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.     

Differential susceptibility of rat and guinea pig colon mucosa DNA to methylation by methylazoxymethyl acetate in vivo

Methylazoxymethanol (MAM) and methylazoxymethyl acetate (MAMOAc) are powerful colon carcinogens in rats, mice and hamsters. In contrast, these agents are not carcinogenic to the colon of the guinea pig. To probe the mechanism responsible for this species difference, we determined the levels of DNA methylation in the livers and colon mucosae of F344 rats and strain-2 guinea pigs after the s.c. administration of 25 mg/kg MAMOAc. While no significant difference was observed between the two species with respect to the degree of liver DNA methylation, the level of O6-methylguanine in guinea pig colon mucosa DNA was 19 times lower than in rat colon mucosa DNA, and the level of 7-methylguanine was below detection limits. However, significant colon mucosa DNA methylation was observed in the guinea pig after the intrarectal administration of 1.25 mg methylnitrosourea. The methylation of colon mucosa DNA in response to MAMOAc in the two species correlated with the activity of alcohol dehydrogenase, an enzyme believed to be involved in the activation of MAM. Thus the resistance of the guinea pig colon to the carcinogenicity of MAM/MAMOAc may be ascribed to the lack of metabolic activation of MAM in this organ.     

DNA methylation in rat stomach and duodenum following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine and the effect of dietary taurocholate

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a high incidenceof carcinomas in the glandular stomach of rats following chronicadministration in the drinking water. We determined the levelof 7-methylguanine and O⁶-mehtylguanine in gastric and duodenalDNA during chronic exposure to MNNG (80 p.p.m.). After considerablefluctuations during the initial 3 weeks, levels of methylpurinesreached a steady state which was approximately three times higherin the pylorus (i.e. the preferential site of tumor induction)thanin the fundus and duodenum, with 7-methylguanine and O⁶-methylguaninevalues in the rangeof 520 and 110 µmol/mol guanine, respectively.When rats were given MNNG in the drinking water at concentrationsranging from 10 to 80 p.p.m. for 3 weeks, levels of methylpurinesreached maximum values already at 10–20 p.p.m. At higherMNNG concentrations, there was no further increase in DNA alkylation.The reason for this lack of dose response remained unclear.Immunohistochemical analyses showed that DNA methylatlon byMNNG is restricted to epithelial cells bordering the luminalsurface. The possibility exists that in this target cell populationthe content of free thiols is a limiting factor for the decompositionof MNNG and its reaction with macromolecules in the gastricmucosa. Addition to the diet of sodium taurocholate, a bileacid previously shown to enhance MNNG-induced stomach carcinogenesis,did not influence the extent of DNA methylation, indicatingthat it acts as a promoter.     

Induction of N-nitrosodimethylamine metabolism in rat liver and lung by ethanol

The metabolic activation of N-nitrosodimethylamine (NDMA) to an active metabolite is important in the manifestation of its carcinogenic effect. The lung and liver were compared for their responses to the induction of NDMA demethylation by 10% ethanol in the drinking water and by repeated bolus injections. Ethanol in the drinking water increased NDMA metabolism several-fold in both the liver and the lung. Repeated ip injections with 0.6 and 3.0 ml ethanol/kg for 7 days also enhanced this activity in a dose-dependent fashion. These results suggest that in the lung, as in the liver, ethanol may influence the metabolic activation of this nitrosamine.     

