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Although several epidemiologic studies have shown that gamma-glutamyltransferase (GGT) is independently associated with cardiovascular disease and all-cause mortality, its relationship with cancer incidence remains widely unexplored. In several experimental models, the ability of cellular GGT to modulate crucial redox-sensitive functions has been established, and it thus may play a role in tumor progression, as has been repeatedly suggested. We prospectively investigated the association between GGT and risk of overall and site-specific cancer incidence in a large population-based cohort of 79,279 healthy Austrian men with serial GGT measurements. Median follow-up was 12.5 years. Adjusted Cox proportional hazards models were calculated to evaluate GGT as an independent predictor for cancer incidence, and nonparametric regression splines were fitted to flexibly capture the dose-response relationship. Elevated GGT significantly increased overall cancer risk, showing a clear dose-response relationship (P for GGT log-unit increase < 0.0001; P for trend < 0.0001). In comparison with the reference GGT concentration (25 units/L), we found adjusted relative risks (95% confidence intervals) equalling 1.19 (1.15-1.22) for GGT concentrations of 60 units/L, 1.32 (1.28-1.36) for 100 units/L, 1.67 (1.60-1.75) for 200 units/L, and 2.30 (2.14-2.47) for 400 units/L. In cancer site-specific models, GGT was significantly associated with malignant neoplasms of digestive organs, the respiratory system/intrathoracic organs, and urinary organs (all P < 0.0001). Age of participants significantly modified the association of GGT and cancer risk (P < 0.001), revealing markedly stronger associations in participants ages

Cisplatin is a potent cytotoxic agent that functions as a bivalent electrophile, forming both interstrand and intrastrand DNA cross-links. Cisplatin-mediated DNA damage results in cell cycle arrest and initiation of apoptotic cell death. Increased cellular glutathione concentrations have been closely correlated with cisplatin resistance but do not reduce the extent of cisplatin-DNA adduct formation. One hypothesis to explain the ability of glutathione to inhibit cisplatin cytotoxicity is that glutathione, through its antioxidant function, plays a role in apoptotic regulatory pathways. We tested this hypothesis using MCF-7 breast cancer cells transfected with the apoptotic inhibitor Bcl-2. Bcl-2 overexpression in MCF-7 cells was associated with a nearly 3-fold increase in cellular glutathione levels and with increased resistance to cell death after cisplatin exposure. Treatment of MCF-7 lines with buthionine sulfoximine, an inhibitor of glutathione synthesis, normalized glutathione levels in Bcl-2 and control transfectants and completely abrogated Bcl-2-mediated cisplatin resistance without affecting Bcl-2 expression. Bcl-2 overexpression and up-regulation of glutathione were not associated with a change in either cisplatin-DNA adduct formation or repair over time. These results suggest that Bcl-2-mediated cisplatin resistance in MCF-7 cells is dependent on up-regulation of glutathione production, which contributes to cell survival by mechanisms independent of cisplatin inactivation or inhibition of DNA adduct formation. A similar dependence on glutathione for Bcl-2-mediated inhibition of cisplatin toxicity was confirmed in a second cell line, the lymphocytic precursor FL5.12. Taken together, these data suggest that apoptotic signaling after genotoxic exposure can be inhibited by the antioxidant activity of glutathione. Inhibition of glutathione synthesis or modulation of glutathione stores in tumors that overexpress Bcl-2 may comprise a novel anticancer strategy.

DRH strain rats were established by inbreeding a closed colony of Donryu rats continuously fed the chemical hepatocarcinogen 3'-methyl-4-dimethylaminoazobenzene for over 10 years. They are highly resistant to chemical induction of liver cancer and preneoplastic lesions. We studied the genetic basis of DRH resistance to preneoplastic lesions by analyzing 108 (F344 x DRH)F2 male rats fed 3'-methyl-4-dimethylaminoazobenzene for 7 weeks. Five parameters of preneoplastic liver lesions were selected for quantitative analysis: (a) number of glutathione S-transferase placental form-positive foci per unit area of liver section; (b) percentage area occupied by the foci; (c) average size of foci; (d) glutathione S-transferase placental form mRNA level; and (e) gamma-glutamyltranspeptidase mRNA level. Furthermore, O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor mRNA levels were quantified. Composite interval mapping analysis showed that there were two remarkably significant clusters of quantitative trait loci affecting preneoplastic liver lesions on chromosomes 1 and 4. These clusters were designated collectively as Drh1 and Drh2, respectively. The functions of the recessive DRH allele of Drh1 and the semidominant DRH allele of Drh2 were to suppress the phenotypes of precancerous lesions. Each cluster showed two to three subpeaks in linkage likelihood plots, suggesting the presence of several closely linked quantitative trait loci affecting preneoplastic lesions. Possible candidate genes at each locus will be discussed. Expression of O6-methylguanine DNA methyltransferase and mannose 6-phosphatase/insulin-like growth factor 2 receptor did not affect DRH resistance to hepatocarcinogenesis, although they were polymorphic between DRH and F344 rats.

