Γ-glutamyl Transpeptidase-positive Foci Research Articles

Suppression of Altered Hepatic Foci Development by a High Fish Oil Diet Compared With a High Corn Oil Diet in Rats

Effects of low corn oil, high corn oil, and high fish oil diets on altered hepatic foci development in female Sprague-Dawley rats were investigated. Rats assigned to Groups 1-4 were initiated with saline as the control and those assigned to Groups 5-7 were initiated with diethylnitrosamine (DEN 15 mg/kg) at 24 hours of age. After weaning, all rats, except those in Group 1, received 500 ppm phenobarbital (PB) in their diet as tumor promoter for three months. Altered hepatic foci development was significantly lower in DEN-initiated rats fed the high fish oil + PB diet than in DEN-initiated rats fed the high corn oil + PB diets. Liver weight and relative liver weight were significantly greater in rats fed the high fish oil + PB diet than in rats fed the other diets, and hepatic biotransformation/detoxification enzyme activities were greater in rats fed the fish oil + PB diets than in rats fed the other diets. These results suggest that the effect of a high fish oil diet on altered hepatic foci may occur through regulation of hepatic biotransformation/detoxification enzyme activities, leading to alteration in the tumor-promoting action of PB. Dietary lipid significantly affected the hepatic phospholipid fatty acid composition of rats. n-3 polyunsaturated fatty acids were incorporated into membrane phospholipid at the expense of n-6 polyunsaturated fatty acids. A high fish oil diet caused greater oxidative stress in rats, as measured by plasma vitamin E level, red blood cell glutathione status, liver lipid peroxidation, and hepatic glutathione reductase activity. Pearson's correlation analysis indicated that the foci number was negatively correlated to the liver thiobarbituric acid-reactive substance and 7-pentoxyresorufin O-dealkylase activity, and the foci area was negatively correlated to the liver thiobarbituric acid-reactive substance activity (p << 0.05) in rats of groups that developed foci. These results suggest that the type of dietary lipid is the more important determinant for γ-glutamyl transpeptidase-positive foci development than the amount of dietary lipid when rats consumed approximately the same amount of calories in all the dietary groups, and the underlying mechanisms may be partially ascribed to the antioxidant/oxidation status and biotransformation/detoxification system of rats.

Effects of Novobiocin on the induction of γ-glutamyltranspeptidase positive foci in the liver of rats treated with diethylnitrosamine

Effects of novobiocin on the induction of gamma-glutamyltranspeptidase(GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, after diethylnitrosamine(DEN) initiation were investigated in Fischer 344 rats. Animals were given DEN at a dose of 20 mg/kg b. w. followed by novobiocin at doses of 50, 100 and 200 mg/kg b. w. The latter two doses, but not 50 mg/kg b. w., significantly inhibited the development of GGT-positive foci, providing evidence of the possible involvement of mono(ADP-ribosyl)ation in the initiation phase of hepatocarcinogenesis in rats.

Synthetic estrogens and tamoxifen as promoters of hepatocarcinogenesis

Previously, we demonstrated that the synthetic estrogens mestranol and ethinyl estradiol (EE) were strong promoters of hepatocarcinogenesis initiated in intact female rats by prior treatment with diethylnitrosamine (J. D. Yager, H. A. Campbell, D. S. Longnecker, B. D. Roebuck, and M. C. Benoit, Cancer Res. 1984; 44:3862–3869). In subsequent studies designed to elucidate possible mechanisms of promotion by EE, we investigated whether the antiestrogen tamoxifen was antagonistic to the effects of EE (J. D. Yager, B. D. Roebuck, T. L. Paluszcyk, and V. A. Memoli, Carcinogenesis 1986; 7:2007–2014). In these and more recent studies we found that tamoxifen inhibited the stimulatory effects of EE on pituitary size, liver DNA synthesis, and, in cultured hepatocytes, the potentiation by EE of epidermal growth factor-induced DNA synthesis. Furthermore, tamoxifen also inhibited the ability of EE to promote hepatocarcinogenesis. However, paradoxically, tamoxifen alone enhanced the appearance of γ-glutamyl transpeptidase positive foci in diethylnitrosamine-initiated livers indicating that it is a promoter of hepatocarcinogenesis.

