Microsomally-mediated denitrosation of nitrosoguanidinium compounds

A major metabolic fate of 1-methyl-2-nitro-1-nitrosoguanidine(MNNG) and nitrosocimetidine (NC) in rodents is denitros ationto generate the unmodified, parent guanidinium compound. MNNGis a potent, locally-acting carcinogen. NC is the nitrosatedderivative of cimetidine, an important clinical drug administeredorally for the treatment of stomach ulcers. Contrary to expectationsbased on the results of various short-term in vitro tests forcarcinogenic potential, NC is not a carcinogen when administeredto rats or mice. Rat liver microsomal enzymes have been foundto be capable of catalyzing the denitrosation of MNNG, NC andan NC analog, 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG)in an NADPIHI-dependent reaction. The denitrosated guanidiniumcompound generated accounts for 50–70% of the nitrosocompound metabolized in a microsomal incubate; nitrite is generatedwith a yield which represents 40–60% of the guanidiniumcompound produced. The cytochrome P450 inhibitors metyrapone,n-octylamine, 1-n-hexylimidazole and ellipticine inhibit theconversion of CyanoDMNG to 1,3-dimethyl-2-cyanoguanidine (CyanoDMG)and nitrite. Microsomal NADPH-cytochrome c reductase activityis not perturbed by this series of organic compound inhibitors.Diethyl maleate at high concentrations weakly stimulates thereaction. The rates of production of the CyanoDMNG degradationproducts CyanoDMG, nitrite and nitrate are markedly diminishedin nitrogen-saturated and in carbon dioxide-saturated microsomalincubates. Preincubating microsomes for 1 h at 37°C priorto substrate and NADPIHI addition has no effect on the denitrosationactivity. Kinetic analysis of the conversion of CyanoDMNG toCyanoDMG indicates a K_m of 1.0 mM and a V_max of 2.7 nmol/min/mgprotein. Microsomes isolated from rats pretreated with the cytochromeP450 inducers pyrazole or phenobarbital show enhanced denitrosationactivity. The denitrosation capacity of hamster liver microsomesis similar to that observed for rat microsomes.     

Evidence for cytosolic glutathione transferase-mediated denitrosation of nitrosocimetidine and 1-methyl-2-nitro-1-nitrosoguanidine

Nitrosocimetidine (NC) and 1-methyl-2-nitro-1-nitrosoguan-idine(MNNG) are closely related N-nitrosamidines. NC is the nitrosatedderivative of cimetidine (Tagamet), an orally administered compoundused extensively in the treatment of gastric ulcers. MNNG isa potent carcinogen capable of initiating tumors close to thesite of administration and used experimentally to produce stomachcancer. It has become evident that the primary metabolic fateof both of these agents is denitrosation. We have discoveredan activity in the cytosol fraction of hamster liver which iscapable of denitrosating these nitrosamidines with an efficiencyapproaching 100%. The activity is heat sensitive and requiresreduced glutathione as a cofactor. Inhibition of the denitrosatingactivity with compounds which inhibit in parallel the conjugationof glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) providesevidence that the activity is glutathione transferase. One moleculeof reduced glutathione is consumed in each denitrosation event.Nitrite is formed as denitrosation proceeds with a yield equivalentto 25–50% of the denitrosated product produced. Glutathionedisulfide is a minor reaction product, representing 3% of thedenitrosation product yield in the MNNG case and 12% in theNC case. Thus far in our survey of N-nitrosamines, N-nifrosamidesand N-nitrosamidines, only the nitrosamidines appear to be vulnerableto the cytosolic denitrosating activity. In an attempt to evaluatethe importance of the glutathione-dependent reaction in theintact hamster, we have depleted glutathione by pretreatmentwith the commonly used agents diethyl maleate (DEM) and L-buthionine-S,R-sulfoximine(L-BSO). Nitroso compound was administered i.v. and the circulatingblood levels of intact and denitrosated compound 5 min afterdosing quantified. NC-and MNNG-derived methylation of organDNA was also monitored. Pretreatment had no effect on the cytosolicdenitrosating or CDNB-conjugating activities. L-BSO pretreatmenthad no apparent effect on the denitrosative metabolism of NCor MNNG. With DEM pretreatment we obtained clear indicationsof a decreased rate of denitrosation and observed a 10-foldincrease in MNNG-derived liver DNA methylation. The differentialeffects of these pretreatments are taken as an indication thatDEM-sensitive proce other than those requiring glutathione dominateN-nitrosamidine denitrosation in the hamster.     