The pattern of gene expression in fetal hepatocytes transformed in culture with a hepatocarcinogen (FRL cells) is studied with respect to a range of markers which are either developmentally regulated and/or shown to be expressed at high levels in hepatoma cells. The relative abundance of the respective mRNAs is determined and immunocytochemistry is used to detect the respective proteins in cultured cells. When compared with its normal counterpart, FRL cells retain the expression of transferrin, alpha 1-acid glycoprotein, gamma-glutamyltranspeptidase, and tyrosine aminotransferase at near normal levels, while expression of the liver-specific isoenzymes of pyruvate kinase (L form) and aldolase (B form) is reduced. The cell lines are different in that they fail to express albumin, alpha-fetoprotein, thiostatin and alpha 2-macroglobulin, and they express high levels of M2-pyruvate kinase and aldolase A, markers often found in abundance in hepatoma cells. Therefore transformation has resulted in different effects on different genes. Furthermore, it is of interest to find that the cells coexpress both forms of the pyruvate kinase isoenzymes which does not occur in the normal developing hepatocyte. These results indicate that it is possible to use this model to study changes which accompany transformation of fetal rat hepatocytes. The resulting cell lines have a stable phenotype and retain the changes which result from transformation even after extended passaging. This facilitates comparisons between the precursor cell and the tumor cell, both of which can be maintained under controlled conditions which exist in culture.

The purpose of this study was to investigate the effect of a two-thirds partial hepatectomy on the expression of gamma-glutamyltranspeptidase (GGT) in parenchymal hepatocytes. Two to 3-month-old Fischer 344 (F344), Wistar-Furth (WF), and WF X F344 F1 rats of both sexes were used in this investigation. Partial hepatectomy in the F344 female rat significantly increased the percent of liver area positive for GGT from 1.3 to 20.4%, a 15-fold increase. In contrast, GGT was not induced by partial hepatectomy in either the female WF or WF X F344 F1 hybrid rat or in the male animals of these three strains of rats. This phenomenon, which was only observed in the F344 female rat, was blocked by oophorectomy 2 weeks prior to partial hepatectomy, indicating that the induction of GGT is in part dependent upon female sex hormones. By transplanting both F344 and WF hepatocytes into the axillary fat pads of isogeneic and WF X F344 F1 rats, we showed that the observed strain difference did not result from a variation in the animal hormonal environment but rather was due to the hepatocyte from the F344 rat being intrinsically more susceptible to GGT induction than that from the WF rat. Further, the extent of the enzyme induction was significantly greater when the F344 liver cells were transplanted into female recipient animals. Since hepatectomy is often used in hepatocarcinogenesis studies, it is important to note that in the F344 female animal, the surgical procedure itself increases hepatic GGT levels in both transplanted hepatocytes and the liver in situ. Thus, the F344 female rat should be used with caution in investigations where the carcinogenic potential of chemicals is based upon the formation of GGT-positive hyperplastic nodules in the liver.

Individual gamma-glutamyl transpeptidase (GGT)-stained cells and cell doublets were rapidly induced in the epithelium of hamster buccal pouch treated with biweekly topical applications of 0.5%, 7,12-dimethylbenz(a)anthracene (DMBA) in mineral oil. The cells were detected histochemically in whole mounts of pouch epithelium harvested as early as 3 days after the first application of this carcinogen. During 3 consecutive weeks of DMBA treatment, progressively larger GGT-stained epithelial cell populations (plaques) up to 0.5 mm in diameter were encountered. Similar GGT-stained lesions were not detected in whole mounts of untreated epithelium and were rarely seen in GGT-stained whole mounts prepared from mineral oil-treated pouch epithelium. In an experiment designed to assess the stability of the GGT-staining pattern, very few plaques could be detected 12 weeks after a 3-week regimen of six DMBA applications. However, data are presented suggesting that a brief series of three DMBA applications reinduced GGT histochemical activity in occult intraepithelial plaques, which had los enzyme activity but had persisted over an 11-week treatment-free interval. The clonal nature of the GGT-stained plaques for their apparent ability to persist in an occult form for several weeks or months lend further support to the hypothesis that these carcinogen-altered cell populations may be potential precursors for the development of squamous epithelial neoplasia.