Toxicological studies on a benzofurane derivative: I. A comparative study with phenobarbital on rat liver

The benzofurane derivative benzbromarone (BBR) previously has led to liver tumor formation after long-term treatment of rats, but no indications of genotoxicity were detected. The present studies were designed to elucidate the mechanism(s) possibly involved in liver tumor formation by BBR. Female Wistar rats were used. Phenobarbital (PB) served as a positive control. (1) Short-term treatment (7 days) with daily doses of 2 to 100 mg/kg BBR led to adaptive responses in the liver, i.e., growth (increases in DNA, RNA, and protein) and induction of monooxygenases. These changes were also observed after feeding BBR for 8, 33, 77, and 102 weeks at doses of 2, 10, and 50 mg/kg/day but tended to weaken with time. Similar effects were obtained with PB fed at 2, 10, or 50 mg/kg/day. However, unlike PB, BBR did not enhance the expression of cytochrome P450-PB as demonstrated by immunostaining of histological liver sections. (2) BBR feeding for 102 weeks, but not for 77 weeks, produced some neoplastic liver nodules and at 50 mg/kg produced one hepatocellular carcinoma (HCC). Thus, BBR was tumorigenic in the present study, but was clearly weaker than PB which had induced liver nodules and HCCs at 77 weeks and even more markedly at 102 weeks. (3) To check for tumor-initiating activity 100 mg/kg BBR was given 14 hr after a two-thirds hepatectomy followed by promotion with PB (50 mg/kg) for 15 weeks. No phenotypically altered liver foci were detected. (4) To test for tumor-promoting activity rats received a single dose of N-nitrosomorpholine (250 mg/kg), and subsequently BBR or PB at doses of 2, 10, and 50 mg/kg/day. While PB markedly enhanced the development of neoplastic nodules and HCCs, BBR had only a weak enhancing effect on the induction of HCC, which was not dose related. γ-glutamyl transpeptidase-positive foci dramatically increased in PB-treated animals, in contrast they showed no response after 2 and 10 mg/kg BBR and even decreased after 50 mg/kg BBR. (5) With PB changes in liver growth, monooxygenase activity, foci expansion, and tumor promotion all correlating with tumorigenesis in a quantitative manner, apparent no-observed-effect-levels are somewhat below 2 mg/kg (or 10 mg/kg for liver enlargement). (6) These studies suggest that BBR belongs to a group of nongenotoxic, growth-stimulating drugs with tumorigenic potential in rat liver. Its effects on the liver are different from those of PB, but seemed to resemble those of peroxisome proliferators, a hypothesis studied in the subsequent papers.

Lack of modifying effects of p-phenylenediamine on induction of γ-glutamyl transpeptidase-positive foci in a medium-term bioassay system using F344 rats

The modifying effects of p-phenylenediamine (p-PD) on liver carcinogenesis were investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg kg , intraperitoneally), rats were given p-PD at dietary levels of 1000, 330 and 110 parts per million (ppm), or 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) at 600 ppm as a positive control for 6 weeks. At week 3 following DEN adminstration, all animals were subjected to partial hepatectomy. Slight retardation of body weight was observed in rats treated with p-PD at all dietary levels. Significant increases in relative liver weight were found in animals treated with 1000 ppm p-PD. Remarkable growth retardation and increased liver weight were found in rats given 3′-Me-DAB. p-PD did not significantly increase the level of γ-glutamyl transpeptidase (γ-GT) positive foci observed after DEN initiation.In contrast, 3′-Me-DAB demonstrated marked enhancing activity as evidenced by significantly increased values for γ-GT positive foci as compared with the controls given DEN alone. The results demonstrate that p-PD does not modify liver carcinogenesis in our in vivo medium-term bioassay system.