Measurement and characterization of the denitrosation of tauromustine and related nitrosoureas by glutathione transferases in liver cytosol from various species

The denitrosation of a novel nitrosourea, l-(2-chloroethyl)-3-[(2-dimethylaminosulfonyl-ethyl-l-nitrosourea,tauro-mustine (TCNU), has been investigated in liver cytosolfrom various species and with a pool of cytosolc rat liver glutathionetransferases (GSTs). In addition, the denitrosation of relatednitrosoureas, l, 3-bis(2-chloroethyl)-l-nitrosourea (BCNU) andl-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) have beeninvestigated. By using radioactive nitrosoureas as substratesand HPLC to detect the denitrosated products, a sensitive assayhas been developed. The activities towards additional substrates,l-chloro-2, 4-dinitrobenzene (CDNB) and trans-stilbene oxide(tSBO) were also measured in the various cytosol fractions.The apparent V_max and_max values for the denitrosation of TCNUin rat liver cytosol were calculated to be 830 pmol/min/mg proteinand 8.4 mM respectively. The rate of denitrosation of TCNU wasfound to be highest in the mouse, followed by the rat and thedog. The induction of GSTs by phenobarbital in rats increasedthe denitrosation of TCNU similar to the activities found towardsCDNB and tSBO as substrates. The highest denitrosation activityby GST was determined towards BCNU, followed by CCNU and TCNU.Our results suggest that the denitrosation of various nitrosoureasvia GST may play an important role in the deactivating processoccurring in vivo and for the efficacy of nitrosoureas usedin chemotherapy.     

Mutational specificity of chromium(VI) compounds in the hprt locus of Chinese hamster ovary-K1 cells.

Chromium(VI) compounds exert their genotoxicity and mutagenicity by complex metabolic reducing pathways that generate a variety of reactive forms of chromium and free radicals. To investigate the molecular nature of chromium-induced mutations, we characterized the entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 27 independent mutants derived from chromium(VI) oxide (CrO3)-treated Chinese hamster ovary-K1 cells, by direct sequencing of PCR-amplified cDNA. Among these mutants, 10 consisted of single base substitutions, five contained two base substitutions, one had four base substitutions, six were splicing mutations, and five exhibited single base pair insertions or deletions. All of the base substitutions and most of the frameshift mutations observed were located at A/T-rich sequences. More than 90% of the base substitutions (22/24) occurred in A.T base pairs. Among them, T-->A and T-->G transversions (18/22) predominated. The mutational hotspots for single and double base substitutions were the 3' thymidine of 5'PuT and thymidines of 5'ATTT sequences respectively. This mutational specificity was also observed in CHO-K1 cells treated with two other chromium(VI) compounds, namely K2Cr2O7 and PbCrO4. Strand bias was noticed in chromium mutagenicity, since 77% of T base substitutions occurred on the non-transcribed strand. This highly sequence-specific mutation spectrum suggests that a particular form of chromium may directly interact with DNA at these hotspot sequences.     

Effect of endogenous glutathione, superoxide dismutases, catalase, and glutathione peroxidase on adriamycin tolerance of Chinese hamster ovary cells

Based on the concept that activated oxygen species are causally involved in Adriamycin toxicity, endogenous antioxidant defenses are expected to be important determinants of cellular Adriamycin tolerance. We have tested this prediction by making use of an oxygen-resistant variant subline of Chinese hamster ovary cells (CHOr), which is characterized by increased levels of glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase. The levels of antioxidant defenses in wild-type CHO (CHOs) cells were within the range reported for human tumor cell lines, except for catalase, which was comparatively high. Oxygen-tolerant CHOr cells, which contained 4.3-fold more catalase activity than CHOs cells, were proportionally more resistant to H2O2, indicating that catalase activity in wild-type CHOs cells was still limiting H2O2 tolerance. The Adriamycin sensitivity of CHOs cells was compared to that of CHOr cells by clonogenic cell survival. After correcting for differential drug uptake in CHOs and CHOr cells, no significant difference in Adriamycin sensitivity could be detected. Furthermore, drug-induced cyanide-resistant oxygen consumption and electron spin resonance data indicated that both cell strains were equally efficient in reducing Adriamycin to its semiquinone radical and in generating activated oxygen species through oxidation-reduction cycling. These results indicate that Adriamycin tolerance of wild-type CHO cells, as determined by clonogenic cell survival, is not limited by endogenous glutathione, copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, or glutathione peroxidase.     

