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.     

Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes.

Activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) produced methylating species and two aldehydes: formaldehyde and 4-oxo-4-(3-pyridyl)-butanal (OPB). We investigated the modulation by glutathione of single-strand breaks (SSB) generated by N-methyl-N-nitrosourea (MNU) and the two aldehydes. Hepatocytes were simultaneously exposed to 0.2 mM MNU and to 0-2.00 mM formaldehyde or OPB for 4 h. Both aldehydes induced SSB in a dose-dependent manner. Formaldehyde and OPB exerted a synergistic effect on the formation of DNA SSB by MNU. It is postulated that both aldehydes interfere with DNA repair processes and thus increase the genotoxic effect of DNA methylating species. We investigated whether glutathione (GSH) could protect DNA from NNK-derived intermediates. Formaldehyde (2 mM) and OPB (2 mM) decreased intracellular GSH contents to 60 and 86% of control respectively. DL-Buthionine-[S,R]-sulfoximine (BSO) treatment reduced the GSH contents of hepatocytes to 19% of control but did not reduce the content of cytochrome P450 nor the metabolism of NNK. The frequency of DNA SSB induced by NNK, formaldehyde or OPB was significantly higher in GSH-depleted hepatocytes. GSH repletion with GSH monoethyl ester returned NNK-induced SSB to its initial frequency. OPB but not NNK nor formaldehyde induced double-strand breaks. We conclude that OPB and formaldehyde inhibit the repair of DNA damage induced by methylating species and that GSH reduces the level of DNA damage induced by NNK-derived reactive metabolites.     

Inhibitory effect of geniposide on aflatoxin B1-induced DNA repair synthesis in primary cultured rat hepatocytes.

We have previously demonstrated that geniposide (GP) inhibits the aflatoxin B1 (AFB1) induced-hepatotoxicity and hepatic DNA binding in rats. To address the mechanism of action, the effects of GP on AFB1-induced DNA repair synthesis and AFB1 biotransformation in cultured rat hepatocytes were investigated. By evaluation of unscheduled DNA synthesis (UDS), GP reduced AFB1-induced DNA repair synthesis in a dose-dependent manner in hepatocyte cultures. GP elevates the metabolism of AFB1 and decreases the formation of AFM1. The enzyme activities of glutathione S-transferase (GST) and GSH-peroxidase (GSH-Px) in AFB1-treated hepatocyte cultures are enhanced in the presence of GP. GP reduces AFB1-induced DNA repair synthesis through an increased AFB1 detoxication metabolism. It provides one possible mechanism for the chemopreventive activity of GP.     

Aflatoxin B1-DNA binding and aflatoxin B1-glutathione conjugation with isolated hepatocytes from rats and hamsters

Binding of aflatoxin B1 (AFB1) to DNA and AFB1-glutathione conjugation during the metabolism of AFB1 have been examined with freshly isolated hepatocytes from male Fischer rats and Syrian hamsters. Even though there was no significant difference in cytochrome P450 and glutathione contents, there were marked differences in the metabolism of AFB1 (33 nM) in hepatocytes from these two species. Thus, AFB1-DNA binding was six-fold higher in the rat than in hamster hepatocytes, whereas AFB1-glutathione conjugation was 12-fold higher in hamster than in rat hepatocytes. The addition of 0.5 mM diethylmaleate had no significant effect in rats, whereas its presence produced a nine-fold increase in AFB1-DNA binding with 85% inhibition of thiol conjugation in hamster hepatocytes. Styrene oxide (1 mM) produced 50% and 25-fold increases in AFB1-DNA binding in rat and hamster hepatocytes, respectively, with corresponding decreases in thiol conjugation. Triethyltin bromide (50 microM) inhibited both processes by 50% in rat hepatocytes, whereas it produced a nine-fold increase in AFB1-DNA binding with a concomitant decrease in thiol conjugation in hamster hepatocytes. These results suggest that glutathione S-transferases play a more significant role in modulating AFB1-DNA binding in hamster than in rat hepatocytes.     

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.     

