Inhibition of dimethylnitrosamine-induced strand breaks in liver DNA and liver cell necrosis by diethyldithiocarbamate.

Diethyldithiocarbamate (DEDTC) prevented dimethylnitrosamine (DMN)-induced strand breaks in liver DNA and liver cell necrosis in male Wistar rats. In contrast, DEDTC did not inhibit the fragmentation of liver DNA caused by several other chemical carcinogens (N-hydroxy-2-acetylaminofluorene, 3-hydroxyxanthine, aflatoxin B1, N-acetoxy-2-acetylaminofluorene, methyl methanesulfonate, methylnitrosourea, and methylazoxy-methanol acetate), whether or not they required metabolic activation. Aminoacetonitrile exerted an action similar to that of DEDTC. The inhibitory effect was transitory, lasting at least for 4 hours, and protection for longer than 4 hours required multiple administrations of DEDTC. DEDTC also inhibited the serum clearance of DMN, methylation of liver DNA, and oxidative demethylation of DMN in the in vitro hepatic microsomal system prepared from either male Wistar rats or from hamsters. Interference of the metabolism of DMN appeared to be the mechanism by which DEDTC arrested DMN-induced biochemical and biologic effects.

Effect of butylated hydroxyanisole pretreatment on in vitro hepatic aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in rats.

The effect of 3(2)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both in vitro hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation has been examined. For these studies, young male F344 rats were fed AIN-76 A diet with or without 0.75% BHA for 2 weeks. There were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated exogenous DNA binding to AFB1 with cytochrome P-450 from control or BHA-treated animals. There were large differences in reduced glutathione S-transferase activity with treated cytosols showing 2.5-fold higher activity than the controls. Hepatic reduced glutathione levels were 25% higher in treated than in controls. Kinetics of cytosolic inhibition of microsome-mediated AFB1-DNA binding and formation of AFB1-SG conjugate when examined at two levels of AFB1 (2 and 10 microM) and a 4-fold range of cytosolic concentrations showed that inhibition of AFB1-DNA binding was greater with cytosol from the treated compared to the controls. However, AFB1-SG conjugation was 3- to 4-fold greater in treated than in controls. Inhibition of AFB1-DNA binding by cytosol was reversed in the presence of 1 mM level of various epoxides with concomitant inhibition of AFB1-SG conjugation. In reconstitution studies with 2 microM AFB1, intact nuclei alone from either group did not yield significant amounts of either DNA binding or AFB1-SG conjugation. However, addition of microsomes from either group to these nuclei generated a large amount of AFB1-DNA binding (82-111 pmol) and a smaller amount of AFB1-SG conjugate (9-28 pmol). The presence of cytosols from the control group reduced AFB1-DNA binding to a much lesser extent than the cytosols from the treated group. However, AFB1-SG conjugation was much higher with the cytosol from treated than with the controls. These reconstitution studies with endogenous DNA show more AFB1-DNA binding with the control than with BHA-treated animals and are in agreement with the studies in vivo. It appears that induced levels of cytosolic reduced glutathione S-transferase modulate AFB1-DNA binding and AFB1 hepatocarcinogenesis.

Effect of butylated hydroxyanisole pretreatment on aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation in isolated hepatocytes from rats.

The effect of 2(3)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione has been examined with isolated hepatocytes and in intact rats. Young male F344 rats were fed AIN-76A diet with or without 0.75% BHA for 2 weeks. Even though there were no significant differences in either cytochrome P-450 or reduced glutathione contents, there were marked differences in AFB1 metabolism in isolated hepatocytes from these two groups. Thus, at the 33 nM AFB1 level, AFB1-DNA binding was 3-fold higher in control compared to BHA-treated hepatocytes whereas AFB1-glutathione conjugation was 5-fold higher in treated compared to controls. Even at higher AFB1 concentrations (2 and 10 microM), DNA binding was 4-6-fold higher in controls whereas thiol conjugation was 5-9-fold higher in treated compared to control hepatocytes. Addition of 0.5-1.0 mM diethylmaleate did not have any significant effect in control hepatocytes whereas its presence produced about 70-100% increase in DNA binding with 65-80% inhibition of thiol conjugation in treated hepatocytes. Addition of 1 mM styrene oxide caused 75-100% and 4-8-fold increase in AFB1-DNA binding in control and treated hepatocytes, respectively, with corresponding decreases in thiol conjugation. In intact rats, BHA treatment reduced hepatic AFB1-DNA binding to 15% of controls with concomitant increase in biliary excretion of AFB1-reduced glutathione conjugate. It appears that the induced cytosolic GSH S-transferases after BHA treatment of rats play a significant role in inhibiting hepatic AFB1-DNA binding and AFB1 hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.