The elevation of bone marrow gamma-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) content in rats carrying mammary carcinoma 5A (MC), reproduced in a short-term (48-h) liquid culture of normal bone marrow cells, was found to be attributable to a blood-borne protein factor with an apparent molecular weight of 60,000. Partial purification, based on the extent of stimulation of GGT expression in this culture, increased the specific activity of the host serum from 1.5 to 40 units and that of MC extracts from 6 to 560 units. Production of the factor by MC in vitro, however, resulted in specific activities of 3000 units in the conditioned medium, and a further 60-fold purification was achieved by DEAE-cellulose, Sephadex G-100, and hydroxylapatite chromatography. The chemical characteristics of the MC-elaborated protein indicate nonidentity to previously isolated colony formation stimulating factors which also induced GGT (and AP) expression by rat bone marrow cells. Most of the AP inducing ability of the MC-serum and MC-conditioned medium copurified with and was still present in preparations with the highest specific activity vis à vis GGT. In mouse (instead of rat) bone marrow cells, however, no AP response accompanied the stimulation of GGT expression by MC (or colony formation stimulating factor) preparations.

The effects of varying the type of dietary fat in the choline-deficient (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of carcinogen-treated rats were investigated, and the results were correlated with the extent of membrane lipid peroxidation induced by the diets. Male Sprague Dawley rats were initiated with a single dose of diethylnitrosamine. Thereafter, groups of rats were fed choline-supplemented or CD diets in which the amount of saturated fat was varied by using hydrogenated vegetable oil (Primex) and corn oil (CO), either alone or in combination. The number and size of GGT-positive foci induced by the CD diet with CO as the sole source of fat were larger than those induced by the diet containing mixtures of Primex and CO. The CD diet with Primex alone was the least effective in inducing GGT-positive foci. Peroxidation of liver microsomal membrane lipids in rats fed regular CD or CD:CO diets was examined by determining the formation of conjugated dienes. The generation of diene conjugate in rats fed a CD:CO diet was evident after 2 days of the diet feeding, and the levels increased at 1 and 2 weeks. No significant diene conjugate was demonstrated in rats fed a regular CD diet for 2 days. However, after 1 and 2 weeks, there was generation of diene conjugate, the levels of which were lower in rats fed the CD diet than those on a CD:CO diet. Addition of an antioxidant, 0.25% butylated hydroxytoluene, to both CD and CD:CO diets abolished the generation of diene conjugate in rat liver microsomal membranes and markedly inhibited the promotion of GGT-positive foci in the liver of diethylnitrosamine-initiated rats. The results suggest that membrane lipid peroxidation in the liver may be related to the promotion of the induction of GGT-positive foci by a CD diet. The enhanced promotion by the inclusion of a higher level of polyunsaturated fat in the diet may be, in part, due to its greater susceptibility to peroxidation.

