Jay I. Goodman

Altered methylation of ras oncogenes in benzidine-induced B6C3F1 mouse liver tumors☆

The B6C3F1 mouse is a hybrid strain which exhibits a high (30%) spontaneous hepatoma incidence and sensitivity to chemical induction of liver tumors. The spontaneous hepatoma incidence of the paternal C3H/He strain is approximately 60%, while that of the maternal C57BL/6 strain is very low. The presence of activated oncogenes, primarily Ha-ras, and to a lesser extent, Ki-ras, has been reported in B6C3F1 mouse liver tumors. Because alterations in a genes capacity for expression, as well as mutation, may be involved in oncogene activation, this investigation was directed toward an examination of a putative control point for transcription, i.e., the methylation state of a gene. Hypomethylation is believed to be necessary, but not sufficient, for transcription. It was therefore hypothesized that alterations in the methylation state of the Ha-ras and Ki-ras oncogenes may facilitate the aberrant expression of these genes in B6C3F1 mouse liver. Restriction enzyme analysis (MspI, HpaII, and HhaI) was used to assess the extent of DNA methylation. MspI digestion of B6C3F1 and C3H/He DNA revealed the absence of a 15-kb Ha-ras band present in MspI-digested C57BL/6 DNA, suggesting that the Ha-ras oncogene of B6C3F1 and C3H/He mouse liver lacks a methylated site. In other respects, the Ha-ras and Ki-ras oncogenes are methylated to a degree which suggests that these oncogenes have a low potential for expression in normal mouse liver. The methylation state of the Ha-ras and Ki-ras oncogenes was also assessed in benzidine-induced hepatomas and adjacent nontumor tissue from B6C3F1 mice. In four out of four cases, the Ha-ras oncogene was hypomethylated in tumor as compared to nontumor tissue and increased expression of the gene was detected in three out of four hepatomas; the Ki-ras oncogene was hypomethylated in two out of four cases. These results suggest that hypomethylation of oncogenes may provide an epigenetic mechanism for facilitating their aberrant expression. The lack of a methylated site observed in the Ha-ras oncogene in B6C3F1 and C3H/He mouse liver may indicate an increased potential for its expression which could, in part, account for the high propensity for hepatoma development in these two strains.

Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene

Male rats were placed on a diet containing 0.05% (w/w) of the hepatic carcinogen 2-acetylaminofluorene (AAF). They ceased to gain weight. However, when the carcinogenic diet was supplemented with butylated hydroxytoluene (BHT) (0.5% w/w), an antioxidant, the animals gained weight at approximately one-half of the normal rate. This observation led to a series of experiments aimed at elucidating the mechanism(s) by which BHT reduced the toxicity of AAF. These initial studies were directed towards the effect of BHT on the extent and duration of the covalent binding of AAF with DNA. BHT feeding was shown to reduce the binding of carcinogen to hepatic DNA. Studies employing cells in culture demonstrated that BHT does not influence either excision repair or post-replication repair of DNA. These data indicate that a potential mechanism of action of BHT is at the anti-initiation level of carcinogen-induced DNA damage.

Hypomethylation of DNA: An epigenetic mechanism that can facilitate the aberrant oncogene expression involved in liver carcinogenesis

Publisher Summary Hypomethylation may be a mechanism underlying the role of cell proliferation in carcinogenesis, and hypomethylation could possibly result from an enzymatic replacement of 5MeC with cytosine, which is not linked to DNA replication. The chapter discusses the mechanisms believed to be involved in altered DNA methylation. The importance of the presence of the DNA-(cytosine-5)- methyltransferase (DNA MTase) gene is discussed in the context of DNA MTase knockout mice, and emphasis is placed on the role that the hypomethylation of DNA may play in tumor promotion. Experimental model used in this research is mouse liver tumorigenesis. It focuses on the oncogenes that are relevant to this system: Ha-ras and raf. Researchers employ the liver tumor prone B6C3F1 (C57BL/6 female x C3H/He male) mouse and make relevant comparisons with the sensitive C3H/He paternal strain and the resistant C57BL/6 maternal strain. The degree of the sensitivity of the parental strains is heritable and is determined largely by a genetic locus, the hepatocarcinogen sensitivity locus. An elegant study involving C3H/HeN↔C57BL/6N chimeric mice demonstrated that the different sensitivities of these strains to tumor development exists in the target cells (e.g., a tumor susceptibility gene) and is not milieu (e.g., hormone) dependent. A unique aspect of research is that it offers the potential to provide insight regarding molecular mechanisms that underlie the promotion of carcinogenesis, while at the same time the results can provide the type of information that is required to take a more rational approach to carcinogen risk assessment. Specifically, it can provide insight regarding dose-response relationships, the existence of thresholds, and species–to–species extrapolation issues.

Alkylation of DNA in specific hepatic chromatin fractions following exposure to methylnitrosourea or dimethylnitrosamine

Abstract This study was designed to assess, qualitatively and quantitatively, the methylation of hepatic DNA isolated from specific chromatin fractions following exposure to carcinogenic alkylating agents, methylnitrosourea (MNU), or dimethylnitrosamine (DMN). Male Sprague-Dawley rats were exposed to 0.15 mmol:kg [ methyl - 3 H]MNU (10 mCi/mmol) or to 0.08 mmol/kg [ methyl - 14 C]DMN (1.0 mCi/mmol) via gastric intubation. Alkylation of hepatic DNA was assessed at 3, 24, 72, and 168 hr postintubation. Hepatic chromatin was fractionated into portions having characteristics of template-active euchromatin (S2) and template-repressed heterochromatin (P2) by digestion with DNase II followed by MgCl 2 precipitation. At times of peak alkylation following the administration of either carcinogen, the S2/P2 DNA alkylation ratio was greater than 1.0 indicating that alkylation was nonrandom. In addition, the S2/P2 DNA alkylation ratio at peak alkylation following MNU treatment was 1.6 times the DMN ratio, suggesting qualitative differences between DMN- versus MNU-induced alkylation.

