Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene

The purpose of this study was to examine the induction of unscheduledDNA synthesis (UDS) by the potent hepatocarcinogen technicalgrade dinitrotoluene (tgDNT; 76% 2, 4-DNT, 19% 2, 6-DNT) usingthe in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344rats were treated by gavage and hepatocytes were isolated byliver perfusion and cultured with [³H]thymidine. UDS was measuredby quantitative autoradiography as net grains/nucleus (NG);5 NG was considered positive. Controls consistently had –3 to – 6 NG. A dose-related increase in UDS was observed12 h after treatment, with 200 mg/kg tgDNT producing 26 NG.A 50-fold increase in the number of cells in S-phase was observedat 48 h after treatment. This increase in S-phase cells couldbe suppressed in the presence of 10–20 mM hydroxyurea(HU), while the same levels of HU did not affect the level ofUDS at 12 h after treatment. 2, 4-DNT produced only a weak response,in contrast to 2, 6-DNT which was a potent inducer of UDS. Treatmentof female rats with tgDNT yielded only modest increases in UDSand DNA replication relative to males. These results are consistentwith the carcinogenicity studies and indicate that tgDNT isa potent genotoxic agent, with 2, 6-DNT contributing the majorportion of the effect.     

Stimulation of DNA synthesis in primary rat hepatocyte cultures by liver tumor promoters: interactions with other growth factors.

Mechanism(s) of tumor promotion in liver by xenobiotics such as hexachlorocyclohexane (HCH), 1,1,1-trichloro-2,2-bis (4-chlorophenyl)ethane (DDT) and phenobarbital (PB) are not understood in detail although growth-stimulatory effects may be significant in their action. As a basis for studying mechanisms of growth control by liver tumor promoters, effects of xenobiotics on DNA synthesis have been examined in primary cultures of normal rat hepatocytes, maintained under fully-defined conditions. The xenobiotics alone were relatively ineffective but they exhibited synergism with epidermal growth factor (EGF), insulin and dexamethasone in stimulating DNA synthesis and were effective in moderate-to-low density cultures but not in confluent monolayers. Under conditions optimized for HCH or pregnenolone 16 alpha-carbonitrile (PCN) (i.e. subconfluent cultures exposed to insulin, EGF and dexamethasone), HCH, PCN, DDT or PB caused a transient stimulation of DNA synthesis, apparent after 2 days in culture. This probably reflected earlier entry of hepatocytes to S-phase. HCH was shown to increase total DNA, total numbers of nuclei and numbers of cells undergoing mitosis per culture. In optimized conditions, HCH or PCN were about additive with norepinephrine, dialyzed serum or pyruvate or with a small effect of tri-iodothyronine in stimulating DNA synthesis. Although conditions optimal for HCH or PCN were not necessarily optimal for detecting growth-stimulatory effects of other xenobiotics or steroids, these culture conditions were shown to support stimulation of DNA synthesis by a variety of known liver tumor promoters including barbiturates, estrogens, progestins, peroxisomal proliferators and bile acids. Several compounds known not to promote liver carcinogenesis failed to stimulate DNA synthesis in similar hepatocyte cultures.     

SHORT COMMUNICATION: Time-Related introduction of DNA repair synthesis in rat hepatocytes following in vivo treatment with cyproterone acetate

Cyproterone acetate (CPA), a synthetic steroid hormone widelyused as a human pharmaceutical, has recently been shown to induceunscheduled DNA synthesis (UDS) in vitro in primary culturesof rat and human hepatocytes. In the present study CPA was evaluatedfor its ability to initiate UDS in the liver of female ratsin vivo by means of the in vivo/in vitro hepatocyte DNA repairtest. Using the standard sampling time of 16 h after singleoral dosing, a dose-related UDS responce at >50 mg CPA/kgbody wt was observed. In order to examine the time course ofCPA induced UDS, different sampling times (4, 16, 48, 72, 96,and 144 h) after a single oral administration of 100 mg CPA/kgbody wt were used. Whereas no UDS was induced in liver cellsisolated 4 h after treatment, continuous DNA repair activitywas observed after 16 h, with a maximum effect of     

Lack of DNA damage or lipid peroxidation measured in vivo in the rat liver following treatment with peroxisomal proliferators