Glutathione depletion as a determinant of sensitivity of human leukemia cells to cyclophosphamide

The role of glutathione (GSH) as a determinant of cellular sensitivity to the cytotoxic and DNA-damaging effects of cyclophosphamide (CP) was studied in a dual culture system of rat hepatocytes and K562 human chronic myeloid leukemia cells, which have elevated aldehyde dehydrogenase activity with a corresponding insensitivity to activated CP. Exposure of K562 cells to 50 microM DL-buthionine-S,R-sulfoximine for 24 h resulted in a depletion of cellular GSH content to 10% of control values without toxicity. Subsequent 1-h exposure of GSH-depleted cells to activated cyclophosphamide, obtained by incubation of CP with suspension cultures of rat hepatocytes, resulted in a 5-fold potentiation of the cytotoxicity of CP. Alkaline elution analysis of cellular DNA demonstrated that the level of apparent interstrand cross-linking was 3 to 4 times higher in GSH-depleted cells than in nondepleted cells. GSH-depleted cells were, in addition, more sensitive to induction of DNA single strand breaks than nondepleted cells. Depletion of GSH content did not increase cellular sensitivity to the cytotoxicity of phosphoramide mustard. Preincubation of K562 cells with 1 mM cysteine for 4 h resulted in an approximately 60% increase in cellular GSH content, which was accompanied by decreased sensitivity to the cytotoxicity of hepatocyte-activated CP. Exposure of nondepleted cells to clinically relevant concentrations of hepatocyte-activated CP resulted in depletion of cellular GSH content. Replenishment of GSH content in these cells was relatively slow following CP exposure. Acrolein was highly effective at depleting cellular GSH content, whereas phosphoramide mustard had no effect on cellular GSH content. The depletion of GSH by intracellularly released acrolein may be important in the mechanism of cytotoxicity of CP.     

Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice

Methylene chloride (MC) induced DNA damage in freshly isolatedhepatocytes from mice and rats, which was detectable as single-strand(ss) breaks by alkaline elution. The lowest in vitro concentrationof MC needed to induce DNA damage in mouse hepatocytes (0.4mM) was much lower than for rat hepatocytes (30 mM), and isclose to the calculated steady- state concentration of MC inthe mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m.by inhalation). DNA ss breaks were also detectable in hepatocyteDNA from mice which had inhaled 4000 p.p.m. MC for 6 h, butnot in bepatocyte DNA from rats similarly exposed. In studieswith hepatocytes cultured overnight in the presence of buthioninesulfoximine to deplete glutathione (GSH), subsequent exposureto MC resulted in less DNA damage in the GSH-depleted cells.This shows that conjugation of MC with GSH is important in itsactivation to DNA-damaging species in the liver. The GSH pathwayof MC metabolism produces two potential DNA- damaging species,formaldehyde and S-chloromethylgluta thione (GSCH Formaldehydeis knovm to cause DNA ss breaks in cells. However, the lowestconcentration of formaldehyde required to induce a significantamount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikelyto be formed following in vitro or in vivo metabolism of MCat concentrations that induce similar amounts of DNA damage.That formaldehyde does not play a role in this DNA damage hasbeen confirmed in experiments with CHO cells exposed to MC andan exogenous activation system from mouse liver (S9 fraction).Formaldehyde was responsible for the DNA– protein cross-linkingeffect of MC, but did not cause the DNA damage leading to ssbreaks. These DNA ss breaks are likely to be caused by GSCH₂ClThe results suggest a genotoxic mechanism for MC carcinogenicityin the mouse liver, and support the proposal that the observedspecies differences in liver carcinogenicity result from differencesin the amount of MC metabolism via the GSH pathway in the targetorgan.     

Grapefruit juice intake does not enhance but rather protects against aflatoxin B1-induced liver DNA damage through a reduction in hepatic CYP3A activity

