 2-acetylaminofluorene (2AAF), the strong liver tumor initiator trans-4-acetylaminostilbene (AAS), the non-carcinogen 4-acetylaminofluorene (4AAF) and, as a control, 3-methylcholanthrene (MC) has been examined. MC and all aromatic amines increased the ODC activity of rat liver from 3.6-fold (MC) to maximal 5.7-fold (AAS). The time course of the maximal induction differs from 2.5 h (2AAF) to 10 h (MC). The cytosolic concentrations of the unchanged compounds parallel the ODC activity. These data indicate that ODC induction in rat liver shows no correlation with tumor promoting properties of the model compounds. It is concluded that the unchanged compound is responsible for the ODC induction.