Effect of feeding a choline-deficient diet on the hepatic nuclear response to tryptophan in the rat

In earlier studies the acute administration of tryptophan (TRP) to rats was reported to induce enhanced in vivo [ 14C]orotate-labeled hepatic nuclear RNA release in vitro. This change was considered to possibly be related to the induction of more and larger γ-glutamyl transpeptidase-positive foci in the livers of rats treated with diethylnitrosamine and fed long-term elevated TRP in a choline-supplemented (CS) but not in a choline-deficient (CD) diet (comparisons with respective controls). In this study we investigated whether feeding a CD compared to a CS diet for 1 week would affect selected hepatic nuclear responses to TRP. Rats fed the CS but not the CD diet and tube-fed TRP 10 min before being killed revealed enhanced labeled hepatic nuclear RNA release in vitro. In all experiments, comparisons were made with the control groups (rats fed the CS or stock diet). When rats were fed elevated TRP (2%) in the diets (CS or CD) for 1 week, labeled hepatic nuclear RNA release was increased with the CS + TRP but not with the CD + TRP diet group. [ 3H]TRP binding to hepatic nuclei in vitro revealed no change in the CS + TRP group, decreased in the CD group, and markedly increased in the CD + TRP group in comparison with the control (CS) group. Hepatic nuclear nucleoside triphosphatase activity was increased only in the CS + TRP group while hepatic nuclear poly(A) polymerase activity was increased in the CS + TRP and in the CD ± TRP groups. Serum cholesterol and triglycerides were decreased in the CD group and increased to control levels in the CD + TRP group.

Influence of level of dietary protein on tryptophan-induced promotional activity in induction of γ-glutamyl transpeptidase-positive foci of rat liver

The influence of varying the dietary protein content on the emergence of γ-glutamyl transpeptidase (GGT)-positive foci in the livers of male rats fed elevated (2%) l-tryptophan (TRP) after being exposed to a hepatocarcinogen was investigated. Subtotal hepatectomies were performed, and 18 hr later the rats were treated with diethylnitrosamine (30 mg/kg). Ten days later four dietary groups were followed for 10 weeks: (1) control diet containing 21% protein (C); (2) control diet containing 5.3% protein (C-LP); (3) C + TRP; and (4) C-LP + TRP. Rats fed the C-LP diet developed heavier livers but fewer and smaller GGT+ foci than did rats fed the C diet. Rats fed elevated TRP diets (C + TRP and C-LP + TRP) developed more and larger GGT+ foci than did rats fed the regular control diets (C and C-LP), indicating that the promotional effect of elevated dietary TRP was similar at the two levels of dietary protein.

Effect of timing of partial hepatectomy on the induction of preneoplastic liver foci in rats given hepatocarcinogens

Beginning 2 weeks after an intraperitoneal injection of diethylnitrosamine (DEN), rats were fed diet containing either 2-acetylaminofluorene (2-AAF), 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), α-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (α-HCH), or phenobarbital (PB) for 6 weeks and subjected to partial hepatectomy (PH) after various periods of chemical administration. The effect of timing of PH was most apparent in rats fed 2-AAF. The size of γ-glutamyl transpeptidase positive foci was largest when PH was performed 1–3 weeks after the start of chemical feeding. Similar tendency was slightly observed with 3′-Me-DAB or α-HCH administration, but not with PB or basal diet feeding. Thus, the enhancing effect of PH on the induction of the preneoplastic liver foci was affected by the type of chemical and the timing of the operation.

Effects of varying fat content of a high tryptophan diet on the induction of γ-glutamyltranspeptidase positive foci in the livers of rats treated with hepatocarcinogen

The influence of varying the dietary fat content on the emergence of γ-glutamyltranspeptidase (GGT)-positive foci in the livers of male rats fed elevated (2%) l-tryptophan (TRP) and exposed to a hepatocarcinogen was investigated. Subtotal hepatectomies were performed, and 18 h later the rats were treated with diethylnitrosamine (DEN) (30 mg/kg). Ten days later 4 dietary groups were followed for 10 weeks: (1) control diet containing 15% fat (C-HF); (2) control diet containing 5% fat (C-LF); (3) C-HF + TRP; (4) C-LF + TRP. Rats fed elevated TRP diets (C-HF + TRP and C-LF + TRP) similarly developed more and larger GGT+ foci than did rats fed the regular TRP diets (C-HF and C-LF), indicating that the promotional effect of elevated dietary TRP was not affected by the fat level (15% vs. 5%).