Influence of grapefruit juice intake on aflatoxin B1 (AFB1)-induced liver DNA damage was examined using a Comet assay in F344 rats given 5 mg/kg AFB1 by gavage. Rats allowed free access to grapefruit juice for 5 days prior to AFB1 administration resulted in clearly reduced DNA damage in liver, to 65% of the level in rats that did not receive grapefruit juice. Furthermore, rats treated with grapefruit juice extract (100 mg/kg per os) for 5 days prior to AFB1 treatment also reduced the DNA damage to 74% of the level in rats that did not receive grapefruit juice. No significant differences in the portal blood and liver concentrations of AFB1 were observed between grapefruit juice intake rats and the controls. In an Ames assay with AFB1 using Salmonella typhimurium TA98, lower numbers of revertant colonies were detected with hepatic microsomes prepared from rats administered grapefruit juice, compared with those from control rats. Microsomal testosterone 6beta-hydroxylation was also lower with rats given grapefruit juice than with control rats. Immunoblot analyses showed a significant decrease in hepatic CYP3A content, but not CYP1A and CYP2C content, in microsomes of grapefruit juice-treated rats than in non-treated rats. No significant difference in hepatic glutathione S-transferase (GST) activity and glutathione content was observed in the two groups. GSTA5 protein was not detected in hepatic cytosol of the two groups. In microsomal systems, grapefruit juice extract inhibited AFB1-induced mutagenesis in the presence of a microsomal activation system from livers of humans as well as rats. These results suggest that grapefruit juice intake suppresses AFB1-induced liver DNA damage through inactivation of the metabolic activation potency for AFB1 in rat liver.     

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)     

Identification of differentially expressed genes in aflatoxin B1- treated cultured primary rat hepatocytes and Fischer 344 rats

Aflatoxin B1 (AFB1), a mutagen and hepatocarcinogen in rats and humans, is a contaminant of the human food supply, particularly in parts of Africa and Asia. AFB1-induced changes in gene expression may play a part in the development of the toxic, immunosuppressive and carcinogenic properties of this fungal metabolite. An understanding of the-role of AFB1 in modulating gene regulation should provide insight regarding mechanisms of AFB1-induced carcinogenesis. We used three PCR- based subtractive techniques to identify AFB1-responsive genes in cultured primary rat hepatocyte RNA: differential display PCR (DD-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH). Each of the three techniques identified AFB1- responsive genes, although no individual cDNA was isolated by more than one technique. Nine cDNAs isolated using DD-PCR, RDA or SSH were found to represent eight genes that are differentially expressed as a result of AFB1 exposure. Genes whose mRNA levels were increased in cultured primary rat hepatocytes after AFB1 treatment were corticosteroid binding globulin (CBG), cytochrome P450 4F1 (CYP4F1), alpha-2 microglobulin, C4b-binding protein (C4BP), serum amyloid A-2 and glutathione S-transferase Yb2 (GST). Transferrin and a small CYP3A-like cDNA had reduced mRNA levels after AFB1 exposure. Full-length CYP3A mRNA levels were increased. When liver RNA from AFB1-treated male F344 rats was evaluated for transferrin, CBG, GST, CYP3A and CYP4F1 expression, a decrease in transferrin mRNA and an increase in CBG, GST, CYP3A and CYP4F1 mRNA levels was also seen. Analysis of the potential function of these genes in maintaining cellular homeostasis suggests that their differential expression could contribute to the toxicity associated with AFB1 exposure.      

Comparison of aflatoxin B1-DNA binding and glutathione conjugate formation by liver preparations from rats of different ages.

The capability of the newborn rat liver to detoxify aflatoxin B1 (AFB1), a potent hepatocarcinogen is not well understood. Our present results show that immature rats are deficient in the hepatic key factors involved in biotransformation of AFB1. The activities of cytosolic glutathione S-transferases and microsomal cytochrome P-450 along with cellular glutathione (GSH) content show postnatal developmental changes. The ability of hepatic subcellular preparation from newborn rats to convert AFB1 to its reactive epoxide form, is reported for the first time in this communication. Epoxidation of [3H]AFB1 in the presence of liver microsomes from different age-groups as measured by its adduct formation to calf thymus DNA in vitro shows that newborn rats are capable of catalyzing only minimal AFB1-DNA binding compared with that of adults. Addition of cytosolic fraction of various age groups to the system suggests that young rats are less efficient in modulating the binding as compared with adults. The amount of AFB1-GSH conjugate formed is also significantly higher when adult GSH S-transferase is involved in the system. These observations show that immature liver is less efficient than a mature organ in handling a chemical carcinogen and the metabolism of AFB1 by neonatal liver differs from that in the adult.     