A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats.

The effect of phenobarbital (PB) pretreatment of rats on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation have been examined in studies in vivo and in vitro. Male Sprague-Dawley rats fed a commercial diet with 0.1% PB in their drinking water for 1 week had total wet liver weight and microsomal protein content about 27% and 38% higher, respectively, than controls. Hepatic cytochrome P-450 content, microsomal cytochrome P-450 mediated AFB1 binding to exogenous DNA and formation of hydroxy metabolites of AFB1 were also about threefold higher in PB-treated rats and cytosolic reduced glutathione S-transferase activities were about doubled. Microsome-mediated AFB1-DNA binding, when examined at 2 microM and 10 microM levels of AFB1, was inhibited two-to threefold more by cytosols of treated rats whereas AFB1-SG conjugation was two- to threefold higher by cytosols of treated rats. In reconstitution experiments with 2 microM AFB1, with intact nuclei serving as a source of endogenous DNA, addition of microsomes from either group generated a large amount of AFB1-DNA binding (68-105 pmol) and a smaller amount of AFB1-SG conjugate (12-21 pmol). The presence of cytosol from the controls reduced AFB1-DNA binding to a much lesser extent than the cytosol from the treated group whereas AFB1-SG conjugation was much higher with the cytosol from the treated group. These results are in agreement with the studies in vivo. In isolated hepatocytes at 33 nM, 2 microM and 10 microM AFB1 levels, AFB1-DNA binding was decreased 50 to 70% by prior PB-treatment whereas AFB1-SG conjugation was two- to threefold higher in treated compared to control hepatocytes. In hepatocytes, addition of 1 mM diethylmaleate increased DNA binding two- to threefold with a corresponding decrease in AFB1-SG conjugation. Addition of 1 mM styrene oxide caused 5- to 10-fold increases in AFB1-DNA binding at levels of AFB1 of 33 nM and 2 microM; but at 10 microM AFB1, increases in AFB1-DNA binding were two- to threefold. In intact rats, PB treatment reduced hepatic AFB1-DNA binding to 30% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. It appears that the induced cytosolic GSH S-transferases after PB treatment of rats plays a significant role in inhibiting hepatic AFB1-DNA binding and hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of beta-naphthoflavone on the metabolism of aflatoxin B1 in hamsters.

The effect of beta-naphthoflavone (BNF) pretreatment of hamsters on the hepatic metabolism of aflatoxin B1 (AFB1) has been examined in studies in vitro and in vivo. Pretreatment with BNF not only increased microsomal cytochrome P-450 by 50-80% but also increased microsome-mediated AFB1 epoxidation as measured by AFB1-DNA binding 2.6 fold without significantly affecting other hydroxylations. Neither cytosolic GSH S-transferases' activities nor AFB1-GSH (AFB1-SG) conjugation were affected. In vivo, hepatic AFB1-DNA binding was also increased about 3-4-fold. These results in contrast to those observed in the rat indicate that induced species of cytochrome P-450 are primarily responsible for higher epoxidation of AFB1 in the hamster.

Urinary excretion of thiol conjugates of aflatoxin B1 in rats and hamsters.

After intraperitoneal injection of [3H]aflatoxin B1 in rats and hamsters, 24-h urinary excretion of aflatoxin B1 metabolites was examined in these species. Total excretion was 10-15% of the injected dose with hamster excreting 50% more than the rat. In both species, 60% of the total excretion was due to glucuronide and sulfate conjugates of hydroxy metabolites. Striking differences were observed in the excretion of both aflatoxin B1-diol and various thiol conjugates. Aflatoxin B1-glutathione and aflatoxin B1-cysteinylglycine were major thiol conjugates in rats and hamsters, respectively. An unknown metabolite believed to be mercapturic acid (N-acetylcysteine conjugate) of aflatoxin B1 was excreted by both species, hamster excreting twice as much as the rat.

Glutathione conjugation of microsome-mediated and synthetic aflatoxin B1-8,9-oxide by purified glutathione S-transferases from rats.

Glutathione (GSH) conjugation of microsome-mediated and synthetic aflatoxin B1 (AFB1)-epoxide and styrene oxide has been investigated with purified GSH S-transferases (GSTs) from rats. Both styrene oxide and AFB1-epoxide were conjugated preferentially by millimicrons GSTs 3-3, 3-4 and 4-4 as compared to alpha GSTs 1-1, 1-2 and 2-2. The highest catalytic activity with styrene oxide conjugation was associated with GST 4-4. The highest catalytic activity with microsome-mediated AFB1-epoxide conjugation was observed with GST 3-3 whereas with the synthetic AFB1-epoxide conjugation was seen with GST 4-4. The catalytic activity of pi GST 7-7 was intermediate to millimicrons and alpha GSTs. It is suggested that GST 3-3 may play an important role in inactivation of AFB1-epoxide generated in vivo in the rat.