Promoting effects of phenobarbital and 3′-methyl-4-dimethylaminoazobenzene on the appearance of γ-glutamyltranspeptidase positive foci in rat liver pretreated with varying doses of diethylnitrosamine

The promotion potential of phenobarbital (PB) and 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) on liver carcinogenesis after initiation with various doses of diethylnitrosamine (DEN) was assessed using an in vivo short term system. Male F344 rats were pretreated with a single intraperitoneal injection of varing doses of DEN (0, 6, 12, 25, 50, 100 or 200 mg/kg body wt), and 2 weeks later were treated with 0.05% PB or 0.06% 3′-Me-DAB for 6 weeks. All animals were subjected to partial hepatectomy 3 weeks after the DEN treatment. Quantitation of γ-glutamyltranspeptidase-positive (γ-GT +) foci revealed a DEN dose-dependent response. Magnitude of promotion by PB and more pronounced by 3′-Me-DAB was, in contrast, strongest at the lower doses of DEN. The results suggest that quantitative diffeences with regard to initiation level may exist, influencing the promotability of initiated cells.

The initiation of γ-glutamyltranspeptidase positive foci in the rat liver by N-hydroxy-2-acetylaminofluorene. The effect of the sulfation inhibitor pentachlorophenol

The role of sulfation of the carcinogen N-hydroxy-2-acetyl-aminofluorene in the initiation phase of liver-tumor formation was studied in an initiation-promotion model modified from the Solt and Farber system (Cancer Res., 43, 188-191, 1983). The effect of the sulfation inhibitor pentachlorophenol on the induction by N-hydroxy-2-acetylaminofluorene of gamma-glutamyltranspeptidase positive foci of cells (regarded as a population of cells from which neoplastic cells will develop) was determined. Inhibition of sulfation did not prevent the induction of gamma-glutamyltranspeptidase positive foci in rat liver; on the contrary, an increase was observed. Since it is known that inhibition of sulfation of N-hydroxy-2-acetylaminofluorene prevents the formation of covalently-bound 2-acetylaminofluorene adducts to proteins, RNA, and DNA in the rat liver (Meerman et al., Carcinogenesis, 2, 413-416, 1981), the results from this study suggest that deacetylated, 2-aminofluorene adducts to DNA are responsible for initiation of gamma-glutamyltranspeptidase positive foci in the rat liver (because formation of these adducts is not affected by pentachlorophenol). After initiation with a single dose of N-hydroxy-2-acetylaminofluorene, proliferation of 'oval cells' was observed only 1 week after administration of the carcinogen. This was not observed in the livers of rats pre-treated with pentachlorophenol in which the largest number of foci develops. These data therefore suggest that 'oval cells' are not involved in the development of gamma-glutamyltranspeptidase positive foci.

Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glucuronyltransferase and inhibition of development of gamma-glutamyl transpeptidase-positive foci in rat liver.

Effects of an antioxidant, butylated (3-tert-butyl-4-) hydroxyanisole (BHA) on the induction of specific molecular forms of glutathione S-transferase (GST), UDP-glucuronyltransferase (UDP-GT) and other glutathione-related enzymes in rat liver were investigated. The development of gamma-glutamyl transpeptidase (gamma-GTP)-positive foci and hyperplastic nodules induced by diethylnitrosamine, 200 mg/kg i.p., followed by 0.02% N-2-fluorenylacetamide (FAA) in diet plus partial hepatectomy was inhibited by the administration of 0.75% BHA in the FAA-containing diet. Inhibition was reflected in decreased area of gamma-GTP-positive foci which correlated with a decrease in gamma-GTP activity measured biochemically. Under the present experimental conditions, total activities of GSTs, especially that of GST-A form, and of UDP-GTs, especially that of the late fetal form (o-GT), were markedly increased, together with glutathione levels in the whole liver, within one week after BHA administration. Without BHA administration the activities of GST-A and o-GT, as well as glutathione levels, were also increased by FAA treatment, primarily localized within gamma-GTP-positive foci. These results suggest that the induction of specific molecular forms of detoxicating enzymes either in enzyme-altered foci or in the whole liver may play an important role in determining the extent of development of preneoplastic nodules from initiated foci under the short term induction conditions used.