Glutathione depletion causes cell growth inhibition and enhanced apoptosis in pancreatic cancer cells

BACKGROUND: Recent studies have demonstrated that various tumors express enhanced levels of the radical scavenger glutathione (GSH). Moreover, there are grounds for claiming that GSH plays a crucial role in cell proliferation and tumor resistance. In the current study, we investigated the relation between cell growth and GSH levels in the pancreatic adenocarcinoma cell line, AsPC-1, and the significance of GSH in tumor resistance to chemotherapy. METHODS: Cell growth in AsPC-1 was initiated through transforming growth factor-alpha (TGF-alpha) or fetal calf serum (FCS). Then, cell cycle, cell proliferation, and cellular GSH content were analyzed at different times in the presence or absence of buthionine sulfoximine (BSO). The impact of GSH on chemotherapy-induced apoptosis was studied using 5-fluorouracil or melphalan in the presence or absence of BSO. Finally, we compared the GSH content of 15 pancreatic tumor specimens with 10 normal pancreatic tissue specimens. RESULTS: Analysis of GSH in pancreatic tissues demonstrated increased GSH levels in cancerous compared with normal tissue (17.5 +/- 2.3 vs. 8. 8 +/- 1.4 nmol/mg protein; P < 0.004). Incubation of AsPC-1 with TGF-alpha or FCS resulted in cell proliferation and cell cycle activity, whereas GSH content was not altered. Incubation of GSH-depleted cells with TGF-alpha did not stimulate cell growth. In addition, GSH-depletion resulted in an increased rate of apoptosis after melphalan (6.3 +/- 0.3 % vs. 11.2 +/- 0.3 %; P < 0.001), but not after 5-fluorouracil treatment. CONCLUSIONS: Taken together, our results show enhanced GSH levels in pancreatic carcinoma and an essential role of GSH in cell proliferation and in resistance of AsPC-1 cells. Therefore, GSH-depletion may improve the efficacy of adjuvant therapy in pancreatic carcinoma.     

Glutathione depletion and cytotoxicity of buthionine sulphoximine and SR2508 in rodent and human cells

SR2508 (1 mM) increases the rate of glutathione (GSH) depletion by L-buthionine-S-R-sulphoximine (BSO) in hypoxic V79 rodent and A549 human cells. Specifically, the GSH content for V79 and A549 cells, after incubating for about 6 hr with 50 and 100 microM BSO, respectively, was lower by at least 10-fold when 1 mM SR2508 was present. In addition, 1 mM SR2508 is extremely toxic to hypoxic cells with lower GSH content. Survival probabilities of GSH-depleted V79 and A549 cells are about 10(-3) after 10 hr incubation with 1 mM SR2508. By itself, 1 mM SR2508 or 50-100 microM BSO decreased cellular viability by about 50% with a 10 hr treatment period. Both the phenomena described above are preferential towards hypoxic cells with minimal effect on aerobic cells.     

Comparative effects of three carcinogens on human, rat and mouse hepatocytes

Ultrastructural changes commonly observed in liver cells of rodents exposed to carcinogens in vivo can be induced in hepatocytes exposed to carcinogens in vitro. Human, rat and mouse hepatocytes in primary culture were treated with actinomycin D, aflatoxin B1 (AFB1) and dimethylnitrosamine (DMN). These cultured hepatocytes were examined for ultrastructural alterations following carcinogen exposure for 24 h. Similar to the effects on liver cells in vivo, the most prominent change was a segregation of the nucleolar components. Human, rat and mouse hepatocytes, dosed with 7.9 X 10(-8) M actinomycin D, developed nucleolar segregation in 86%, 98% and 55% of cells, respectively. When incubated with 3.2 X 10(-6) M AFB1, 60% of human and 84% of rat hepatocytes developed nucleolar segregation. However, exposures of mouse hepatocytes less than or equal to 3.2 X 10(-5) M of AFB1 failed to induce segregation of the nucleolus. DMN administered at a dose of 2.0 X 10(-2) M caused segregation in 11% of the rat hepatocytes and in 60% of the mouse hepatocytes. Distinct nucleolar segregation did not occur in human hepatocytes until they were exposed to a concentration of 5.0 X 10(-2) M DMN (31%). Actinomycin D, AFB1, DMN, as well as other compounds that bind to DNA and interfere with template activity cause nucleolar segregation. Morphologic changes observed in cultured rat and mouse hepatocytes correlate well with in vivo experiments with regard to the relative sensitivity of rats and mice to toxicological effects of these carcinogens. Thus, hepatocyte cultures may provide a realistic system to determine the sensitivity of human liver cells to carcinogens.     

