Induction of gamma-glutamyltranspeptidase-positive foci in rat liver by deoxycholic acid

Initiating activities of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), were examined in terms of the induction of gamma-glutamyltranspeptidase (GGT)-positive foci in rat liver. A rapid induction model of enzyme-altered foci was utilized. GGT-positive foci were clearly induced by DCA ranging from 0.05% to 0.5% in the diet. In contrast, the induction of GGT-positive foci by LCA was less pronounced and was not dose-dependent. These results suggest that DCA possesses initiating activity.     

Induction and promotion of gamma-glutamyltranspeptidase-positive foci in the rat liver by methylglyoxal

The effect of pre-(initiation) and post-(promotion) administration of methylglyoxal (MG) on the induction of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of F344 male rats was investigated. GGT-positive foci were produced in dose-related amounts by 0.05 and 0.2% MG in drinking water, either when administered to uninitiated rats or when given in the promotion phase.     

Lack of initiating and promoting activity of thiourea in rat liver foci bioassay

Thiourea (TU) fails to enhance the incidence of foci deficient in adenosine-5'-triphosphatase (ATPase) either by initiation or by promotion in a rat liver foci bioassay. To weanling female Sprague-Dawley rats, 3 x 200 or 3 x 500 mg/kg body wt of TU, respectively, were applied for initiation. One week later Clophen A 50, a technical mixture of polychlorinated biphenyls (PCBs) 2 x 10 mg/kg body wt were given twice weekly as promoting agent for 11 weeks. For promotion 0.2% of TU was administered with the drinking water for 51 days to rats of both sexes, and 0.1% and 0.05% of TU, respectively, for 70 days to females after initiation with 1 x 8 mg/kg body wt of diethylnitrosamine (DEN).     

Induction of rat liver gamma-glutamyltranspeptidase-positive foci by oral administration of 1-nitropyrene

In the present study, the question of whether the ubiquitous environmental pollutant, 1-nitropyrene (1-NP), can induce gamma-glutamyltranspeptidase (GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, when given orally to F344 rats was examined. Significant induction of GGT-positive foci was observed with all the doses of 1-NP (1000, 500, 250 and 100 mg/kg body wt) used in the present experiment after 6 repeated intragastric (i.g.) intubations when accompanied by partial hepatectomy (PH) performed midway, but not after a single i.g. 1000 mg/kg body wt intubation plus PH. The potential for induction of foci by 1-NP, however, was far less than that by benzo[a]pyrene (B[a]P). The results thus suggested a weak but substantial initiation activity for 1-NP in the rat liver when given orally, particularly with repeated doses.     

Synergistic effect of a choline-devoid diet and phenobarbital in promoting the emergence of foci of gamma-glutamyltranspeptidase-positive hepatocytes in the liver of carcinogen-treated rats

The effect of feeding phenobarbital (PHB) with a choline-devoid (CD) diet on the emergence of foci of gamma-glutamyltranspeptidase (GGT)-positive hepatocytes in the liver of carcinogen-treated rats was investigated. Male Sprague-Dawley rats were given a single dose of diethylnitrosamine (50 mg/kg) 18 hr after a partial hepatectomy and 10 days later were placed on a plain choline-supplemented (CS) diet, a plain CD diet, or the CS and CD diets containing 0.06% PHB. Groups of rats were killed after 5 and 7 weeks of feeding each of the four diets, the livers were taken, and the number and size of foci of GGT-positive hepatocytes were determined. In rats fed the CS + PHB diet, the number of foci per sq cm of liver section was greater than that in rats fed the plain CS diet but smaller than that in rats fed the plain CD diet. Addition of PHB to the CD diet resulted in twice as many foci as in the plain CD diet and foci larger than those resulting from the plain CD diet. THe hepatocytes in the foci of rats fed th CD and CD + PHB diets showed, uniformly, not only GGT positively but also a relative absence of fatty change. The results indicate that PHB and a CD diet, when combined, have a synergistic effect in promoting the evolution of liver cells, initiated by a chemical carcinogen, to foci of altered GGT-positive hepatocytes. This promoting regimen may become useful in studies concerned with the initiation and promotion stages of liver carcinogenesis.     

