2-Acetylaminofluorene-mediated alteration in the level of liver arylsulfotransferase IV during rat hepatocarcinogenesis.

Rat liver cytosolic sulfotransferase activity forms the highly reactive sulfuric acid ester of N-hydroxy-2-acetylaminofluorene (N-OH-2AAF), an ultimate carcinogen in 2-acetylaminofluorene (2AAF) hepatocarcinogenesis. A previous report demonstrated that 2AAF-induced liver hyperplastic nodules displayed a persistent loss of cytosolic N-OH-2AAF sulfotransferase activity following a hepatocarcinogenesis-producing regimen of 2AAF administration. As an initial step in examining the mechanism responsible for lowering N-OH-2AAF sulfotransferase activity, a monospecific polyclonal antibody to aryl sulfotransferase IV (AST IV) was produced and used in the assessment of AST IV as a candidate enzyme for liver cytosolic N-OH-2AAF sulfotransferase activity. Studies comparing the levels of N-OH-2AAF sulfotransferase activity of highly purified AST IV and rat liver cytosols with corresponding immunochemical analysis of AST IV contents demonstrated that there was sufficient AST IV activity in liver cytosols to indicate that it was the primary enzyme catalyzing cytosolic N-OH-2AAF sulfation. A subsequent immunochemical survey of nine extrahepatic tissues showed no detectable AST IV content and indicated that AST IV expression may be tissue specific. An immunochemical comparison of AST IV levels in control liver cytosols (high in sulfotransferase activity) with cytosols from 2AAF-derived hyperplastic nodules (low in sulfotransferase activity) or liver tumors (no sulfotransferase activity) showed low or no detectable levels, respectively, of AST IV. In addition, an immunochemical analysis of four rat hepatoma cell lines showed they contained no detectable levels of AST IV. These results suggested a strong correlation existed between a decrease in AST IV expression and tumor development. When the liver cytosols of rats taken from early, intermediate, and late stages of 2AAF carcinogenesis were analyzed for the development of a persistent loss of N-OH-2AAF sulfotransferase activity, a parallel loss of cytosolic N-OH-2AAF sulfotransferase activity and AST IV content was observed in rats which had proceeded from a stage of low risk to high risk for liver cancer. These findings indicated that (a) AST IV, a liver-specific enzyme, was the principle enzyme comprising cytosolic N-OH-2AAF sulfotransferase activity and (b) the decrease in sulfotransferase activity in nodules and tumors resulted from a decrease in the level of AST IV expression. Furthermore, it is suggested that a persistent decrease in AST IV expression may reflect a role for AST IV as part of a resistance phenotype in which transforming liver cells are able to escape the cytotoxic effects of highly reactive 2AAF metabolites and progress to cancer.

Modulation of hepatic mRNA translation activity and specific expression of arylsulfotransferase IV during acetylaminofluorene-induced rat hepatocarcinogenesis.

Enzymatic sulfation of N-hydroxylated arylamines by mammalian hepatic cytosol sulfotransferases (AST; EC 2.8.2.1) is an important metabolic step which generates ultimate carcinogens. The metabolic activity of AST IV, the putative isozymic form of AST primarily responsible for catalyzing N-hydroxy-2-acetylaminofluorene sulfation, is modulated during 2-acetylaminofluorene (AAF)-induced rat hepatocarcinogenesis. To characterize the molecular mechanisms regulating the differential expression of AST IV, we have assessed polyadenylated mRNA derived from the livers of Sprague-Dawley rats undergoing different stages of AAF hepatocarcinogenesis for general in vitro translation capacity and specific expression of AST IV and albumin. Following 1 and 3 cycles of a cyclical feeding regimen (3 weeks 0.05% AAF, then 1 week basal diet), the mRNA capacity for translation was lowered and the expression of AST IV and albumin was down-regulated about 2-fold each but recovered to normal levels when treated rats were subsequently placed on basal diet for 3 continuous weeks. Cytosolic albumin levels were determined by Western blot analysis to be lowered about 1.5-2-fold. In contrast, however, mRNA from rats on basal diets for 3 weeks subsequent to cycle 5 of the feeding regimen recovered only about 50% of the capacity for AST IV expression, although overall translation capacity and albumin expression returned to normal levels. This pattern of reversible expression, followed by irreversible expression of AST IV at early and late stages of AAF hepatocarcinogenesis, respectively, provides the first evidence correlating the modulation of hepatic mRNA capacity for AST IV expression with differential cytosolic AST IV activity in the AAF hepatocarcinogenesis model. The results further suggest that during early stages in hepatocarcinogenesis modulation of mRNA protein synthesis functions may be a critical factor in AAF-mediated lowering of AST IV expression, while other persistent genetic lesions are likely playing a more significant role at the late stages of the carcinogenic process leading to neoplastic transformation of initiated hepatocytes.