Inhibition of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase and ornithine decarboxylase activities by glutathione level-raising agents and selenium-containing compounds

The present study was undertaken to determine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter known to inhibit superoxide dismutase (SOD) (superoxide: superoxide oxidoreductase, EC 1.15.1.1) and catalase (CAT) (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) activities, on mouse epidermal glutathione (GSH) peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) activity in vivo and in vitro. TPA led to a rapid and transient increase in GSH peroxidase specific activity within 30 min followed by a decrease from 1 to 12 h. Incubation of isolated epidermal cells with GSH level-raising agents and/or selenium-containing compounds increased remarkably basal GSH peroxidase activity, and thus, abolished totally the prolonged inhibitory effects of TPA on this enzyme. The inhibitory effects of 0.2 mM cysteine (Cys) or 0.5 mM GSH and 2.5 microM Na2 SeO3 or 50 microM selenocystamine on TPA-decreased GSH peroxidase activity were additive, in relation with their additive inhibitory effects on TPA-induced ornithine decarboxylase (ODC) (L-ornithine carboxylase, EC 4.1.1.17) activity. These data support the hypothesis that the stimulators of the GSH-dependent antioxidant protective system of the epidermal cells may inhibit the oxidative challenge linked to skin tumor promotion by TPA.     

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.     

P-Glycoprotein expression in human, mouse, hamster and rat hepatocytes in primary culture

P-Glycoprotein (P-gp), the multidrug resistance (MDR) gene product,has previously been shown to be functional and overexpressedin adult rat hepatocytes during primary culture. In the presentstudy, we examined P-gp expression in human, mouse and hamsterhepatocytes cultured in conditions similar to those used forrat liver cells. Northern blotting and doxorubicin P-gp-mediatedefflux analyses revealed that liver parenchymal cells from thesethree species exhibited only limited increased MDR mRNA levelswith no intracellular decreased drug retention, thus suggestingthat the marked functional P-gp induction observed in rat hepatocytescould reflect a species-specific response of these cells toan unfamiliar environment. An 8 h exposure to cycloheximide,which strongly induced MDR mRNAs in rat liver cells, also increased,although to a far lesser extent, MDR mRNA levels in human, mouseand hamster hepatocytes, indicating that P-gp expression innormal liver parenchymal cells could be at least partly negativelyregulated by a labile protein factor.     

Effects of organosulfur compounds from garlic and onions on benzo[a]pyrene-induced neoplasia and glutathione S-transferase activity in the mouse

In the present study, eight organosulfur compounds from garlic and onions were studied for their inhibitory effects on benzo[a]pyrene (BP)-induced neoplasia of forestomach and lung of female A/J mice when administered 96 and 48 h prior to carcinogen challenge. These compounds had one, two or three linearly connected sulfur atoms. They included the four allyl group-containing derivatives: allyl methyl trisulfide (AMT), allyl methyl disulfide (AMD), diallyl trisulfide (DAT), and diallyl sulfide (DAS), and also four corresponding saturated compounds in which propyl groups were substituted for the allyl groups. All four allylic compounds inhibited BP-induced neoplasia of the forestomach. The saturated analogs were almost without inhibitory activity, indicating the importance of the allyl groups. DAT, which contains two allyl groups, was more potent than AMT, which contains only one allyl group, thus providing further evidence for the role of allyl groups in the inhibitory effects observed. DAS and AMD, but not DAT or AMT, inhibited pulmonary adenoma formation. The fact that in the lung the monosulfide and disulfide inhibited, but the trisulfide did not inhibit, indicates that the number of sulfur atoms in the molecule can control the organ sites at which protection against carcinogenesis will occur. All four allylic compounds induced increased glutathione S-transferase (GST) activity in the forestomach, but varied in their capacity to induce GST in lung, liver and small bowel. Their saturated analogs produced little or no induction. In evaluating relationships between diet and cancer, it would be useful to consider the possible role of garlic and onion organosulfur compounds as protective agents. In addition, further studies of this class of chemicals might lead to the identification and development of useful new chemopreventive compounds.     