Modulation by indomethacin or prostaglandin E2 of the incidence of diethylnitrosamine-induced gamma-glutamyltranspeptidase-positive foci in rat liver

We investigated the effect of a pretreatment with indomethacin (IMC, ip 3.6 mg/kg body weight (bw)) or dimethylprostaglandin E2 (PGE2, ip 10 micrograms/kg bw) on the incidence and development of gamma-glutamyltranspeptidase (GGT)-positive foci of altered hepatocytes, scored 8 or 14 weeks after ip injection of diethylnitrosamine (DENA, 50 mg/kg bw) to rats submitted to two-thirds hepatectomy (PH) or sham operation (Sh). IMC reduced by about 4 times the incidence of DENA-induced GGT-positive foci per cm3 of liver tissue in sham-operated as well as in two-thirds hepatectomized rats, compared to the respective unpretreated controls. In contrast, PGE2 pretreatment increased the incidence of DENA-induced foci in both groups, this effect, in terms of absolute numbers of foci, being additive to that of PH alone. IMC pretreatment resulted in foci with lower average size in the Sh but not in the PH animals, whereas with PGE2 pretreatment the mean volume of the foci was increased in the two groups of rats. At the dose used, IMC did not modify the proliferative response of hepatocytes to PH, and PGE2 did not stimulate proliferation in the sham-operated animals. Altogether, these results indicate that: 1, the incidence of DENA-induced foci can be negatively modulated by interfering with the prostaglandins pathway through a mechanism that does not involve an action either on proliferative activity or on any other process that would be specific to the post-hepatectomy regenerative state; 2, positive modulation of the incidence of DENA-induced foci does not necessarily require stimulation of proliferation.     

Inhibition by pentachlorophenol of the initiating and promoting activities of 1'-hydroxysafrole for the formation of enzyme-altered foci and tumors in rat liver

The hepatocarcinogen 1'-hydroxysafrole (HOS) exhibited weakinitiating activity and strong promoting activity for the inductionof enzyme-altered foci and tumors in rat liver. Thus, administrationof a single dose of HOS to rats 18 h after a 70% hepatectomy,followed by administration of phenobar-bital (PB) in the dietfor 6 months, induced a low, but statistically significant,number of foci of enzyme-altered cells. This treatment did notresult in gross liver tumors, even when the PB treatment wascontinued for 16 months. Large numbers of enzyme-altered focideveloped when HOS was administered in the diet at levels of0.05–0.25% to rats previously administered a single doseof N,N-diethylnitros-amine (DEN) 24 h after a 70% hepatectomy.Similarly, rats given a single dose of DEN 24 h after a partialhepatectomy and then fed 0.10 or 0.25% of HOS in the diet for10 months developed a high incidence of hepatocellular carcinomas.In the absence of pretreatment with DEN, dietary administrationfor at least 4 months of 0.10 or 0.25% of HOS induced significantnumbers of enzyme-altered foci; these data and liver tumor inductionby continuous feeding of HOS, in the absence of pretreatmentwith DEN, provide additional evidence for an initiating, aswell as a promoting, activity of HOS in rat liver. Concurrentadministration of the hepatic sulfotransferase inhibitor pentachlorophenolwith HOS in each of the above assays almost completely inhibitedthe initiating and promoting activities of HOS for the formationof enzyme-altered foci and tumors; these data strongly suggestthat both the initiating and promoting activities are mediatedby the sulfuric acid ester, 1'-sulfooxysafrole. HOS also exhibitedinitiating activity in adult mouse liver. Thus, dietary administrationof 0.25% of HOS for only 1 month, followed by administrationof the hepatic tumor promoter 1,4-bis[2-(3,5-dichloropyridyloxy)]benzeneresulted in a high incidence and multiplicity of hepatomas by10 months. In the absence of the promoter, administration ofHOS for only 1 month induced no hepatomas; 1,4-bis[2-(3,5-dichloropyridyloxy)]benzenealone induced only a low incidence. In mice not given the promoter,continuous administration of HOS     

Dose-dependent promoting activity of chloroform in rat liver foci bioassay

Chloroform enhances dose-dependently the number of preneoplastic foci in livers of weanling female Sprague-Dawley rats. The preneoplastic foci were induced with a single dose of 8 mg diethylnitrosamine (DEN)/kg body wt. Thereafter chloroform was applied twice weekly for 11 consecutive weeks in doses of 100, 200 and 400 mg/kg body wt, respectively. This treatment raised the number of adenosine-5'-triphosphatase (ATPase)-deficient foci up to 5-fold, that of gamma-glutamyltranspeptidase (GGTase) and glycogen-positive foci 13- and 10-fold, respectively, after 12 weeks; 25 mg caused no effect compared to DEN-treated controls. In contrast, daily doses of chloroform only, 200 and 400 mg/kg body wt for 33 days, and 800 mg/kg body wt for 20 days given to 3-4-week-old female Sprague-Dawley rats did not lead to island formation, measured after 12 weeks, indicating a promoting rather than an initiating potency.     