This study was undertaken to investigate the hypothesis linkingperoxisome proliferation with the production of reactive oxygenspecies and subsequent DNA damage. Hepatic peroxisomal proliferationwas induced in male Wistar-derived rats by the administrationof clofibrate, methyl clofenapate, di(2-ethylhexyl)phthalate,or its metabolite mono (2-ethylhexyl)phthalate (MEHP) for periodsof up to 28 days. Genotoxicity was monitored using an alkalineelution technique to assay for DNA strandbreaks and cytotoxicitywas monitored by measuring lipid peroxidation. Both parametersmight be expected to be elevated if peroxisome proliferationis accompanied by an elevated level of oxygen free radicalswithin the cell. Enzyme measurements made on the livers of thetreated rats showed that peroxisomal palmitoyl CoA oxidase activitywas markedly increased over control whereas peroxisomal catalaseactivity was not. In addition, both the cytosolic glutathioneperoxidase and superoxide dismutase activities were found tobe lowered in the treated animals by up to 50 and 20% respectively.Despite such changes in enzyme activity, no evidence for increasesin DNA strandbreaks or lipid peroxidation was obtained withany of the chemicals at any of the time points examined. DNAstrandbreaks were also assayed on hepatocytes treated in culturewith MEHP (0.5 mM) for 3 days and then exposed to inhibitorsof DNA repair for 2 h immediately before assay. Again, no significantincrease over controls was observed. Our data suggest that anyincrease in radical production in the livers of rats exposedto peroxisome proliferators is not large enough to give riseto a biologically significant degree of DNA damage and thatthe mechanism whereby such chemicals produce liver tumours incertain rodent species may be one other than simply DNA damagedue to increased production of radical species.     

DNA adduct formation and unscheduled DNA synthesis in rat esophagus in vivo after treatment with N-methyl-N-nitrosourea

An in vivo method for assessment of DNA adduct formation and unscheduled DNA synthesis (UDS) in the esophagus of rats was devised. Small ventral incisions were made in the neck and upper abdomen regions of 6 week old F344 rats and ligation of the esophagus with thread at the two extreme ends performed to make an esophageal pouch. For the DNA adduct formation study, a solution (0.5 ml) containing various concentrations of N-[3H]methyl-N-nitrosourea ([3H]MNU) was injected into the pouch. DNA binding levels were calculated from radioactivity of the isolated DNA and dose-dependent DNA adduct formation could be detected 2 h after the treatment with MNU. By HPLC analysis, both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) adducts were identified in the esophageal DNA, the ratio of 7-mGua/O6-mGua being 5.7-12:1. For UDS measurement, a solution containing MNU plus [3H]thymidine (200 microCi/ml) was similarly injected into the pouch. UDS was dose-dependently demonstrated as silver grains over the nuclei of the epithelial cells by autoradiography. The results thus showed that MNU, when injected into the esophageal lumen, can penetrate the surface mucosa, react with the epithelial cell DNA and induce DNA adduct formation and DNA repair synthesis dose-dependently.     

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment

Peroxisome proliferator hepatocarcinogens lack genotoxic activityin numerous in vitro assays using non-target cells which donot respond with peroxisome proliferation. Therefore, the effectof in vivo treatment with WY-14,643 on DNA repair was quantitatedin rat hepatocytes, the target cell for carcinogenesis. PalmitoylCoA oxidase and carnitine acetyltransferase activities in isolatedhepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavagefor up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethylhexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicatingperoxisome proliferation had occurred. DNA repair as unscheduledDNA synthesis (UDS) was measured autoradiographically as netnuclear grains following thymidine incorporation in primaryhepatocyte cultures. Treatment of rats with WY-14,643 (gavageor feeding) or DEHP (feeding) did not induce UDS. Addition of2-acetylaminofluorene to replicate cultures demonstrated thatWY-14,643 or DEHP treatment did not prevent repair response.Additional cultures were treated with H₂O₂ (0.8 mM H₂O₂ 3 xat 1-h intervals) to evaluate the ability of UDS to detect anyrepair which may be induced by peroxisomal metabolism. H₂O₂did not induce UDS at this concentration, nor did it prevent2-acetylaminofluorene- induced repair. UDS was, however, observedin a separate experiment using a higher concentration of H₂O₂.In summary, a highly carcinogenic peroxisome proliferator didnot induce UDS in the target cell for carcinogenesis in spiteof peroxisome proliferation following in vivo treatment.     