Altered mouse bone marrow glutathione and glutathione transferase levels in response to cytotoxins

The mouse bone marrow has been used as a model for the investigation of the response of cells to cytotoxins and carcinogens. The effects of cyclophosphamide, 1-beta-D-arabinofuranosylcytosine, and X-irradiation on the levels of glutathione and glutathione transferases have been studied. A high dose of cyclophosphamide (500 mg/kg) caused a significant depletion of glutathione levels in marrow, liver, and blood. A lower dose, 75 mg/kg, caused a similar depletion but only in marrow and liver. In this case, 5 to 7 days following treatment, the glutathione content of surviving cells was 1.8- to 3-fold higher than in controls. Glutathione transferase activity was also increased at this time (2- to 3-fold). 1-beta-D-Arabinofuranosylcytosine and X-irradiation also caused a depletion of marrow glutathione and glutathione transferase levels followed increased cellular levels (approximately 2-fold) 3 to 4 days later. Animals given cyclophosphamide (75 mg/kg) survived an otherwise lethal dose of this compound administered 5 to 7 days later. The time course of this effect closely paralleled the higher glutathione and glutathione transferase levels, suggesting a correlation between these effects.     

Use of monochlorobimane for glutathione measurements in hamster and human tumor cell lines

The use of monochlorobimane (MCIB) as a fluorescence label for glutathione (GSH) quantitation was investigated in human tumor cell lines. When MCIB was used with a hamster fibroblast cell line under conditions where GSH was either depleted or elevated, an excellent correlation between bimane-GSH fluorescence and the standard cyclic GSH reductase assay (Tietze's) was accomplished. When the MCIB technique was applied to a human lung adenocarcinoma cell line, little or no GSH labeling was noted even at MCIB levels 10X higher than that used for the hamster line. HPLC analysis suggested that the source of the problem may be the affinity for MCIB to glutathione S-transferase. By using higher dye concentrations and longer staining times, adequate staining was possible. While the MCIB technique may have problems quantitating GSH levels between cell types, the possibility of examining GSH heterogeneity in solid tumor biopsies remains feasible.     

Kinetic isotope effect on the demethylation and denitrosation of N-nitrosodimethylamine in vitro

Deuteration of N-nitrosodimethylamine (NDMA) has been shown to decrease the carcinogenicity of this compound. This result is believed to be due to a kinetic isotope effect on the metabolic activation of this carcinogen, but conflicting views exist concerning whether the isotope substitution affects the Km or Vmax of the reaction. In order to elucidate the molecular basis of these observations, as well as the mechanisms of the demethylation and denitrosation reactions, the metabolism of NDMA and deuterated NDMA (NDMA-d6) was studied using acetone-induced rat-liver microsomes. The demethylation of NDMA displayed a Km of 0.06 mM and a Vmax of 7.9 nmol/min per mg protein. Deuteration of NDMA increased the Km value by five fold but did not appreciably affect the Vmax. The denitrosation of NDMA also displayed a Km of 0.06 mM, but the Vmax was 0.83 nmol/min per mg; deuteration again increased the Kmax several fold but had no effect on the Vmax. The results indicate that deuteration inhibits the metabolism of NDMA by increasing the Km but not the Vmax and suggest that there is a close relationship between the demethylation and denitrosation reactions.     

Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues

This study was performed to elucidate the lung glutathione-related defense potential in tumoral tissues. Reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase (GR), selenium-dependent (SeGPx) and total glutathione peroxidase (tGPx), and glutathione S-transferase (GST) activities in 38 tumoral lung tissues and 17 normal lung tissues were determined to obtain a comprehensive profile of the lung glutathione and glutathione-related enzymes in cancer. The enzyme levels in tumoral tissues (n = 38) were found to be significantly higher (P < 0.05) than those in normal tissues (n = 17). Reduced glutathione levels, and not oxidized glutathione levels, were found to be higher in normal tissues than those in tumoral tissues. We found no statistically significant difference between the adenocarcinoma and squamous cell carcinoma groups for any of the parameters studied.     

Dietary energy restriction-induced modulation of protein kinase C ζ isozyme in the hamster pancreas