Effect of glutathione levels on aflatoxin B1-DNA binding in livers and kidneys of male rats and hamsters pretreated with buthionine sulfoximine and dimethylmaleate.

The effects of pretreatment of buthionine sulfoximine (BSO) alone or in combination with diethylmaleate (DEM) on glutathione (GSH) levels and aflatoxin B1 (AFB1)-DNA binding have been examined in livers and kidneys of young male Fischer rats and Syrian golden hamsters 2 h after an intraperitoneal injection of [3H]AFB1 (0.4 mg/kg body wt.). Animals were treated with BSO (4 mmol/kg body wt.) alone at 4 h and 2 h or with DEM (3 mmol/kg body wt.) at 4 h and BSO at 2 h before AFB1 injection. Hepatic AFB1-DNA binding was about 29.0 and 6.0 pmol/mg DNA in rats and hamsters, respectively. In rats, BSO increased AFB1-DNA binding by about 40% with a drop in GSH by 70%. Treatment with DEM-BSO increased AFB1-DNA binding by about 80% with a concomitant drop in GSH in both species. In hamsters, BSO increased AFB1-DNA binding by only 10% with a 50% drop in GSH. The kidneys of both species have lower GSH levels and AFB1-DNA binding than their respective liver tissues. The effect of BSO alone or in combination with DEM on both GSH levels and AFB1-DNA binding are comparable even though BSO alone is less effective in both species. The role of modulation of GSH levels on AFB1-DNA binding and hence biological effects of AFB1 in these two species is discussed.

Effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated rats, hamsters and guinea pigs.

The effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated (100 mg/kg body wt. 24 h before sacrifice) rats, hamsters and guinea pigs was investigated. At low substrate concentration (2 mM), liver microsomes from pretreated rats and hamsters showed 30-50% lower demethylation activity than their respective controls. No such difference was found in the guinea pig. At high substrate concentration (100 mM), all 3 pretreated species showed 50-100% higher enzyme activity than their respective controls. Enzyme activities among the 3 species showed the following order of activity: hamster greater than guinea pig greater than rat.

Protein bound carboxyl-methyl ester as a precursor of methanol formation during oxidation of dimethylnitrosamine in vitro.

Protein modification with dimethylnitrosamine was studied in vitro in the presence of hamster liver microsomal fraction. Incorporation of radioactive methyl groups from dimethylnitrosamine into the exogenously added protein was dependent on the microsomal mixed function oxidase system. The methylation yielded chemically labile and stable products. The former was completely hydrolyzed by the mild alkaline treatment, pH 7.4, 100 degrees C, for 5 min and the hydrolytic product was identified as methanol indicating that the activated methyl groups from dimethylnitrosamine were incorporated into a protein as a carboxyl-methyl ester. Thus, it is suggested that methanol, recovered as one of the products during the biodegradation of dimethylnitrosamine [8], is derived, at least in part, from protein carboxyl-methyl ester which is unstable under physiological conditions.

Factors influencing the activity of dimethylnitrosamine demethylase in hamster, rat and chicken liver microsomes.

In vitro metabolism of dimethylnitrosamine (DMN) by liver microsomal fractions of hamster, rat and chicken revealed that the three species under certain assay conditions, were capable of metabolizing DMN at different rates (hamster greater than rat greater than chicken). The magnitude of the demethylase activity was found to be dependent on the nature of the buffer, the concentration of cytochrome P-450 (P-450) and the concentration of the substrate DMN. Enzyme activity was higher in Hepes buffer than in the phosphate buffer. Concentrations of phosphate higher than 20 mM inhibited the activity of the rat and chicken enzymes. This effect of phosphate was not a consequence of increase in ionic strength since KCl over a wide range of concentration failed to inhibit the activity.

Effect of butylated hydroxyanisole on in vivo and in vitro hepatic aflatoxin B1-DNA binding in rats.