Stimulation of DNA synthesis and c-fos mRNA expression in primary rat hepatocytes by estrogens

The mechanism(s) of tumour promotion in liver by estrogens is not well understood although growth stimulation is known to be one important element of their action. As a basis for studying mechanisms of growth control by estrogens, effects of both natural and synthetic estrogens on DNA synthesis and protooncogene c-fos mRNA expression were examined in primary cultures of normal rat hepatocytes. 17beta-Estradiol (E(2)) alone was stimulatory and exhibited dramatic synergism with epidermal growth factor (EGF) in stimulating DNA synthesis. All estrogens tested (natural, synthetic, steroidal and non-steroidal) exhibited an ability to stimulate hepatocyte DNA synthesis. This appears to correlate with their ability to induce c-fos mRNA expression. In contrast to a non-estrogenic liver tumour promoter, phenobarbital, insulin is not permissive for the growth-stimulatory action of E(2). Dexamethasone, which is required for stimulation of DNA synthesis by the non-estrogenic tumour promoter alpha-hexachlorocyclohexane and tetradecanoylphorbol acetate, completely blocked E(2)-stimulated DNA synthesis. Such differential requirements for auxiliary factors suggests that estrogen and other non-estrogenic liver tumour promoters act via distinct mechanisms in stimulating hepatocyte DNA synthesis. E(2) alone had no effect, but when in combination with EGF significantly induced c-fos mRNA expression at early times in culture (maximal at 10 h in culture). Such findings, coupled with the observations that (i) E(2) and EGF were synergistic in growth stimulation, (ii) estrogen receptor levels are higher at early times in culture and (iii) the growth-stimulatory ability of E(2) is limited to 4-24 h in culture, support the notion that in hepatocytes E(2) acts via the estrogen receptor to transactivate c-fos expression (an interaction with EGF), which ultimately culminates in enhanced DNA synthesis. Dexamethasone did not block E(2)-induced c-fos gene expression, suggesting that it acts in a pathway(s) distal to activation of fos gene expression. The possible inhibitory mechanisms of action of dexamethasone on E(2)-stimulated DNA synthesis are discussed.     

A liver DNA synthesis promoter induced in rat plasma by injection of dimethylnitrosamine (DMNA) or thioacetamide

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.     

Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures.

Unscheduled DNA synthesis was observed in primary rat liver cell cultures treated with members of five different classes of chemical procarcinogens requiring enzymatic activation as well as with a direct-acting carcinogen. In total, ten carcinogens and one related analog not commonly accepted as carcinogenic were active, while one weak carcinogen and four noncarcinogens were inactive. The production of unscheduled DNA synthesis by this spectrum of chemical carcinogens indicates that these cultures have substantially retained the metabolic capability of liver for activating diverse procarcinogens. Thus, such cultures may be useful for detecting the ability of chemicals to interact with DNA and, thereby, assigning them priority for consideration as potential cancer-causing agents.     

Nafenopin-induced rat liver peroxisome proliferation reduces DNA methylation by N-nitrosodimethylamine in vivo

The hypolipidaemic drug nafenopin (NAF) has been shown to enhancethe hepatocarcinogenic effect of N-nitrosodimethylamine (NDMA)and N-nitrosodiethylamine in rats. We have investigated whetherthe NAF-induced peroxisome proliferation in hepatocytes interfereswith NDMA's metabolism and interaction with DNA. Adult maleWistar rats received a single i.p. injection of [¹⁴C]NDMA (2mg/kg) and were killed 4 h later. DNA was isolated from liverand kidney, hydrolysed in 0.1 N HCI and analysed by Sephasorbchromatography. In rats pre-treated with NAF (0.2% in the dietover a period of 3 weeks), the concentration of N7-methylguaninein hepatic DNA (µmol/mol guanine) was 46% below controlvalues. This is probably due to the greater amount of targetDNA, as NAF caused a marked hepatomegaly with a 50% increasein total liver DNA content. Concentrations of N7-methylguaninein kidney DNA were twice as high in NAF-pre-treated animalswhen compared to control rats. This is unlikely to result froma shift in the metabolism of NDMA from liver to other rat tissuessince the time course and extent of the conversion of [¹⁴C]NDMAto ¹⁴CO₂ and ¹⁴C-labelled urinary metabolites were identicalin NAF-treated and control animals. There was no indicationthat NAF inhibits the activity of the hepatic O⁶-alkylguanine-DNAalkyltransferase.     