Effect of ethanol on dimethylnitrosamine activation and DNA synthesis in rat liver

Male Wistar rats were treated for 2 weeks with 20% ethanol inthe drinking water. Twenty four hours after the terminationof treatment animals were injected with different doses of [¹⁴C]dimethylnitrosamineand killed 4 h thereafter. The amounts of 7-methylguanine andO⁶-methylguanine present in liver DNA were determined. Therewas no significant difference in the levels of either DNA alkylationproduct between untreated controls and animals pre-treated withethanol. The O⁶-methylguanine/7-methylguanine ratio was alsounchanged. Specific radioactivity levels in the cellular proteinof [¹⁴C]dimethylnitrosamine injected animals were slightly loweredafter ethanol pre-treatment. In contrast, incorporation of labelledguanine into liver DNA was greatly enhanced at all DMN dosesin ethanol pre-treated animals indicating an increase in DNAsynthesis. This enhancement of DNA synthesis was confirmed atdifferent ethanol doses, given either in the drinking wateror by stomach tube, by the increase in specific radioactivityof liver DNA and the increase in the number of labelled nucleifollowing [³H]thymidine pulse-labelling.     

Effects of timing and quantity of chronic dietary ethanol consumption on azoxymethane-induced colonic carcinogenesis and azoxymethane metabolism in Fischer 344 rats

Epidemiological studies have shown an association between consumption of alcoholic beverages and carcinoma of the large bowel, but studies in experimental models of colonic carcinogenesis have yielded conflicting results. We assessed the effects on azoxymethane-induced colonic carcinogenesis of both timing of chronic dietary ethanol consumption relative to carcinogen administration and quantity of ethanol consumption. Ten-week-old male Fischer 344 rats were given 11%, 22%, or 33% of calories as reagent ethanol or no ethanol by pair feeding with Lieber-DeCarli-type liquid diets providing comparable total carbohydrates, proteins, fats, and calories. Ten weekly s.c. injections of the bowel carcinogen azoxymethane (AOM), 7 mg/kg, were given to all rats in weeks 1-10. Three experimental groups were given their respective ethanol diet during acclimatization and AOM administration (preinduction and induction phases) and then were given the no-ethanol diet from week 11 until sacrifice in week 26 (postinduction phase). Three other groups received the no-ethanol diet during acclimatization and AOM administration and then were changed to their respective ethanol diet until sacrifice. The control AOM group received the no-ethanol diet throughout the study. Suppression of colonic tumorigenesis occurred in the groups with high levels of chronic dietary ethanol consumption during acclimatization and AOM administration: in the 33% and 22% diet groups, the prevalence of colonic tumors was 3% and 20% as compared with 50% in control (P less than 0.001 and P less than 0.02, respectively). Tumorigenesis in the left colon was more affected than in the right colon, as tumor prevalence in the left colon was decreased in both the 33% and 22% diet groups (0% in both versus 24% in control, P less than 0.005), whereas prevalence in the right colon was decreased only in the 33% diet group (3% versus 38%, P less than 0.001). By contrast, prevalence of colonic tumors in the 11% diet group was not significantly different from control. Chronic dietary ethanol consumption after AOM administration had no effect on tumor outcome, regardless of quantity of consumption. In an analogous study of [14C]AOM metabolism in rats fed the 33% diet during acclimatization and AOM administration, 14CO2 was exhaled at a slower rate than in rats fed no-ethanol diet (P = 0.05), indicating suppression of AOM metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of acute and chronic ethanol intake on metastasis formation in rat liver

The effect of acute ethanol intoxication and prolonged intake of ethanol on metastasis formation in the liver was studied in the rat. The tumour cells used were derived from a pharmacologically induced colon carcinoma (NGW 1) and were injected intraportally. Acute ethanol intoxication at the time of injection was associated with a liver weight of 13.29 +/- 0.89 gm two weeks later, compared to 8.43 +/- 0.51 gm in controls. Daily intake of ethanol for eight weeks resulted in a mild reduction of platelets. The liver weight in two groups was 7.31 +/- 0.50 gm and 12.19 +/- 1.40 gm three weeks after intraportal injection of cancer cells as compared to 9.53 +/- 1.11 gm and 19.63 +/- 1.67 gm in controls. The results indirectly supported the hypothesis that acute ethanol intoxication causes increased platelet activity and confirmed earlier results showing the importance of platelets for metastasis formation.     