Dietary restriction in experimental animals enhances life span, delays disease, inhibits immunological perturbations, and ameliorates cancer. Protein kinase C(a) isozymes mediate signals generated by hormones, growth factors, and neurotransmitters for cell proliferation and differentiation. The results of our study showed that a C-terminally directed anti-PKC ζ antibody detected an 81–kDa band in the pancreases of control and energy-restricted hamsters. Syrian golden hamsters were fed energy-restricted diets formulated such that the hamsters received 90%(10% energy restriction(a) ), 80%(20% ER), or 60%(40% ER) of the total energy consumed by control hamsters, with the energy reduced proportionally from fat and carbohydrate. ER decreased PKC ζ isozyme levels by 40–75% in hamsters fed 10, 20, and 40% ER diets for 8 wk. PKC ζ isozyme expression was decreased by 75–80% in hamsters fed ER diets for 15 wk. Although ER caused significant decreases in PKC ζ isozyme levels compared with those of control hamsters at both time points, the relative differences in PKC ζ levels between the dietary ER groups(10, 20, and 40%) were small and not significant. A significant decrease in the body weights of ER animals compared with those of controls was observed at both time points. No differences in tomato lectin and phytohemagglutinin reactivity were observed between control animals and animals fed 10, 20, and 40% ER diets. Furthermore, the cellular expression of PKC ζ in the hamster pancreas did not differ among hamsters fed the various ER diets. These observations may be important for understanding not only the role of dietary ER in pancreatic cancers but also PKC ζ signal transduction mechanisms in normal pancreatic physiology.© 1995 Wiley-Liss, Inc     

Role of glutathione peroxidase in the radiation response of mouse kidney

Glutathione peroxidase (GSH-Px) has been implicated in mediating the radioprotective effects of glutathione (GSH). This hypothesis was tested in vivo by determining the effect of GSH-Px depletion on the radiation response of murine kidneys. Renal GSH-Px levels were depleted to 17% of control values by feeding animals a selenium deficient diet for 6 weeks; this had no significant effect on renal levels of GSH or GSH-S-transferase (GST). However, we also tested the effect of direct depletion of GSH to 3-4% of control values, using a combination of DL-buthionine sulphoximine (BSO) and diethyl maleate (DEM). Kidneys with normal or depleted levels of GSH-Px and/or GSH were irradiated with 240kVp X rays (2 fractions, 7 days apart to minimize intestinal injury). Mice breathed 7% oxygen during irradiation. Renal damage was assessed at 20, 25, and 32 weeks after the first fraction of X rays, in terms of reduced hematocrit and renal clearance of 51Cr-EDTA. Depletion of GSH-Px levels to 17% of control did not alter renal radiosensitivity, but depletion of GSH to 3-4% of control values radiosensitized the kidney by a factor of 1.4. Depletion of both GSH and GSH-Px together did not radiosensitize the kidney any more than was achieved by GSH depletion alone.     

Effects of disulfiram, diethyldithiocarbamate, bisethylxanthogen, and benzyl isothiocyanate on glutathione transferase activities in mouse organs

Four sulfur compounds known to inhibit tumorigenic effects of chemical carcinogens were administered to female CD-1 mice at 0.5% of the diet for 14 days, and their effects on cytosolic glutathione transferase (EC 2.5.1.18) specific activities were examined in liver, lung, kidney, urinary bladder, forestomach, proximal small intestine, and colon. Disulfiram, sodium diethyldithiocarbamate, bisethylxanthogen, and benzyl isothiocyanate elevated glutathione transferase specific activities in most of the organs examined. The four sulfur compounds differed in the extents and organ specificities of their effects on these enzyme activities. In the liver, bisethylxanthogen and benzyl isothiocyanate increased glutathione transferase activities to at least 3 times control levels and caused differential increases in the isozyme patterns observed after isoelectric focusing of the cytosols. Bisethylxanthogen also increased immunoreactive glutathione transferase in liver cytosol. Recrystallized disulfiram was less effective in enhancing hepatic glutathione transferase activities than was commercial (97%) disulfiram. Among the six extrahepatic organs examined, the small intestine and the forestomach exhibited the greatest response of glutathione transferase activities to each of the four sulfur compounds. Benzyl isothiocyanate was most effective in these "portal of entry" organs but less effective than bisethylxanthogen in the other extrahepatic organs examined. Bisethylxanthogen elicited significant increases in glutathione transferase activities in liver, lung, and small intestine even when administered at 0.01% to 0.05% of the diet, suggesting that this compound may have considerable potential as an inhibitor of carcinogens susceptible to enzymatic conjugation with glutathione.     