Lipid peroxidation of liver microsome membranes induced by choline-deficient diets and its relationship to the diet-induced promotion of the induction of gamma-glutamyltranspeptidase-positive foci

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.     

Effect of six edible plants on the development of AFB1-induced gamma-glutamyltranspeptidase-positive hepatocyte foci in rats

Six edible plants, green tea (GT), black tea (BT), Lentinus edodes (berk) Sing (LE), Hericium erinaceus (Bull. ex Fr.) Pers. (HE), Mixture of Ganoderma Lucidum (Ley ss ex Fr.) Karst et Ganoderma Japanium (Fr.) Lloyd (MGLJ) and mung bean (MB), were tested for the effect on the development of AFB1-induced gamma-glutamyltranspeptidase positive hepatocyte foci (gamma-GT foci) using an in vivo short-term test model in rats. The rats received intraperitoneally 12 doses of initiator AFB1, 400 micrograms/kg per dose for 2 successive weeks. Two weeks after the initiation, the rats were submitted to a modified "Solt-Farber promotion program", i.e., a two weeks' feeding of a diet containing 0.015% acetylaminofluorene plus a two-third partial hepatectomy (PH) on day 7. The rats were sacrificed 10 days after PH and the livers were processed to gamma-glutamyltranspeptidase staining. The tested substances were powdered and mixed with the basal diet at the concentration level of 30% for MB and 5% for the others. The rats were fed with the diet-containing tested substances from 10 days before the AFB1 initiation to 3 days after the AFB1 conclusion. Consequently, the liver of the rats which had consumed GT showed significantly less and smaller gamma-GT foci, and those which had consumed BT, HE and LE showed somewhat less and significantly smaller foci than the control groups. It is indicated that the four diets have an inhibiting effect on AFB1-induced gamma-GT foci in different degrees. MB and MGLJ show no significant influence on the foci.     

Synergistic effects of low-dose hepatocarcinogens in induction of glutathione S-transferase P-positive foci in the rat liver

The effects of combined administration of hepatocarcinogens at low doses on the development of glutathione S-transferase P-form (GST-P)-positive foci of rat liver were examined utilizing a bioassay model which consists of a single injection of diethylnitrosamine (DEN, 200 mg/kg, ip), two-thirds partial hepatectomy at week 3 and a 6-week administration of test compounds. The chemicals used, 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), phenobarbital (PB), thioacetamide (TAA), N-ethyl-N-hydroxyethylnitrosamine (EHEN), benzo[a]pyrene (B[a]P), carbazole, and alpha-hexachlorocyclohexane (alpha-HCH) were incorporated in the diet, except for EHEN which was dissolved in the drinking water, at levels of 1/6 of the doses usually used. The combinations were: I) 2-AAF, 3'-Me-DAB, PB, TAA, EHEN and B[a]P, II) 2-AAF, 3'-Me-DAB and PB, III) TAA, EHEN and B[a]P, IV) 2-AAF, 3'-Me-DAB, carbazole, TAA, EHEN and alpha-HCH, V) 2-AAF, 3'-Me-DAB and carbazole, and VI) TAA, EHEN and alpha-HCH. All combinations, except for II, caused an increase in the area of the foci as evaluated by the ratios of areas in the combined administration groups to the sum totals of 3 or 6 individual data: I) 1.75, II) 0.81, III) 2.01, IV) 3.62, V) 1.34 and VI) 2.91. The non-synergistic effect in combination II might be related to PB induction of hepatic microsomal enzymes leading to enhanced enzymatical detoxification of 2-AAF and 3'-Me-DAB. The present results indicate that exposure to several chemicals of similar organotropism, even at doses lower than the apparent carcinogenic levels, might be critical to the carcinogenic process.     

Screen of five alkyl carbamates for initiating and promoting activity in rat liver.

Five alkyl carbamates, methyl, ethyl, propyl, hydroxypropyl and ethylhexyl, were tested for initiating and promoting activity in rat liver. The test for initiating activity consisted of administering the carbamate by gavage to male Sprague--Dawley rats at either 6 or 18 h after a 2/3 partial hepatectomy. One week later, the rats received 500 ppm sodium phenobarbital in their drinking water until killed 10 weeks later. None of the carbamates at 1/5 the LD50 induced gamma-glutamyltranspeptidase (GGT)-positive foci, indicating the lack of initiating activity. The tumor promoting activity test consisted of initiation with 80 mmol/kg diethylnitrosamine administered 18 h after a partial hepatectomy. One week later, the rats received one of the the carbamates at either 1/10 or 1/20 the LD50 5 days per week until sacrificed 10 weeks later. The alkyl carbamates increased the volume of the foci, the percent of the liver occupied by the foci, the number of foci/cm3 (except ethylhexyl carbamate), and the number of foci/liver (except ethylhexyl carbamate). These results suggest that the alkyl carbamates are tumor promoters and not tumor initiators in rat liver.     