The relationship between N-nitrosodimethylamine metabolism and DNA methylation in isolated rat hepatocytes

The metabolism of N-nitrosodimethylamine (NDMA) and its methylationof DNA were simultaneously determined in hepatocytes isolatedfrom untreated and saline- and pyrazole-treated male Sprague-Dawleyrats. Metabolism of NDMA was directly measured by monitoringits disappearance via gas chromatography coupled with a sensitiveand specific detector for N-nitrosamines. DNA methylation wasdetermined in the same cells employed in the metabolism studiesusing a monoclonal antibody-based competitive ELISA procedurespecific for O⁶-methyldeoxyguanosine (6-Me-dG). The apparentK_m and V_max, for NDMA metabolism are 61 µM and 56 pmol/min/10⁶cells respectively for hepatocytes isolated from untreated rats.It was found that the addition of pyrazole to the in vitro hepatocyteincubations caused a dose-dependent inhibition of both metabolismand DNA methylation. However, when DNA methylation is expressedas a function of NDMA metabolized, there is no significant differencebetween hepatocyte incubations without or with pyrazole, withan average value of 79 nmol 6-Me-dG/mol dG/nmol NDMA metabolized.Based on the pyrazole inhibition studies, cyto-chrome P450IIE1is responsible for at least 60% of the DNA methylation in rathepatocytes. In pyrazole-pretreated rats there was an inconsistentincrease in NDMA metabolism, but when metabolism was elevatedso was DNA methylation. In contrast, microsomes isolated frompyra zole-pretreated rats consistently showed elevated metabolismof NDMA. Based on the simultaneous determination of adduct levelsand metabolism, there is 1 6-Me-dG adduct formed/133 000 NDMAmolecules metabolized in the uninduced hepatocytes.     

DNA methylation in specific cells of rat liver by N-nitrosodimethylamine and N-nitrosomethylbenzylamine

Dose-response curves for the O6-methylation of guanine in the hepatic DNA of Wistar and Sprague-Dawley rats were determined after administration of N-nitrosomethylbenzylamine (NMBzA) or N-nitrosodimethylamine (NDMA). Similar results were obtained for both rat strains but methylation of hepatic DNA by NDMA was approximately 9-fold more efficient than with NMBzA when doses were compared on a molar basis. Comparison by immunohistochemical analysis of the distribution of nuclei containing O6-methylguanine within the liver lobules showed that both agents tended to alkylate cells close to the central veins at the lower doses. With increasing doses, the band width of alkylated cells around the central vein increased, spreading in the case of NDMA virtually into the portal zones, whereas with NMBzA the zone of alkylated nuclei reached little more than halfway from the central vein to the portal zone. These differences in the distribution of alkylated cells may explain the differing hepatic responses to these two nitrosamines.     

Effects of neonatal and adult castration on the in vitro metabolism of steroids and xenobiotics in rat liver

Previous studies in our laboratory have shown that the sex-differentiated metabolism of 4-androstene-3,17-dione and of several other steroid hormones in adult rat liver is "feminized" following neonatal castration of male rats, due to an influence via the hypothalamo-pituitary-liver axis. The metabolism of many xenobiotics is also sex differentiated, and an important question is whether endocrine ablations might alter hepatic carcinogen metabolism in a way explaining, for example, the decreased tendency of castrated male rats [Y.C. Toh, In: Shanmagarathnam et al. (eds.), Liver Cancer, Cancer Problems in Asian Countries, Proceedings of the Second Asian Cancer Conference, pp. 167-171. Singapore: Singapore Cancer Society, 1976] to form liver tumors following 2-acetylaminofluorene treatment. The results presented in this paper clearly show that neonatal castration of male rats, much more efficiently than adult castration, feminizes the cytochrome P-450-dependent, sex-differentiated, liver microsomal formation of 7-hydroxy-2-acetylaminofluorene, 9-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 1-hydroxy-2-acetylaminofluorene, and N-hydroxy-2-acetylaminofluorene from 2-acetylaminofluorene as well as the total microsomal formation of benzo(a)pyrene metabolites (male greater than female). O-Deethylation of 7-ethoxyresorufin was neither sex differentiated nor affected by castration. The capacity for in vitro sulfation of N-hydroxy-2-acetylaminofluorene in the postmicrosomal supernatant, markedly sex differentiated in the rat (male greater than female), was completely feminized by neonatal but not by adult castration. The results suggest that the influence of endocrine ablations on chemical carcinogenesis in rat liver might be mediated via the hypothalamo-pituitary regulation of certain pathways of hepatic xenobiotic metabolism.     