Effects of combined treatments with selenium, glutathione, and vitamin E on glutathione peroxidase activity, ornithine decarboxylase induction, and complete and multistage carcinogenesis in mouse skin

Several structurally different tumor promoters altered to various degrees both glutathione (GSH) peroxidase (EC 1.11.1.9) and ornithine decarboxylase (ODC, L-ornithine carboxy-lyase, EC 4.1.1.17) activities in mouse epidermis in vivo. At 5 h after their application to the skin, the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the stage 2 promoter mezerein were the most potent in inhibiting GSH peroxidase activity and inducing ODC activity. In comparison, the effects of anthralin, phorbol-12,13-didecanoate, benzoyl peroxide, H2O2, and phorbol-12,13-dibenzoate were much smaller, whereas the nontumor promoter phorbol, the hyperplastic agent ethyl phenylpropiolate, and the stage 1 promoter 4-O-methyl TPA did not alter GSH peroxidase and ODC activities. Various treatments including i.p. injections of 40 micrograms of Na2SeO3 and 100 mumol of GSH and/or topical applications of 40 mumol of D-alpha-tocopherol (vitamin E) 20 or 15 min, respectively, before tumor promoter treatment inhibited in an additive manner the effects of either TPA or mezerein on both GSH peroxidase activity and ODC induction. Moreover, these Na2SeO3, GSH, and/or vitamin E treatments inhibited in the same additive manner the tumor-promoting activity of TPA in the initiation-promotion protocol. However, when tested in the 2-stage promotion protocol with 4 doses of TPA followed by twice weekly applications of mezerein, Na2SeO3 plus vitamin E and GSH plus vitamin E treatments inhibited remarkably the tumor-promoting activity of mezerein but were ineffective in the first stage of promotion. The sequence and magnitude for the effects of 7,12-dimethylbenz[alpha]anthracene (DMBA) on GSH peroxidase and ODC activities were very different from those of the tumor promoters. In contrast with their antitumor-promoting activity, the treatments with Na2SeO3 plus vitamin E and GSH plus vitamin E failed to inhibit the carcinogenicity of a single large dose of DMBA and even enhanced the induction of skin tumors by repeated applications of subcarcinogenic doses of DMBA. These results suggest that the promoting component of DMBA carcinogenesis may be different from that of TPA. Moreover, the anticarcinogenicity of Na2SeO3, GSH, and vitamin E may be linked to their ability to facilitate or enhance the activity of the natural GSH-dependent antioxidant protective system of the epidermal cells during the later stages of skin tumor promotion.     

Differential specificity of monochlorobimane for isozymes of human and rodent glutathione S-transferases

Monochlorobimane (MCB) has been used as a glutathione (GSH) specific fluorescent probe capable of delineating GSH heterogeneity in cellular systems. Generally, low concentrations of MCB (less than 50 microM) have been used to quantitatively label GSH in rodent cell lines. Incubation of the hamster cell lines, CHO AB1 and V79, with 10 microM MCB labeled 75 and 39% of the reduced GSH pool, respectively. In contrast, incubation of 7 different human cell lines with 10 microM MCB labeled less than 4% of the total reduced GSH pool. The human cell lines required 1000 microM MCB to label an average of 73% of the GSH pool (range, 60-88%). When using 1000 microM MCB to label GSH, flow cytometry results from 7 different cell lines (human and rodent) were in good agreement with high performance liquid chromatography and standard spectrophotometric analysis with regards to a rank ordering of the GSH content determined for each cell line. The human glutathione S-transferases B2B2, B1B2, psi, pi, and the rat transferases 1-2, 3-3, and 3-4 were isolated and purified for steady state kinetic analysis with MCB and GSH as the primary substrates. The human basic transferases, B1B2 and B2B2, had Km values for MCB of 354 and 283 microM and Vmax values of 33.3 and 34.6 mumol bimane-GSH/min/mg protein, respectively. The rat basic transferase 1-2 showed similar kinetic results with a Km of 199 microM and a Vmax of 35.5 mumol bimane-GSH/min/mg protein. The human neutral transferase (psi) had a Km for MCB of 204 microM with a Vmax of 6.5 mumol bimane-GSH/min/mg protein. In contrast, MCB has a high affinity for the rat neutral transferase with a Km of 2.6 microM and a Vmax of 35.1 mumol bimane-GSH/min/mg protein. The human acidic transferase (pi), the predominate transferase found in most human tumor cell lines, has a Km of 264 microM for MCB and a Vmax of 1.99 mumol bimane-GSH/min/mg protein. The kcat/Km values indicated that MCB is an excellent substrate for the rat neutral transferases while the human pi glutathione S-transferase showed the least reactivity. Collectively the data indicate that MCB fails to label GSH at lower concentrations (less than 50 microM) in human cell lines because of the reduced affinity of MCB for the human transferases and possibly also due to differences in glutathione S-transferase isozyme expression between rodent and human cell lines.