Suppression of natural killer and lymphocyte functions associated with carcinogen-induced premalignant hyperplastic nodules in rat liver.

The effects of chemical carcinogenesis to produce premalignant hyperplastic nodules in rat liver on concomitant immune function were studied. Induction of hyperplastic nodules in Fischer rats was accomplished using a combined regimen of diethylnitrosamine, 2-acetylaminofluorene, and partial hepatectomy. Hyperplastic nodules were detected in carcinogen-treated rats from 5 to 23 weeks as confirmed by gross pathology, histopathology, and significantly elevated liver gamma-glutamyltransferase activity. Suppression of natural killer activity of either peritoneal or peripheral blood lymphoid, but not splenic, cells for YAC-1 target cells occurred during 5 to 20 weeks in carcinogen-treated rats. Spleen and blood lymphocyte mitogenic responses to concanavalin A and pokeweed mitogen were also suppressed at most intervals from 8 through 20 weeks. Control groups given individual carcinogen or partial hepatectomy alone or in dual combination were not suppressed in their immune function and failed to develop hyperplastic foci or changes in liver gamma-glutamyltransferase. Our findings indicate that immunosuppression of natural killer and lymphocyte mitogenic functions occurs for a protracted period concurrently with the development of the premalignant hyperplastic state in rat liver. The data suggest a potential role for immune competency during the onset of malignant neoplasia.

Characterization of a complementary DNA for rat liver aryl sulfotransferase IV and use in evaluating the hepatic gene transcript levels of rats at various stages of 2-acetylaminofluorene-induced hepatocarcinogenesis.

A complementary DNA (cDNA) for rat hepatic aryl sulfotransferase IV (AST IV) was isolated, characterized, and used as a hybridization probe to evaluate the molecular basis for the differential expression of AST IV during 2-acetylaminofluorine (2AAF)-induced hepatocarcinogensis. The AST IV cDNA clone was obtained by immunochemical screening of a male Sprague-Dawley rat liver cDNA library. The AST IV cDNA was found to be 1.3 kilobases long and to encode a fusion protein which was reactive with an antibody to AST IV and enzymatically able to generate the sulfuric acid ester of N-hydroxy-2AAF. Sequence analysis of the AST IV cDNA showed it to be 1127 residues in length and to have essentially complete homology with PST-I cDNA, a previously reported (S. Ozawa, et al., Nucleic Acids Res., 18: 4001, 1990), 1028-base cDNA for an uncharacterized rat liver aryl sulfotransferase. Comparison of the PST-I/AST IV cDNA-deduced amino acid sequence with data from a partial (51%) amino acid sequence analysis of purified AST IV showed complete amino acid homology, confirming the identity of the cDNA and establishing that AST IV was an N-blocked, 291-amino acid protein with a molecular mass of 33,909 daltons. The AST IV cDNA sequence differed from the PST-I cDNA in two principal ways: the 5' end lacked 18 coding bases, and the 3' end contained a 190-base extention in the untranslated region, including a consensus sequence for signalling polyadenylation. Studies of AST IV gene transcript levels showed that the livers of rats fed 2AAF for 3 wk (early stage hepatocarcinogenesis) and hyperplastic nodules from the livers of rats fed 2AAF for 19 wk (intermediate stage hepatocarcinogenesis) displayed transcript levels similar to those of livers from normal rats. This contrasted with the 60 to 70% lower than normal capacity of the mRNA fractions to express AST IV observed during in vitro translation. These results indicated that modulation of AST IV expression at early and intermediate stages of hepatocarcinogenesis involved regulatory mechanisms at the translational level. In contrast, mRNA fractions isolated from some 2AAF-induced liver tumors or from known chemical carcinogen-derived rat hepatoma cell lines showed losses of both AST IV transcript level and in vitro translation capacity, suggesting that regulation at the transcriptional level may become important at late stages of 2AAF-induced hepatocarcinogenesis. These results indicated that the molecular mechanisms for the 2AAF-mediated down regulation of AST IV expression during 2AAF-induced hepatocarcinogenesis involved alterations in regulation at both translational and transcriptional levels.