Effect of methylazoxymethanol acetate on rat liver nuclear and nucleolar RNA synthesis

The effect of methylazoxymethanol acetate on rat liver nuclear and nucleolar RNA synthesis is investigated at various doses (5 to 50 mg/100 g body weight) and for various lengths of time (1 to 24 hr). The results show that this carcinogen is a potent inhibitor of both nuclear and nucleolar RNA synthesis. Like other carcinogens studied previously in this laboratory, e.g., N-hydroxy-2-acetylaminofluorene, aflatoxin B1, and actinomycin D, methylazoxymethanol acetate inhibits RNA synthesis at multiple sites. It impairs chromatin template function and selectively inhibits the activity of RNA polymerase II. Experimental evidence suggests the mechanism of inhibition of RNA polymerase II activity is due to a decrease in catalytic efficiency rather than in the total number of the enzyme. In addition, it is found that methylazoxymethanol acetate induces a dramatic condensation of nucleoplasmic chromatin.     

Effects of dietary curcumin on glutathione S-transferase and malondialdehyde-DNA adducts in rat liver and colon mucosa: relationship with drug levels.

Curcumin prevents colon cancer in rodent models. It inhibits lipid peroxidation and cyclooxygenase-2 (COX-2) expression and induces glutathione S-transferase (GST) enzymes. We tested the hypothesis that 14 days of dietary curcumin (2%) affects biomarkers relevant to cancer chemoprevention in the rat. Levels of inducible COX-2, as reflected by prostaglandin E(2) production by blood leukocytes, were measured ex vivo. Total GST activity and adducts of malondialdehyde with DNA (M(1)G), which reflect endogenous lipid peroxidation, were measured in colon mucosa, liver, and blood leukocytes. Curcumin and its metabolites were analyzed by high-performance liquid chromatography in plasma, and its pharmacokinetics were compared following a diet containing 2% curcumin versus intragastric (i.g.) administration of curcumin suspended in an amphiphilic solvent. The curcumin diet did not alter any of the markers in the blood but increased hepatic GST by 16% and decreased colon M(1)G levels by 36% when compared with controls. Administration of carbon tetrachloride during the treatment period increased colon M(1)G levels, and this increase was prevented by dietary curcumin. Dietary curcumin yielded low drug levels in the plasma, between 0 and 12 nM, whereas tissue concentrations of curcumin in liver and colon mucosa were 0.1--0.9 nmol/g and 0.2--1.8 micromol/g, respectively. In comparison with dietary administration, suspended curcumin given i.g. resulted in more curcumin in the plasma but much less in the colon mucosa. The results show that curcumin mixed with the diet achieves drug levels in the colon and liver sufficient to explain the pharmacological activities observed and suggest that this mode of administration may be preferable for the chemoprevention of colon cancer.     

Nonrandom nature of in vivo methylation of dimethylnitrosamine and the subsequent removal of methylated products from rat liver chromatin DNA.

This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.     

Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis

The aim of this study was to examine the significance of aberrant DNA methylation, the participation of which in genetic instability is controversial, in hepatocarcinogenesis. The DNA methylation status of the region around the promoter of the E-cadherin tumor suppressor gene, which is located on 16q22.1, and the allelic status at the D16S421 locus, which is adjacent to the E-cadherin locus, were examined using microdissected liver specimens from 38 hepatocellular carcinoma (HCC) patients. Almost all of the non-cancerous liver tissues showed histological findings compatible with chronic hepatitis and cirrhosis, which are considered to be precancerous conditions. DNA hypermethylation was detected in 61% of the non-cancerous liver tissues. The incidence of DNA hypermethylation in the non-cancerous liver tissues of patients with HCCs also showing DNA hypermethylation (72%) was significantly higher than that of patients without DNA hypermethylation in their HCCs (53%, P<0.05). Loss of heterozygosity (LOH) at the D16S421 locus was detected in 35% of the non-cancerous liver tissues. The incidence of LOH in the non-cancerous liver tissues of patients with HCCs also showing LOH was 78%, whereas LOH was not detected in non-cancerous liver tissues of patients without LOH in their HCCs. Fifty-two percent of the non-cancerous liver tissues showed both or neither of DNA hypermethylation and LOH; the incidence of DNA hypermethylation alone in noncancerous liver tissue was 41%. The incidence of LOH alone in non-cancerous liver tissue (7%) was significantly lower compared to those of the former two cases (P<0.0001). These data suggest that aberrant DNA methylation participates in the precancerous stage of hepatocarcinogenesis by preceding, or causing, LOH.     

Effects of chronic dietary beer and ethanol consumption on experimental colonic carcinogenesis by azoxymethane in rats

Epidemiological studies have shown an association between consumption of alcoholic beverages, particularly beer, and carcinoma of the large bowel, especially the rectum. We studied the effects of chronic dietary beer and ethanol consumption on experimental colonic carcinogenesis, fecal bile acid and neutral sterol levels, fecal bacterial flora, and colonic epithelial DNA synthesis. Ten-week-old male Fischer 344 rats were pair fed throughout the study with Lieber-DeCarli-type liquid diets providing comparable total carbohydrates, proteins, fats, and calories. The diets provided 23 or 12% of calories as alcohol in beer (Hi-Beer and Lo-Beer groups), 18 or 9% of calories as reagent ethanol (Hi-EtOH and Lo-EtOH groups), or no alcohol (control group). After 3 weeks of dietary acclimatization, 10 weekly s.c. injections of the bowel carcinogen azoxymethane, 7 mg/kg, were given (weeks 1-10). At necropsy in week 26, the high alcohol groups (Hi-Beer and Hi-EtOH) showed a significantly reduced incidence of tumors in the right colon (42 and 46% versus 81% in control, P less than 0.01 and P = 0.02) but no effect on left colonic tumorigenesis. By contrast, the low alcohol groups (Lo-Beer and Lo-EtOH) showed a trend toward increased incidence and proportion of tumors in the left colon (incidence of 42 and 35% versus 15% in control, P = 0.06 for Lo-Beer; 28 and 30% of tumors in left colon versus 11%, P = 0.08 and P = 0.07) but no effect on right colonic tumorigenesis. Numbers of right colonic tumors were inversely correlated with alcohol consumption of all rats (r = -0.350, P less than 0.001), but left colonic tumors were not correlated. Fecal bile acid and neutral sterol levels, fecal bacterial counts, and colonic epithelial DNA synthesis did not correlate with the effects of alcohol consumption on colonic tumorigenesis. Our findings suggest that: modulation of experimental colonic tumorigenesis by chronic dietary beer and ethanol consumption was due to alcohol rather than other beverage constituents; tumorigenesis in the right and left colon was affected differentially by the levels of alcohol consumption, reflecting complex interactions among the potential mechanisms for alcohol effects in the model used.     