Hepatocytes isolated as a relatively pure population from normal adult rats were maintained in primary monolayer culture for 4 to 7 days on plastic dishes. Factors affecting activity of gamma-glutamyltranspeptidase (GGT) in culture were investigated to establish a basis for in vitro studies on carcinogen-induced changes in GGT. Freshly plated cultures contained few GGT-positive cells (0.4%) and very low total GGT activity, but with all media tested, activity increased progressively with time in culture, the extent of increase depending on medium composition. For hepatocytes in modified Waymouth medium alone (control cultures), there was a slow rise in activity after a 1- to 2-day lag period. This increase was enhanced up to 10-fold in the presence of 3 microM dexamethasone for cells on plastic but only 2- to 3-fold for cells on collagen gels. The time- and dose-dependent action of dexamethasone was prevented by cycloheximide, partly blocked by actinomycin D, and reversed after several hr by removing the steroid, when GGT activity decreased with a maximum half-life of 60 to 80 hr. These observations suggest that dexamethasone caused reversible enzyme induction dependent on continuing RNA and protein synthesis. Cell maintenance with fetal calf serum or with N6,O2'-dibutyryl adenosine 3':5'-monophosphate or glucagon markedly reduced the extent of induction by dexamethasone and slightly lowered activity in control cultures. Maintenance at lower pH within the range of 7.2 to 7.8 or at higher glucose concentrations within the range of 2.8 to 28 mM also resulted in markedly lower GGT activities in control or dexamethasone-induced cultures. Glucose repression was independent on insulin concentration. The reversible induction and repression of GGT may reflect broad changes in hepatocyte gene expression rather than specific controls of GGT function. Since GGT in cultured hepatocytes is subject to regulation by a variety of noncarcinogens, caution is required in its use as a preneoplastic marker. Nevertheless, the identification of culture media which preserve low activity may allow studies on carcinogen-induced changes in GGT as a probe of the early events in vitro hepatocarcinogenesis.

The effect of dietary administration of 0.5% ethoxyquin (EQ) on the in vivo induction of enzymes and effect on aflatoxin B1 (AFB1)-DNA binding in liver and the consequent in vitro metabolism of AFB1 by male Fischer F344 rat liver-derived fractions have been examined. EQ increased microsomal cytochrome P-450s, in particular those isozymes classed as phenobarbital inducible, and the in vitro rate of metabolism of AFB1. The formation of the presumed detoxified metabolites, aflatoxins M1 and Q1, was enhanced to a greater extent than was the formation of the active metabolite, aflatoxin B1-8,9 epoxide (assessed by the level of aflatoxin B1-8,9-dihydrodiol). Prolonged feeding with EQ was accompanied eventually by a reduction in the initially elevated cytochrome P-450 content, but this was not reflected in any significant decrease in the rate of AFB1 metabolism in vitro. EQ increased the glutathione S-transferase activity of the liver cytosol fractions as assessed with the model substrate 1-chloro-2,4-dinitrobenzene. The capacity of these fractions specifically to catalyze the conjugation of AFB1 with glutathione was induced to a far greater extent than was the conjugation of 1-chloro-2,4-dinitrobenzene. gamma-Glutamyl transpeptidase was induced in the periportal areas of the liver lobule. Reduced in vivo binding of [3H]AFB1 to DNA of liver and kidney was found to result from EQ treatment. It is concluded that the reduced hepatocarcinogenesis which results from feeding EQ simultaneously with AFB1 is due to the reduction in DNA-adduct formation which in turn is due at least in part to increased detoxifying metabolism in the microsomal, cytosolic, and plasma membrane compartments of the liver cells.

Mouse epidermal basal cells can be selectively cultivated in medium with 0.02 to 0.09 mM Ca2+ and can be induced to differentiate by medium containing 1.2 mM Ca2+. Basal cell cultures were studied to determine if all cells in this population responded identically to the skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies on the induction of the enzyme epidermal transglutaminase by TPA demonstrated a 2- to 4-fold increase in activity within 12 hr of exposure. This activity increase paralleled morphological differentiation in approximately 50% of the basal cell population, and differentiating cells sloughed from the culture dish within 24 to 48 hr as transglutaminase activity returned to basal levels. The cells which remained were resistant to induced differentiation by 1.2 mM Ca2+ medium, in that they failed to demonstrate increased transglutaminase activity or decreased thymidine incorporation, both characteristics of control basal cells induced to differentiate by 1.2 mM Ca2+. Cells remaining after a single exposure to TPA did not respond to a second exposure with an induction of transglutaminase if the interval between exposures was 4 days. TPA-pretreated cells did not undergo a transient decrease in thymidine incorporation (characteristic of control cells) when exposed to TPA a second time but instead were directly stimulated to proliferate by the phorbol ester, indicating that such cells were not refractory to the promoter. When the treatment-free interval after TPA was extended from 4 to 10 days, transglutaminase inducibility was restored in basal cells to either TPA or 1.2 mM Ca2+ as inducers. These results indicate that heterogeneity exits within the epidermal cell population and that exposure to phorbol esters induces differentiation in some cells, while stimulating proliferation in others. Such heterogeneous responses would cause a selective redistribution of the epidermal cell population and could lead to clonal expansion of initiated cells.