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver.

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [3H]Thd, [14C]orotate or with inorganic 32Pi. Expression of incorporated radioactivity as log10% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [3H]Thd was asymmetrically distributed among the isostichs, i.e., 3H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., T/C ratios for the first 10 isostichs averaged 1.43 +/- 0.08 and 1.28 +/- 0.05, depending on the method of analysis; furthermore, the T/C ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [3H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [3H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecular weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [14C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [3H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of 14C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from 32Pi, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since 32P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of 32P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the 'replitase' concept for channeling dNTPs to replicating forks.

Differences in the distribution of phosphate content in the ribosomal subunit proteins of free and membrane-bound ribosomes from normal and regenerating rat liver.

Proteins of membrane-bound ribosomes from normal liver contained 60-70% more phosphate than did proteins from free ribosomes. This difference was not a reflection of the phosphate contents of respective 40 S subunits. Instead, it was owing to the presence of high levels of phosphorylated proteins in the 60 S subunits, i.e., phosphate contents equal to or greater than those for 40 S subunits. The proteins of membrane-bound 60 S subunits contained twice the phosphate as free 60 S subunits. In regenerating rat liver, membrane-bound ribosomes had increased phosphate in the proteins of the 40 S subunits and decreased phosphate in proteins of the 60 S subunit when compared to controls for normal rat liver. No significant changes occurred in the proteins of free ribosomes from regenerating rat liver. These findings are discussed with respect to (a) the importance of assessing total phosphate contents of proteins in the study of ribosomal protein phosphorylation, and (b) the possible involvement of ribosomal protein phosphorylation in the segregation of ribosomes into free and membrane-bound populations and the regulation of these distributions to meet changes in the translational demands of the cell.

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver.

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-14C) orotic acid and inorganic (32P] phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.

Genomic structure of rat liver aryl sulfotransferase IV-encoding gene.

This report contains the first description of the genomic structure for a sulfotransferase (ST). The gene (ASTIV) encodes rat hepatic aryl ST IV, also known as tyrosine-ester ST (EC 2.8.2.9). A phage genomic clone containing 70% of the 3' AST gene coding sequence was isolated after screening a rat genomic library with an ASTIV cDNA. The remaining 5' sequence was determined from a PCR product obtained from rat genomic DNA and ASTIV cDNA-specific primers. ASTIV spans 3.5 kb and contains eight exons and seven introns. The fourth intron of this gene contains sequences homologous to rodent B1 repetitive elements and an Alu repeat found in rat. An alignment of the primary structures of ten different ST revealed several conserved regions, as well as a putative binding site for the cofactor for enzymatic sulfation reactions, 3'-phosphoadenosine-5'-phosphosulfate.

Nuclear matrix associated poly(ADP-ribose) metabolism in regenerating rat liver.