Butylated hydroxyanisole (BHA) pretreatment of male rats has been examined for its effect on in vivo and in vitro hepatic aflatoxin B1-DNA binding (AFB1-DNA) in these animals. No difference either in cytochrome P-450 content or microsome-mediated AFB1-DNA was observed between livers from control and treated rats. However, cytosols from treated animals showed severalfold more inhibition of microsome mediated AFB1 binding to either exogenous or endogenous DNA than cytosols from controls. Presence of 1 mM level of either trichloropropene oxide or styrene oxide partially reversed the cytosolic inhibition of binding. Intraperitoneal administration of AFB1 2h before killing produced 50% less AFB1 binding to nuclear DNA in treated than in control animals. The role of induced glutathione S-transferases in treated rats in modulating hepatic AFB1-DNA binding is discussed.

Differential effects on the metabolism of dimethylnitrosamine and aflatoxin B1 by hepatic microsomes from senescent rats.

The ability of hepatic microsomes from senescent rats to metabolize the two potent hepatocarcinogens dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) was investigated. Seven and 24-month-old male Sprague-Dawley rats were used. Liver weights, and microsomal protein per gram tissue weight were higher, whereas cytochrome P-450 and cytochrome b5 were significantly lower in older rats. Glutathione S-transferases and NADPH cytochrome c reductase activities were dramatically reduced in senescent rats. There was no difference in the formation of formaldehyde from DMN in vitro (31 vs. 34 pmol/nmol P-450) between the young and old rats. In contrast, increased microsome mediated binding of AFB1 to DNA was observed in older rats (116 vs. 228 pmol/nmol P-450) suggesting the possibility of either quantitative or qualitative changes in P-450 species. Additionally the cytoplasmic GSH S-transferases from older rats affected lower inhibition of binding of AFB1 to DNA. These results indicated differential abilities in the hepatic microsomal metabolism of these two carcinogens which may cause differential effects of these carcinogens in senescent rats.

Effect of purified rat and hamster hepatic glutathione S-transferases on the microsome mediated binding of aflatoxin B1 to DNA.

Rat and hamster liver cytosolic glutathione (GSH) S-transferases purified by GSH-affinity chromatography have been examined for their effects on the microsome mediated binding of aflatoxin B1 (AFB1) to DNA and on the conjugation of AFB1-2,3-epoxide with GSH. Like previous studies with cytosolic preparations (Raj et al. (1984) Carcinogenesis 5, 879), our present study with purified GSH S-transferases showed 2-3-fold more inhibitory activity of AFB1-DNA binding with hamster than that with the rat. Concomitant with the inhibition of AFB1-DNA binding, increase in AFB1-GSH conjugation occurred. Subunit compositions of GSH S-transferases indicate preponderance of Yb and Ya subunits in the hamster and rat, respectively. The role of GSH S-transferases in modulating AFB1-DNA binding and AFB1 induced hepatocarcinogenesis is discussed.

Species and sex differences of aflatoxin B1-induced glutathione S-transferase placental form in single hepatocytes.

Species and sex differences of aflatoxin B1 (AFB1)-induced glutathione S-transferase placental form (GST-P) positive single hepatocytes have been investigated 48 h after an intraperitoneal injection of AFB1 to young male and female Fischer rats (2 mg AFB1/kg body wt) and male Syrian golden hamsters (6 mg AFB1/kg body wt). The presence of GST-P positive hepatocytes was examined by the immunohistochemical method. Male rats formed three times as many AFB1-induced GST-P positive hepatocytes as females. Pretreatment of both male and female rats with an inhibitor of GSH synthesis, buthionine sulfoximine (BSO) (4 mmol/kg body wt), 2 h and 4 h before AFB1 injection increased AFB1-induced GST-P positive hepatocytes by about 120% above the controls. Male hamsters formed several-fold less AFB1-induced GST-P positive hepatocytes than male rats. Pretreatment with BSO did not increase AFB1-induced GST-P positive hepatocytes in hamsters even though it produced an increase in hepatic necrosis. It appears that GSH and GSH S-transferases play an important role in modulating hepatic AFB1-DNA binding and AFB1-induced GST-P positive hepatocytes in rats and hamsters.

Antibody inhibition of oxidative activation and inactivation of the carcinogen 2-acetylaminofluorene by purified hepatic cytochrome P-450 from 3-methylcholanthrene pretreated rats.

Immunochemical studies on metabolic N- and ring hydroxylation of 2-acetylaminofluorene (AAF) were performed with total cytochrome P-450 isozymes and with highly purified P-450 D isozyme isolated from liver microsomes from 3-methylcholanthrene (MC)-pretreated rats. In a reconstituted system with rat P-450 D, addition of antibodies against beta-naphthoflavone (BNF) induced rat P-450 B at 15 mg IgG/nmol P-450 inhibited both oxidations completely. With total P-450 isozymes in a reconstituted system, antibody against BNF-rat-P-450 B at 20 mg IgG/nmol P-450 inhibited both oxidations up to 70-80% only. At these concentrations, preimmune antibody or phenobarbital (PB) induced antibody against rat-P-450 B showed no inhibition of AAF oxidations. These results suggest that P-450 D is the predominant cytochrome P-450 isozyme responsible for AAF N- and ring-oxidations in liver microsomes from MC-pretreated rats. Other P-450 isozymes are also suggested to be involved in AAF oxidations.