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.     

Effect of allixin, a phytoalexin produced by garlic, on mutagenesis, DNA-binding and metabolism of aflatoxin B1

Allixin, a phytoalexin isolated from garlic, was examined for its effects on aflatoxin B1(AFB1)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver S9 fraction as the metabolic activation system. The effects of allixin on the binding of [3H]AFB1 to calf thymus DNA and on the formation of metabolites of [3H]AFB1 were also determined. Allixin showed a dose-related inhibition of Histidine+ revertants induced by AFB1. Allixin at 75 micrograms/ml inhibited [3H]AFB1 binding to calf thymus DNA and reduced formation of AFB1-DNA adducts. In addition, allixin exhibited a concentration-dependent inhibition of the formation of organosoluble metabolites and the glutathione conjugates of [3H]AFB1. The data indicate that the effect of allixin on AFB1-induced mutagenesis and binding of metabolites to DNA may be mediated through an inhibition of microsomal P-450 enzymes. Allixin may thus be useful in the chemoprevention of cancer.     

The importance of glutathione and glutathione transferase for somatic mutations in Drosophila melanogaster induced in vivo by 1,2-dichloroethane

Two principal pathways of metabolism of the carcinogenic compoundl,2-dichloroethane (DCE) have been proposed. One is a mixedfunction oxidase dependent pathway requiring oxygen and NADPH.The other pathway depends on the presence of glutathione (GSH)and glutathione transferase (GST). The aim of this study wasto investigate the role of the latter pathway for the in vivomutagenicity of DCE in the somatic wing spot test in Drosophilamelanogaster. DCE caused a dose-dependent increase of wing spots.In order to investigate the role of cellular GSH for the mutagenicity,the level of GSH was decreased by 24 h pretreatment with buthioninesulfoximine (BSO), an efficient inhibitor of GSH synthesis.This pretreatment decreased the GSH level to 6% as comparedto the control. The pretreatment also resulted in a significantdecrease of the mutagenicity of DCE. Treat ment of the larvaewith phenobarbiturate (PB) resulted in 200% induction of cytosolicGST, and a corresponding increase in the DCE mutagenicity. Theseresults indicate that the important pathway in vivo for themutagenicity of DCE is dependent on GSH and GST. A similar experimentalprotocol was used to study interactions between aflatoxin B₁(AFB) and GSH and GST. No effect of the treatment with BSO onthe mutagenicity of AFB was observed, while pretreatment withPB caused a decrease of the mutagenicity of AFB.     

Susceptibility to aflatoxin B1-induced carcinogenesis correlates with tissue-specific differences in DNA repair activity in mouse and in rat

To investigate the mechanisms responsible for species- and tissue-specific differences in susceptibility to aflatoxin B(1) (AFB(1))-induced carcinogenesis, DNA repair activities of nuclear extracts from whole mouse lung and liver and rat liver were compared, and the ability of in vivo treatment of mice with AFB(1) to alter repair of AFB(1)-DNA damage was determined. Plasmid DNA containing AFB(1)-N(7)-guanine or AFB(1)-formamidopyrimidine adducts were used as substrates for the in vitro determination of DNA repair synthesis activity, detected as incorporation of radiolabeled nucleotides. Liver extracts from CD-1 mice repaired AFB(1)-N(7)-guanine and AFB(1)-formamidopyrimidine adducts 5- and 30-fold more effectively than did mouse lung, and approximately 6- and 4-fold more effectively than did liver extracts from Sprague-Dawley rats. The susceptibility of mouse lung and rat liver to AFB(1)-induced carcinogenesis correlated with lower DNA repair activity of these tissues relative to mouse liver. Lung extracts prepared from mice treated with a single tumorigenic dose of 50 mg/kg AFB(1) i.p. and euthanized 2 hours post-dosing showed minimal incision and repair synthesis activities relative to extracts from vehicle-treated mice. Conversely, repair activity towards AFB(1)-N(7)-guanine damage was approximately 3.5-fold higher in liver of AFB(1)-treated mice relative to control. This is the first study to show that in vivo treatment with AFB(1) can lead to a tissue-specific induction in DNA repair. The results suggest that lower DNA repair activity, sensitivity of mouse lung to inhibition by AFB(1), and selective induction of repair in liver contribute to the susceptibility of mice to AFB(1)-induced lung tumorigenesis relative to hepatocarcinogenesis.     