The rat liver foci bioassay: I. Age-dependence of induction by vinyl chloride of ATPase-deficient foci

The age-dependence of the induction of pre-neoplastic enzyme-alteredhepatic foci was investigated. Rats were exposed (8 h/day, 7days/week) to 2000 p.p.m. vinyl chloride (VC) either ‘transplacentally’(exposure of pregnant females), or immediately after birth fordifferent time intervals (5, 11, 17, 47, 83 days) or from anage of 7 or 21 days onwards. The animals were then kept withoutfurther treatment; livers were evaluated for ATPase-deficientfoci at the age of 4 months. ‘Transplacental’ exposureand exposure from day 1 through 5 caused no increase over controlsin ATPase-deficient foci, probably due to the lack of hepatocellularproliferation and the low rate of VC metabolism at this developmentalstage. However, fod area was steeply increased when newbornrats were exposed for 11 and 17 days; but this was not furtherenhanced by a 47- or 83-day exposure. Only a few ATPasedeflcientfoci occurred when exposure started 21 days after birth. Exposureof adult rats did not result in more ATPasedeficient fod thanwere seen in untreated controls; control values could not beincreased by a proceeding partial hepatectomy. The results indicatethat the induction of pre-neoplastic hepatocellular lesionsin rats by VC is restricted to a well defined period ({smalltilde}day 7–21) in the early lifetime of the animals.This period of highest sensitivity is characterized by the beginningof rapid liver growth.     

Influence of dietary tryptophan on the induction of gamma-glutamyltranspeptidase-positive foci in the livers of rats treated with hepatocarcinogen

The ingestion of an elevated level (2%) of L-tryptophan (TRP) in a purified diet was investigated to determine whether it would influence the induction of gamma-glutamyltranspeptidase (GGT)-positive foci in the livers of rats exposed to a hepatocarcinogen. Subtotal hepatectomies were performed, and 18 h later, the rats were given injections i.p. of diethylnitrosamine (30 mg/kg). Ten days later, groups of male rats were placed on choline-supplemented (CS), CS + TRP, choline-deficient (CD), or CD + TRP diets for 10 wk. In two separate experiments, the rats fed the CS + TRP diet or the CD diet developed more and larger GGT + foci than did rats fed the CS diet. Rats fed the CD + TRP diet revealed similar changes to those found in rats fed the CD diet. The liver weights of the rats fed the CD or the CD + TRP diet were greater than those of rats fed the CS or the CS + TRP diet. Hepatic GGT activity was somewhat elevated in rats fed the CS + TRP diet and markedly elevated in rats fed the CD or the CD + TRP diet. Hepatic ornithine decarboxylase activity was increased in rats fed the CD + TRP diet. The results suggest that increased dietary tryptophan has a promoting effect on liver carcinogenesis as measured by the induction of GGT + foci in the livers of rats exposed to diethylnitrosamine. A potentiating effect by tryptophan was not observed in the livers of rats fed a CD diet.     

Effects of rat strain, diet composition, and phenobarbital on hepatic gamma-glutamyl transpeptidase histochemistry and on the induction of altered hepatocyte foci and hepatic tumors by diethylnitrosamine

To extend our ongoing characterization of modulatory influences on hepatic tumorigenesis, we examined effects of rat strain (Sprague-Dawley versus Fischer), diet composition (semipurified diet versus standard nonpurified laboratory chow), and dietary phenobarbital on the production of gamma-glutamyl transpeptidase (GGT)-positive hepatocyte foci and hepatic tumors initiated by diethylnitrosamine. In addition to GGT-positive foci, we observed, under certain conditions, the appearance of extensive hepatic GGT staining not associated with focal lesions. This elevated nonfocal GGT was found in rats of both strains fed the nonpurified rather than the purified diet, but the level of staining was higher in Fischer than in Sprague-Dawley rats. Enhancement of this nonfocal staining by dietary phenobarbital appeared insignificant. By comparison, frequencies of GGT-positive foci were generally higher in rats fed the semipurified rather than the nonpurified diet, and the frequencies of GGT-positive foci were invariably higher in Sprague-Dawley than in Fischer rats. Moreover, dietary phenobarbital generally enhanced focus production. Assessments of focus and tumor yields among these experimental groups showed that differences in focus frequencies did not correspond closely to differences in subsequent tumor formation. These results document the need to consider the influences of diet and rat strain on experimental end points in designing protocols for hepatocarcinogenesis studies, especially those involving GGT histochemistry. The data also raise questions about the mechanistic relevance of GGT induction to hepatocarcinogenesis and support our prior evidence against the putative lineal relationship between foci and tumors.     