Acute effects of dietary cholic acid and methylazoxymethanol acetate on colon epithelial cell proliferation; metabolism of bile salts and neutral sterols in conventional and germfree SD rats

The acute effects of cholic acid ingestion on methylazoxymethanol acetate [(MAM) CAS: 592-62-1]-treated conventional and germfree rats were investigated. Male SD rats were divided into 4 treatment groups. The first group received control chow; the second group, control chow plus 0.5% cholic acid; the third group, control chow plus MAM; and the fourth group, control chow plus 0.5% cholic acid plus MAM. Fecal bile acids, cholesterol, cholesteral degradation products, and neutral sterols, as well as labeling indices and numbers of epithelial cells per crypt column, were measured after 6 weeks of treatment. The administration of MAM to germfree groups diminished both fecal bulk and the amount of fecal water. MAM did not affect the fecal bile acid composition. Analysis of the fecal bile acids in conventional rats fed cholic acid demonstrated that half of the bile acids were in a form of deoxycholic acid. In the germfree groups fed cholic acid, 90% of the bile acids appeared unaltered in the feces. Neither in the germfree nor in the conventional groups was an effect seen of MAM on the output of fecal neutral sterols. The addition of cholic acid to the food decreased the output of neutral sterols both in the conventional (P less than .001) and in the germfree (P less than .02) animals. The germfree animals showed a reduced amount of neutral steroid excretion (P less than .01) when compared to the findings for the conventional groups. MAM had no influence on the fecal cholesterol or coprostanol output. The consumption of 0.5% cholic acid decreased the total output of cholesterol (P less than .05). The excretion of coprostanol was significantly diminished in the conventional rats fed cholic acid (P less than .001). No difference in labeling indices was observed between conventional and germfree rats, whether treated with cholic acid, MAM, or cholic acid plus MAM. However, all germfree groups showed less epithelial cells per crypt column (P less than .001) than did conventional animals.     

S-Adenosylmethionine metabolism and DNA methylation in hydrazine-treated rats

The treatment of rats with hepatotoxic doses of hydrazine (NH₂-NH₂)induces the rapid formation of 7-methylguanine and O⁶-methylguaninein liver DNA. The methyl moiety in these reactions might bederived from the cellular S-adenosyl-methionine pool becauseradioactivity administered to these rats as methionine rapidlyappears in the DNA as methylated guanine. An increased incorporationof radioactivity into 5-methylcytosine was previously reportedfollowed by subsequent suppression. This increased radiolabelingof 5-methylcytosine coincided with time of maximal DNA guaninemethylation. To determine the nature of S-adenosyl-methioninemetabolism during the period of DNA methylation induced by hydrazinetreatment, and to determine if the increased radiolabeling of5-methylcytosine at this time reflected an actual increase in5-methylcytosine synthesis, liver DNA synthesis and S-adenosylmethioninelevels and turnover were assayed. Liver S-adenosylmethionineconcentrations varied slightly between control rats and hydrazine-treatedrats during the first five hours after hydrazine ad ministration,and no difference was detectable in the incorporation of administered[³H]methionine into S-adenosylmethionine. Because S-adenosylmethioninespecific radio activity in hydrazine-treated rats was not differentfrom control rats, the previously observed increased radiolabelingof 5-methylcytosine appeared to represent an actual increasein synthesis. This conclusion was supported by finding thatin corporation of radioactive thymidine into DNA was also acceleratedimmediately following hydrazine administration, again followedby a decrease. 5-Methylcytosine synthesis, therefore, appearsto follow DNA synthesis during hydrazine toxicity, and formationof 7-methylguanine and O⁶-methylguanine in liver DNA of hydrazine-treatedrats occurs during a short period of increased DNA synthesisand 5-methylcytosine formation very early in hydrazine toxicity.     