Induction of hepatic cell proliferation and unscheduled DNA synthesis in mouse hepatocytes following in vivo treatment

We have modified the in vivo-in vitro hepatocyte DNA repairassay for measurement of unscheduled DNA synthesis (UDS) andhepatic cell proliferation in B6C3F1 mice. Dimethylnitros-amineand methylmethane sulfonate produced significant increases inUDS in both rats and mice. 2-Acetylaminofluorene induced a significantincrease in UDS in rats, but not in mice. The mouse hepatocarcinogens,carbon tetrachloride, trichloroethylene, polybrominated biphenylsand 2,6-dichloro-p-phenylenediamine all failed to induce UDSin male and female mice, but all induced significant elevationsin hepatic cell proliferation. Increased cell turnover in theliver may therefore be an important mechanism in hepatocarcinogenicityin the mouse.     

Stimulation of liver DNA synthesis by a factor from ascites tumor 6C3HED

C3H mice inoculated with 6C3HED ascites tumor show a gradual increase in the incorporation of ³²P into liver DNA. Cell-free supernatant fluid from this tumor has the capacity to stimulate increased incorporation of ³²P and ³H-labelled thymidine into liver DNA of mice not bearing the tumor. This liver-stimulating activity (LSA) was destroyed by heating to 60° C for 30 min and by pronase but not by RNase or DNase. The time of maximum stimulation of liver DNA synthesis was on the 4th day after injection of LSA. A 70-fold purification of LSA was accomplished using sephadex chromatography. It is suggested that LSA is the factor responsible for the increased liver DNA synthesis seen in tumor-bearing animals, but the mechanism of this action has yet to be explained.     

Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters.

Parenchymal liver cells were isolated from human liver pieces of surgical waste as well as from rat livers. DNA synthetic activity was measured after different times in primary culture by [3H]thymidine incorporation and autoradiography. Labeling of control cultures of human hepatocytes at densities between 8,000 and 15,000 cells/cm2 was very low (0.4 to 1.3%). Human recombinant epidermal growth factor increased labeling 2- to 4-fold (P less than 0.01). Treatment with known inducers of liver growth in rats, namely, cyproterone acetate, alpha-hexachlorocyclohexane, nafenopin, phenobarbital, and rifampicin did not increase the number of labeled human liver cells. In some of the experiments, a 24-h exposure to the chemicals of rat or human hepatocytes was followed by a 24-h treatment with epidermal growth factor (EGF). In rat hepatocytes, incorporation rates were significantly increased. Cyproterone acetate and EGF acted in an additive manner, alpha-hexachlorocyclohexane and EGF were clearly overadditive, and phenobarbital had little effect. In human hepatocytes, little alteration in labeling indices was found; in some cases labeling was, rather, found to be lower than in cultures treated with EGF alone. These results show that human hepatocytes cultured in vitro are sensitive to stimulation of DNA synthesis by EGF; they differ from rat hepatocytes in their response to some drugs which show liver growth-promoting activity in rodents.     

DNA damage and repair in intact animals. Single-stranded regions in rat liver DNA following administration of dimethylnitrosamine.

Rat liver DNA was radioactively labelled by administration of [3H]thymidine following partial hepatectomy. Two weeks later, the rats were treated with the carcinogen, dimethylnitrosamine. DNA was isolated and fractionated by elution from benzoylated DEAE-cellulose with NaCl and caffeine solution. The caffeine-eluted fraction was increased by administration of dimethylnitrosamine. This increase was proportional to the dose of carcinogen injected and persisted for at least 24 h after administration of the carcinogen. These data, together with the results of hydroxyapatite chromatography, suggest that the DNA contains short single-stranded sections associated with much longer regions of native DNA.     

Immunocytochemical localization of DNA adducts in rat tissues following treatment with N-nitrosomethylbenzylamine

Immunocytochemical visualization of O6-methylguanosine (meGua) and 7-meGua shows that DNA methylation by N-nitrosomethylbenzylamine(NMBzA) occurs not only in the target organs for tumour induction by this nitrosamine, the oesophagus and (occasionally) the tongue, but also in other tissues (liver, lung, trachea, tracheal glands and nasal cavity) for which no tumour induction by NMBzA has been reported. Thus, the organotropic carcinogenic action of NMBzA cannot be exclusively ascribed to differences in levels of DNA methylation. Additional determinants of the cancer risk in extra-oesophageal tissues could be the small size of the NMBzA-activating target cell population and a low proliferative activity.