We have examined a possible role for protein poly(ADP-ribosylation) during in vivo DNA replication by studying the metabolism of poly(ADP-ribose) in the nuclear matrix fraction from normal and regenerating rat liver. This fraction contains the newly replicated DNA and thus allows for the examination of the events closely associated with the replication process. It was found that 55% of the total nuclear protein-bound poly(ADP-ribose) and 15-35% of the total nuclear poly(ADP-ribose)-polymerase activity were tightly associated with this subnuclear compartment in normal liver. Surgical removal of two-thirds of the liver initiated a time-dependent decrease in nuclear matrix associated polymers of ADP-ribose and poly(ADP-ribose) polymerase activity which reached a minimum of 40% of control livers after 24 h, before returning to normal levels at 41 h post-partial hepatectomy. In contrast, the total levels of poly(ADP-ribose) in intact liver and the total polymerase activity of isolated nuclei exhibited a 2-fold increase over basal levels. These results are consistent with the conclusion that the nuclear matrix is a major poly(ADP-ribosylation) site within the nucleus and that this metabolic reaction may be closely connected with the events modulating DNA replication in this fraction.

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A.

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.

Quantitative analysis of changes in cell proliferation and apoptosis during preneoplastic and neoplastic stages of hepatocarcinogenesis in rat.

In situ markers for quantitative analysis of cell proliferation and apoptotic cell death have been used to evaluate both the proliferation level and net growth potential of preneoplastic nodules and malignant tumor tissues from rats experimentally induced for hepatocarcinogenesis by the dietary administration of 2-acetylaminofluorene. The findings show that although tumors have a much higher level of cell proliferation than preneoplastic liver nodules, the nodules have a higher potential for net growth when apoptosis is taken into account. These results support a role for a decrease in apoptosis during the promotion stage of carcinogenesis.

Carcinogen-induced alteration in liver epidermal growth factor receptor distribution during the promotion stage of hepatocarcinogenesis in rat.

Changes in hepatic membrane binding capacity for epidermal growth factor (EGF) were assessed during 2-acetylaminofluorine (2-AAF)-induced hepatocarcinogenesis. An overall decrease in membrane EGF binding levels was observed throughout the period of 2-AAF administration. Immunochemical studies indicated the decreases in EGF binding levels were paralleled by decreases in tissue EGF receptor levels. Immunohistochemical studies showed that the losses in hepatic EGF receptor levels were not uniform throughout the liver during the early, promotion stage of cancer development. During the promotion stage, preneoplastic liver nodules were found to display a higher level of EGF receptor than surrounding hepatic tissue. This resistance to 2-AAF-mediated down-regulation of EGF receptor may confer a proliferative advantage to the developing nodule relative to the surrounding liver tissue. Similar immunochemical and immunohistochemical analysis of hepatocarcinomas at late stages of 2-AAF-induced hepatocarcinogenesis indicated a uniform loss of EGF receptor in tumor and surrounding tissue. These studies indicate that carcinogen-mediated changes in EGF binding levels are different during the multistage process of hepatocarcinogenesis, and that resistance to down-regulation of EGF receptor among preneoplastic nodules may have a role in providing a selective growth advantage to initiated cell populations. EGF receptor may be useful as a dynamic marker assessing the development of hepatic tumors.

Changes in rat liver N-hydroxy-2-acetylaminofluorene aryl sulfotransferase activity at early and late stages of hepatocarcinogenesis resulting from dietary administration of 2-acetylaminofluorene.

The ability of 2-acetylaminofluorene (AAF) to mediate a loss in N-hydroxy-AAF (N-OH-AAF) aryl sulfotransferase activity when fed to male Sprague-Dawley rats was examined at early and late stages of hepatocarcinogenesis. Administration of 0.05% AAF in the diet for 1 week caused liver N-OH-AAF aryl sulfotransferase activity to decrease to 15 +/- 5% of that for liver from non-carcinogen-fed rats, and the activity remained low throughout 19 weeks of AAF feeding. When rats were fed AAF diet for 3 weeks, then placed on a control diet, liver N-OH-AAF aryl sulfotransferase activity returned to normal levels within 3 weeks. In contrast, when rats were fed AAF for 19 weeks, then placed on control diet for an additional 10 weeks, little or no recovery of N-OH-AAF aryl sulfotransferase activity was observed in cytosols from whole livers or isolated hyperplastic nodules, respectively. These findings suggest two types of AAF-mediated decreases in sulfotransferase activity: (a) a decrease observed early in the initial stages of AAF feeding which returns to normal levels when AAF is removed from diet, and (b) a persistent decrease in activity following long term AAF administration.