Inhibition of microsome-mediated binding of aflatoxin B1 to DNA by glutathione S-transferase.

Hamster liver microsome-mediated [14C]aflatoxin B1 binding to DNA is inhibited by 105,000 X g liver supernatant fraction. Absolute requirement of glutathione for the inhibitory activity of the dialyzed supernatant and irreversible loss of the inhibitory activity by the heat-treated supernatant indicate involvement of glutathione S-transferase. Concomitant with inhibition of aflatoxin B1-DNA binding, the formation of an aflatoxin B1-glutathione conjugate is indicated.

Alkylation of protein by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro.

Methylation of horse heart cytochrome c has been examined in vitro with [methyl-14C]methanesulfonate (MMS) and [1-methyl-14C]-1-nitrosourea (MNU) as alkylating agents. Analysis of protein hydrolyzates by an automatic amino acid analyzer indicates that, at pH 9.0 with MMS, epsilon-N-monomethyl-lysine is found to be the only major methylated basic amino acid. On the other hand, the identity of the predominant basic amino acid residue which is [methyl-14C]-labeled by MNU cannot be determined at present. Peptide mapping of chymotryptic digests of cytochrome c after reaction with MMS reveals a lack of specificity in methylation of a specific lysine residue in this hemoprotein.

p53 protein expression in patients with hepatocellular carcinoma from the high incidence area of Guangxi, Southern China.

Mutation of the p53 gene has been reported in hepatocellular carcinoma (HCC) occurring worldwide. The most frequent p53 mutation has been found in HCCs in regions with high hepatitis B virus (HBV) infection and intake of aflatoxin B1 (AFB1). The aim of our study was to examine p53 protein expression in HCCs from a high incidence area of Guangxi, Southern China, where HBV infection and dietary intake of AFB1 are high. Immunohistochemical staining of p53 protein was carried out using a polyclonal rabbit antibody (CM-1). Serial sections were also stained for hepatitis B surface antigen and core antigen. p53 Protein expression was detected in 13 (43.3%) of the 30 HCCs. Expression of p53 was found in 25.0% (1/4) of the < or = 5.0 cm diameter HCCs, in 36.8% (7/19) of the 5.1-10.0 cm diameter HCCs and in 71.4% (5/7) of the >10.0 cm diameter HCCs. Expression of p53 was observed more in moderately and poorly differentiated than in the well differentiated HCCs and more frequently seen in HCCs from younger patients. These data indicate that there is a close association between p53 protein expression and tumor size, histological grade and age of patients. Twenty-seven out of 30 cases (90.0%) were positive for HBV. No significant association between p53 expression and sex. HBV infection, cirrhosis or alpha-fetoprotein has been found.

Inhibition of aflatoxin B1-induced initiation of hepatocarcinogenesis in the rat by green tea.

Several studies have demonstrated that green tea (GT) inhibits various chemically induced cancers in experimental animals. In the present study, effect of GT has been examined on the initiation of aflatoxin B1 (AFB1)-induced hepatocarcinogenesis in the rat. Young male Fischer rats were given AIN-76A diet with or without 0.5% instant GT powder in their drinking water for 2 or 4 weeks. Initiation was examined by hepatic AFB1-DNA binding in vivo, AFB1 metabolism in vitro and by the appearance of AFB1-induced glutathione S-transferase placental form (GST-P)-positive hepatocytes detected by immunohistochemical method. There was no influence of GT feeding on microsome-mediated AFB1 binding to exogenous DNA. However, GT feeding enhanced microsome-mediated formation of non-toxic hydroxylated metabolites of AFB1 by 2-3-fold. Hepatic nuclear AFB1-DNA binding in vivo was significantly inhibited by about 20-30% in animals pretreated with GT: AFB1-induced GST-P positive single hepatocytes were inhibited significantly by 60-70% in rats pretreated with GT. These results suggest that feeding of GT inhibits initiation of AFB1-induced hepatocarcinogenesis in the rat by modulation of AFB1 metabolism, thereby inhibiting AFB1-DNA binding and AFB1-induced GST-P-positive hepatocytes.