Effects of chronic ethanol consumption on the metabolism and carcinogenicity of N'-nitrosonornicotine in F344 rats

The effects of chronic ethanol consumption on the carcinogenicity and metabolism of N'-nitrosonornicotine (NNN) in male F344 rats have been investigated. Groups of 26 to 30 rats were maintained on either a control liquid diet (Groups 1, 3, and 5) or an ethanol-containing liquid diet (Groups 2, 4, and 6) for 4 weeks prior to and during treatment with NNN. The carcinogen was injected s.c. (10 mg/kg, Groups 3 and 4) three times weekly or added to the liquid diet (17.5 mg/liter, Groups 5 and 6). The total dose was 1 mmol of NNN per rat. Control rats (Groups 1 and 2) received s.c. injections of 0.9% NaCl solution. The nasal mucosa was the main target tissue of NNN in Groups 3 and 4, but both the nasal mucosa and esophagus were major target tissues in Groups 5 and 6. In rats treated s.c. with NNN (Groups 3 and 4), ethanol consumption had no effect on the distribution and incidence of nasal cavity tumors. In rats treated with NNN added to the control liquid diet or to the ethanol-containing liquid diet, the number of tumors of the nasal cavity was 18 in Group 5 and 26 in Group 6 (p less than 0.05). In contrast, the number of rats with esophageal tumors was 25 in Group 5 and 20 in Group 6 (p less than 0.05). The effects of ethanol on the enzyme system which activates NNN were studied in rats which had been maintained on an ethanol-containing liquid diet for 4 weeks. Explants of nasal mucosae, lingual mucosae, esophagi , and livers were cultured in vitro with NNN. Nasal mucosae of ethanol-consuming rats had a 1.5-fold higher (p less than 0.05) alpha-carbon-hydroxylating activity than did those of control rats. Activating enzymes in the lingual mucosae, esophagi , and livers were not induced by ethanol. The results show that the increased susceptibility of the rat nasal mucosa to the carcinogenic effects of NNN added to an ethanol-containing diet could be due in part to an induction of activating enzymes by ethanol. However, since chronic ethanol consumption had no apparent effect on the incidence of nasal cavity tumors in rats treated by s.c. injection of NNN, factors other than enzyme induction are important in determining the effects of ethanol on NNN carcinogenicity.     

金雀异黄素的体内与体外抗结肠癌作用及其机制

目的 探讨金雀异黄素(Gen)对结肠癌细胞增殖、凋亡和相关蛋白表达的影响,及其抗结肠癌的作用机制.方法 采用四甲基偶氮唑蓝(MTT)法检测结肠癌细胞增殖情况,光镜和电镜下观察细胞形态学和超微结构变化,流式细胞术检测Gen对结肠癌细胞周期和凋亡的影响,流式细胞术和免疫组化法检测结肠癌细胞凋亡相关蛋白的表达情况.结果 MTT法检测结果显示,Gen对SW480细胞增殖具有抑制作用,呈明显的剂量-效应和时间-效应关系,抑制率以80μg/ml Gen作用72 h为最高(60.2%).光镜下,药物作用后细胞皱缩、破裂,胞浆中出现空泡.透射电镜下,细胞体积缩小,核染色质高度浓缩,形成沿核膜收缩的新月状小体.流式细胞术检测结果显示,0、20、40、80μg/ml Gen作用SW480细胞48 h后,增殖细胞核抗原(PCNA)蛋白的荧光指数(F1)值分别为1.49±0.02、1.28±0.04、1.14 4±0.03和0.93±0.08,血管内皮生长因子(VEGF)蛋白的F1值分别为1.75 4±0.02、1.34 4±0.06、1.32 4±0.04和1.23 4±0.04,p21蛋白的F1值分别1.26±0.05、1.36 4±0.06、1.61 4±0.03和1.73±0.03,20、40、80 μg/ml Gen组与μg/ml Gen组比较,差异有统计学意义(P<0.05或P<0.01).免疫组化检测结果显示,Gen处理组中PCNA和VEGF蛋白的表达降低,p21蛋白表达升高,与对照组差异有统计学意义(P<0.05).结论 Gen抑制结肠癌细胞的生长与诱导细胞凋亡、阻滞细胞周期于G_2/M期有关.Gen抗结肠癌的作用机制可能与下调PCNA和VEGF蛋白的表达、上调p21蛋白的表达有关.