Assessment of rat liver microsomal epoxide hydrolase as a marker of hepatocarcinogenesis.

The influence of eleven xenobiotics on the activity and amount of hepatic microsomal epoxide hydrolase was determined. Activity was assayed using three different substrates after rats were fed, throughout 3 weeks, diets containing one of six hepatocarcinogens, viz. 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, 4'-fluoro-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1 and ethionine. Five hepatocarcinogens induced activity 4- to 10-fold; ethionine was relatively ineffective as an inducer. Two non-carcinogenic analogues of hepatocarcinogens, viz. fluorene and p-aminoazobenzene, caused no appreciable increase in enzyme activity, but phenobarbital, barbital and 1-naphthylisothiocyanate induced activity 2- to 3-fold. All eleven xenobiotics increased the amount of microsomal epoxide hydrolase 2- to 9-fold when examined immunochemically using either a radial diffusion assay or an enzyme-linked immunosorbent assay (ELISA). Serum glutamic oxaloacetic acid transaminase activity was not appreciably elevated by feeding ten of the xenobiotics, suggesting that inductions were not owing to toxicity. Using ELISA, microsomal epoxide hydrolase was detected in post-microsomal (PM) supernatant fractions from control rat liver, thus confirming an earlier report by Gill et al. [Carcinogenesis 3, 1307 (1982)]. The eleven xenobiotics induced the amount of ELISA-detectable antigen in PM supernatant fractions by 3- to 34-fold. Longer centrifugation of PM supernatant fractions yielded a pellet fraction that contained 92 +/- 1.2% of the ELISA-detectable antigen irrespective of the xenobiotic regimen. Relationships between xenobiotic induction of microsomal epoxide hydrolase activity and amount and hepatocarcinogenesis are discussed.

Molecular approach to rapid assessment of p53 tumor suppressor mutations in esophageal tumors from stained histological slides.

The analysis of the tumor suppressor gene, p53, is of fundamental importance in prognosis and staging in many cancers; however, the molecular techniques required to analyze this gene have been expensive, time consuming, and unrelatable to the histological appearance of the samples. This research explored one model of clinically testing for specific mutations in the p53 gene by scraping selected areas of stained histological slides and analyzing for "hot-spot" p53 mutations. Selectively removing samples from the stained histological slide will be of special value in examining suspicious regions in adenomas, potential metastatic regions, and the margins of resected area. A polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis approach in which naturally occurring or primer-mediated mutagenesis-induced restriction enzyme sites were utilized to test seven hot-spot mutations. These assays were able to detect one mutated sequence in 100, and therefore, were sufficiently sensitive to be used with very heterogeneous tumors. Several of the assays could be multiplexed to reduce the number of PCRs necessary to screen for the seven mutational hot spots. Furthermore, an exact determination of the base change could be obtained by direct sequencing of the PCR products. Although this form of analysis may be applicable only to certain types of cancers (e.g., bladder, brain, colon, esophageal, gastric, thyroid, and ovarian tumors), this approach can obtain detailed mutational information from specific regions of a histological slide in a cost-effective and timely manner.

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA.

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.

Molecular mechanisms for the regulation of aryl sulfotransferase IV expression during 2-acetylaminofluorene-induced hepatocarcinogenesis in rat.