Effects of 3-aminobenzamide on the induction of gamma-glutamyl-transpeptidase-positive foci by various chemicals in rat liver

The effects of 3-aminobenzamide (ABA), a representative inhibitor of poly(ADP-ribose)polymerase, on the induction of gamma-glutamyl-transpeptidase (GGT)-positive foci, an early lesion occurring during hepatocarcinogenesis, in rat liver by various chemicals were studied in Fischer 344 and Wistar rats. ABA exhibited an enhancing effect on the induction of GGT-positive foci by benzo[a]pyrene, but no effects on induction by a methylating agent, N-methyl-N-nitrosourea, in both rat strains. In contrast, the induction of GGT-positive foci by another methylating agent, 1,2-dimethylhydrazine, was enhanced by ABA in Wistar rats, but not affected in Fischer rats. There were no effects of ABA on the induction of GGT-positive foci by N-bis(2-hydroxypropyl) nitrosamine in Wistar rats. These results indicate a differential effect of ABA on the induction of GGT-positive foci by different chemicals in different rat strains.     

Comparison of enhancement of GGTase-positive foci and induction of ornithine decarboxylase in rat liver by barbiturates

The induction of ornithine decarboxylase (ODC) by barbituratesand the ability of barbiturates to enhance neoplastic progressionof chemically initiated cancer was examined in rat liver. Allseven barbiturates induced ODC with barbital (7.7 fold increase)and phenobarbital (5.7 fold increase) demonstrating the mostpotent activity. Maximum induction of ODC by phenobarbital wasobtained in 18 h. Barbital (500–5000 p.p.m.) and phenobarbital(500 p.p.m.) administered in the drinking water enhanced theappearance of diethylnitrosamine (DENA)-initiated -glutamyltranspeptidase(GGTase)-positive foci. Amobarbital, hexabarbital and pentabarbitaldid not enhance the appearance of GGTase-positive foci. In theabsence of previous initiation by DENA, the enhancing regimenof the barbiturates did not cause the appearance of GGTase-positivefoci. Barbiturates induced ODC activity in rat liver and enhancedthe incidence of DENA initiated GGTase-positive foci.     

Rapid lipid peroxidation in the nuclear fraction of rat liver induced by a diet deficient in choline and methionine

A diet deficient in choline and methionine, known to produce hepatocellular carcinoma in the absence of any added chemical carcinogen, induced lipid peroxidation in the nuclear fraction of the liver when fed to male Fischer 344 rats. This lipid peroxidation was detected within 1 day of feeding the diet by the appearance of diene conjugates and increased progressively up to 3 days. It was prevented completely by the addition of choline chloride to the diet. The close proximity of DNA may make it a possible target for attack by free radicals.     

Lack of rapid initiating, promoting or sequential syncarcinogenic effects of di(2-ethylhexyl)phthalate in rat liver carcinogenesis

The effect of prolonged dietary administration of the peroxisomeproliferating plasticizer di(2-ethylhexy1)phthalate (DEHP wasstudied on liver carcinogenesis initiated by N-2- fltuorenylmxhmide(FAA) and with that of the neoplasm-promoter phenobarbital (PB).Also, DEHP was studied as an initiator by giving it in placeof FAA before PB. Male rats were fed FAA for 7 weeks to inducebepatocellular altered foci, and were subsequently given nochemical, 12 000 p.p.m. DEHP or 500 p.p.m. PB for 24 weeks inthe diet. In the rats fed DEHP, substantial hepatomegaly andperoxisome proliferation were induced. No evidence of indudionof hepatacellular altered foci or hepatic neoplasms was foundeither when DEHP was given alone for 24 weeks or for 7 weeksfollowed by PB. Also, DEHP fed for 24 weeks had no promotingeffect on liver altered foci that were induced by FAA and producedlittle or no enhancement of the occurrence of FAA-induced liverneoplasms. In contrast, PB exerted a marked enhancing effecton foci and substantially increased the incidence and multiplicityof liver neoplasms. Thus, the findings demonstrate that DEHPdid not have either a rapid initiating activity, a significantsequential syncarcinogenic activity, or a promoting effect onliver carcinogenesis under conditions in which numerous agentswith such activities have been identified.