Non-random interaction of N-2-acetylaminofluorene and its N-hydroxy metabolite with DNA of rat hepatic parenchymal and non-parenchymal cell nuclei following in vivo administration of carcinogen

Treatment of rats with the hepatocarcinogen, N-2-acetylaminofluorene (AAF) results in development of malignant tumors derived primarily from hepatic parenchymal cells. Following administration of AAF, or its N-hydroxy derivative, in vivo nuclei from parenchymal cell and non-parenchymal cell populations (NI and NII nuclei populations, respectively) were isolated and treated with the endonuclease DNase I. The binding of carcinogen residues to the DNA of nuclease-accessible vs. nuclease-resistant regions of chromatin was evaluated on the basis of the selectivity of DNase I for transcriptionally active DNA. Under the experimental conditions employed DNase I digested approx. 50-60% of the genome of NI nuclei while only 10-20% of the DNA from non-parenchymal cell nuclei (NII) was susceptible to this enzyme. When the DNA of NI and NII nuclei were nick translated following limited digestion with DNase I, a greater degree of transcriptional activity (nuclease accessibility) was found in parenchymal cell nuclei (NI). Following a single injection of rats with [ring-3H]AAF or its N-hydroxy metabolite (N-[ring-3H]-OH-AAF) (1.8 mumol carcinogen/100 g), adducts were preferentially associated with DNA of DNase I resistant regions of target cell nuclei (NI), while preferentially associated with nuclease-accessible regions of non-target cell nuclei (NII). Damage following a single injection persisted for up to 3 days in DNase I-resistant DNA of NI nuclei, carcinogen adducts were rapidly lost from DNase I-accessible DNA of NII nuclei. These studies stress the importance of investigating specificity of carcinogens for particular regions of the genome of cell subpopulations within the target organ.

Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions.

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.

A comparison of the utilization of thymine and thymidine for the synthesis of DNA-thymine in novikoff hepatoma cells☆

Abstract A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10 −7 M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the K m was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells.

Hepatic DNA repair synthesis in rats fed 3′-methyl-4-dimethylaminoazobenzene

Summary Rats, whose liver DNA was prelabeled with BdUrd-14C, were placed on a diet containing 0.05% of the hepatic carcinogen 3′-methyl-4-dimethylaminoazobenzene. At various times during the second day on the carcinogenic diet animals received an injection of dThd-3H and were sacrificed 1 hr later, hepatic DNA was extracted and analyzed by density equilibrium centrifugation in alkaline CsCl gradients. The results of these studies indicate that non-semiconservative DNA synthesis (DNA repair synthesis is an early consequence of carcinogen exposure and that the amount of DNA repair synthesis occurring at a given time depends, in part, on the animals feeding schedule.

DNA-purine methylation in hepatic chromatin following exposure to dimethylnitrosamine or methylnitrosourea.

The investigations reported in this paper were designed to analyze the patterns of DNA-purine methylation in hepatic chromatin following in vivo exposure to the carcinogenic alkylating agents dimethylnitrosamine (DMN) or methylnitrosourea (MNU). Male Sprague-Dawley rats were exposed to [14C]DMN (8 mumoles, 1.0 microCi per mumole per 100 g) or [3H]MNU (15 mumoles, 10 microCi per mumole per 100 g) via gastric intubation. Hepatic chromatin was fractionated into portions having characteristics of template-active euchromatin (S2) and template-repressed heterochromatin (P2) by digestion with DNase II followed by MgCl2 precipitation. Specific DNA purines were identified at 24 hr post-intubation using an isocratic high pressure liquid chromatographic system. A qualitatively similar pattern of 7-methylguanine, O6-methylguanine, 1-methyladenine and 3-methyladenine alkylation was observed in DNA from total chromatin versus heterochromatin at 24 hr following exposure to either carcinogen. These assessments were made at times following carcinogen exposure which produced maximal quantitative differences in alkylation of euchromatin versus heterochromatin DNA. Similar patterns of DNA purine alkylation were observed in total chromatin and heterochromatin. These observations suggest that, once the reactive species is generated and access to chromatin DNA occurs, a similar pattern of DNA-purine alkylation is produced in different regions of the genome.

Spontaneous mutation at codon 61 of the Ha-ras gene in the nascent liver of B6C3F1, C3H/He and C57BL/6 mice.

DNA was isolated from the liver of young B6C3F1, C3H/He and C57BL/6 mice, 6-9 weeks old. A portion of exon 2 of Ha-ras was amplified by PCR allele-specific amplification. The PCR product was identified by (a) size, (b) presence of a diagnostic restriction site, and (c) direct sequencing. Our results indicate that nascent mouse liver bears a subpopulation of cells which contain a mutation in codon 61 of Ha-ras, specifically an A to G transition at position 2. Therefore, the detection of this mutation in chemically induced mouse liver tumors does not demonstrate that the chemical in question acts as a mutagen. It might act by a nongenotoxic mechanism, i.e., by facilitating a clonal expression of cells bearing this spontaneous mutation.