The dietary administration of 2-acetylaminofluorene to male rats to induce hepatocarcinogenesis causes a reversible as well as persistent down-modulation of N-hydroxy-2AAF sulfotransferase activity. Studies are presented which indicate that several molecular mechanisms may be involved in the down-regulation of sulfotransferase activity and expression. These include carcinogen-mediated inactivation of sulfotransferase mRNA or protein, interference with hormonal regulation of sulfotransferase expression, and, mutation of the sulfotransferase gene.

Hypomethylation of the rat aryl sulfotransferase IV gene and amplification of a DNA sequence during multistage 2-acetylaminofluorene hepatocarcinogenesis.

Rat hepatic aryl sulfotransferase IV (AST IV), which catalyses sulfuric acid esterification of N-hydroxy-2-acetylaminofluorene to its ultimate carcinogenic form, is differentially expressed during multistep 2-acetylaminofluorene (AAF) hepatocarcinogenesis. Two molecular mechanisms associated with this effect involve modulation of mRNA translational capacity at the early stages, and gene transcription at the late stages of the carcinogenic process. To characterize further the molecular mechanisms that may be involved in the transient regulation of the enzyme expression, an AST IV cDNA was used to assess the change in methylation profile and restriction fragment length polymorphism (RFLP) in the gene domain of genomic DNA derived from rats at different stages of carcinogenesis. The onset of hypomethylation of the AST IV gene domain and amplification of a 5.3-kb DNA sequence was found to correlate with the stage in AAF hepatocarcinogenesis, where rats begin to exhibit irreversible loss in hepatic enzyme expression and the liver becomes committed to hepatoma formation. This represents the first observation of both altered methylation status of AST IV gene domain and amplification of a DNA sequence whose expression may play a role in the genesis and/or progression of neoplastic transformation of initiated cells during AAF hepatocarcinogenesis.

Sulfation of carcinogenic aromatic hydroxylamines and hydroxamic acids by rat and human sulfotransferases: substrate specificity, developmental aspects and sex differences.

Sulfation of the carcinogen N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and structurally related hydroxamic acids by rat and human sulfotransferases was studied. There was a clear sex and age difference in the sulfation of N-OH-AAF and the other hydroxamic acids by rat liver cytosols; adult male rats had the highest sulfation activity. Experiments with purified aryl sulfotransferase IV (AST IV) indicated that the high expression of this enzyme in male rat liver may be responsible for these differences. No such sex or age difference was found for the sulfation of aromatic hydroxylamines. In cytosols of adult human livers, sulfation activity towards aromatic hydroxamic acids and hydroxylamines was clearly present, but activities were much lower than in rat liver cytosols. Sulfation activity towards these compounds was also found in fetal and neonatal liver and adrenals. These compounds probably are sulfated by several different sulfotransferases in humans.

Alterations in epidermal growth factor binding to hepatic membranes following dietary exposure of rats to known hepatocarcinogens.

Administration of the chemical carcinogen 2-acetylaminofluorene (2-AAF) has previously been shown to lower hepatic epidermal growth factor (EGF) binding levels during chemically induced hepatocarcinogenesis. To further characterize the specificity of this response, EGF binding levels for liver microsomes were determined after a 3-week administration of subacute doses of 2-AAF and five other known hepatocarcinogens: 3'-methyl-4-dimethylaminoazobenzene (3'Me-DAB), 2-AAF, aflatoxin B1 (AFB1), thioacetamide (TA), ethionine, benzidine (Benz), as well as four non-hepatocarcinogens: fluorene, p-aminoazobenzene, 4-acetylaminofluorene (4-AAF), and 3-methylcholanthrene. Five of six of the hepatocarcinogens tested (3'Me-DAB, 2-AAF, TA, AFB1 and Benz) caused significant lowering of EGF binding levels, and one of the four non-hepatocarcinogens (4-AAF) caused significant lowering of EGF binding levels. Paired feeding studies indicated that the decreases in EGF binding levels were not a result of differences in net diet consumption. These findings show that decreases in EGF binding capacity are caused by a diverse group of known hepatocarcinogenic compounds at an early stage in the carcinogenesis process.