Formation of the DNA adduct S-[2-(N7-guanyl)ethyl]glutathione from ethylene dibromide: effects of modulation of glutathione and glutathione S-transferase levels and lack of a role for sulfation

Hepatic S-[2-(N7-guanyl)ethyl]glutathione DNA adducts were determined in several strains of rats and mice after i.p. injection of a dose of 37 mg ethylene dibromide/kg body wt. More adducts were formed in rats than in mice, while no difference was noted among strains within each species. Removal of adducts in liver DNA was relatively slow in all animals tested. On the contrary, in vitro incubation of calf thymus DNA with ethylene dibromide and either rat cytosol or mouse cytosol gave rise to similar amounts of adduct, yet mouse cytosol showed much higher glutathione (GSH) S-transferase activity toward 1-chloro-2,4-dinitrobenzene. Human cytosol also activated ethylene dibromide, with the extent of conjugation being approximately half that of rat cytosol. Pretreatment of rats with phenobarbital or beta-naphthoflavone induced GSH S-transferases but did not increase the in vivo formation of DNA adducts, suggesting that concomitant induction of cytochrome P450 might abolish the effect of induction of GSH S-transferase by increasing the oxidation of ethylene dibromide. Butylated hydroxytoluene induced GSH S-transferase and also markedly increased DNA adduct levels. Disulfiram, a known cytochrome P450 inhibitor, significantly increased the formation of DNA adducts whereas it did not affect GSH S-transferase activity. Depletion of GSH by pretreatment of rats with diethylmaleate or buthionine sulfoximine resulted in decreased in vivo DNA adduct levels and the degree of reduction was well correlated with the extent of GSH depletion. In vitro incubation of tritiated S-(2-hydroxyethyl)GSH with calf thymus DNA in the presence of 3'-phosphoadenosine-5'-phosphosulfate and rat liver cytosol did not result in significant binding to DNA, suggesting that sulfation of the alcohol does not readily occur to add a leaving group and regenerate an episulfonium ion. These results suggest that induction of the Phase II enzyme GSH S-transferase can be detrimental in the case of ethylene dibromide and that decreases in GSH levels reduce DNA alkylation in rats.     

Comparative ultrastructural effects of aflatoxin B1 on mouse, rat, and human hepatocytes in primary culture

Human, rat, and mouse hepatocytes in primary culture were treated with aflatoxin B1 (AFB1) and examined for ultrastructural alterations. As early as 1 h following in vitro exposure to AFB1, there were ultrastructural changes in the nuclei of rat and human hepatocytes. The most prominent change in the nuclei was a segregation of nucleolar components that resembled the segregation in liver cells of rats exposed to AFB1 in vivo. The nucleolar segregations were developed by incubating rat hepatocytes for 24 h in a medium containing as little as 0.01 micrograms of AFB1 per ml. The minimum concentration to induce the same change in human hepatocytes was 0.1 micrograms/ml. No distinct nucleolar alteration was observed in mouse hepatocytes incubated in a medium containing 10 micrograms of AFB1 per ml. Irregular nuclear chromatin condensation also developed in the cells exposed to a higher concentration of AFB1, whereas little damage was observed in mitochondria and lysosomes. The similarity in morphological changes between our in vitro model and in vivo models previously investigated indicates that the hepatocytes in primary culture maintain the biological properties necessary for carcinogen responses similar to liver cells in vivo. In addition, the morphological changes in cultured rat and mouse hepatocytes induced by AFB1 correlate with in vivo experiments insofar as mice are relatively resistant, whereas rats are sensitive to AFB1 carcinogenesis. Thus, cultured hepatocyte systems may be a valuable tool to study genetic damage which may lead to hepatocellular carcinomas in human and animal livers.