The effect of pre- and post-treatment with phenobarbital on the extent of γ-glutamyl transpeptidase positive foci induced in rat liver by N-nitrosomorpholine

Male Sprague-Dawley rats were treated with a combination of N-nitrosomorpholine (NNM) and phenobarbital and the extent of foci positive for γ-glutamyl transpeptidase (γ-GT), as demonstrated histochemically in liver, was quantitated. Phenobarbital (0.05% in the diet) was given either alone, before, or after a 7-week period of treatment with NNM (80 ppm in the drinking water). When given after the carcinogen treatment, phenobarbital enhanced the formation of γ-GT-positive foci in liver compared with rats treated with NNM alone. No such increase, however, was observed when phenobarbital was administered before treatment with NNM. Phenobarbital alone did not produce any apparent amount of foci in liver. These data suggest that the enhancement of carcinogenic response mediated by phenobarbital as observed under the present experimental conditions is only operative in the presence of pre-existing initiated hepatocytes.

Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of gamma-glutamyl transpeptidase-positive foci development in the liver of rats.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were evaluated for possible promoting activity for urinary bladder carcinogenesis in male F344 rats initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). The rats were treated with 0.01 or 0.05% BBN in the drinking water for 4 weeks and then administered 2% BHA or 1% BHT in the diet for 32 weeks. Surviving rats were killed at the end of week 36 of the experiment. The incidences of cancer and papilloma and the average number of cancers, papillomas and papillary or nodular hyperplasias (PN hyperplasias) per 10 cm of basement membrane were significantly increased in the group receiving BHA following initiation by 0.05% BBN compared with the group given BBN only. BHT also significantly increased these lesions of the bladder, but not the average number of cancers, in rats treated with 0.05% BBN. The ability of four antioxidants, BHA, BHT, sodium L-ascorbate (ascorbate) and ethoxyquin, to promote the induction of gamma-glutamyltranspeptidase (gamma-GT)-positive foci initiated by diethylnitrosamine (DENA) in the liver of F344 rats was tested. Rats were given a single i.p. injection of 200 mg/kg body weight of DENA, and 2 weeks later the animals were exposed to 2% BHA, 1% BHT, 5% ascorbate or 1% ethoxyquin, respectively, in the diet for 6 weeks. All animals were subjected to partial hepatectomy at the end of week 3. The number of gamma-GT-positive foci in the groups fed either BHA, BHT or ethoxyquin after DENA were significantly decreased compared with the control group. These findings show that BHA and BHT are promoters for the urinary bladder carcinogenesis initiated by BBN, but that these and other antioxidants significantly inhibit the induction of gamma-GT-positive foci in the liver.

Effects of varying the fat content of a choline-devoid diet on promotion of the emergence of γ-glutamyltranspeptidase positive foci in the liver of carcinogen-treated rats

Previous studies have shown that feeding a choline-devoid (CD) diet to rats is an effective promoter of liver carcinogenesis. In the present studies, we investigated the effects of CD diets containing different levels of fat (high, 15%; low, 4%) on the induction of foci of γ-glutamyltranspeptidase (GGT) positive hepatocytes in the liver of rats initiated with a single dose of diethylnitrosamine. In rats fed the high-fat CD diet for 4–6 weeks, a greater number of foci was induced than in rats fed similarly the low-fat CD diet. However, liver DNA synthesis and the rate of hepatocyte mitosis were not significantly different in rats fed the 2 CD diets, but were significantly higher than in rats fed a choline-supplemented diet. These results indicate that beside stimulation of liver cell proliferation, other factor(s) determine the efficacy with which a CD diet exerts its promoting action.

Enhancing effect of 3-aminobenzamide on induction of γ-glutamyl transpeptidase positive foci in rat liver

Enhancing effect of 3-aminobenzamide on induction of γ-glutamyl transpeptidase positive foci in rat liver