Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis,the induction and growth of -glutamyltranspeptidase (GGT)-positivefoci and their evolution into persistent nodules, were analyzedin the liver of rats treated with diethylnitrosamine (DEN).The induction of GGT-positive foci was inhibited by a diet containing0.36–1.5% VE given after initiation with 200 mg/kg bodyweight (b.w.) DEN for 6 weeks with partial hepatectomy (PH)on week 3. The numbers and areas of GGT-positive foci were enhancedby diets containing 036 and 0.72% VE, given for 1 week afterinitiation with 10 mg/kg b.w. DEN and PH, followed by selectionby 0.02/ 2-acetylaminofluorene (AAF) and carbon tetrachloride(CCl⁴), but these were not enhanced by a diet containing 1.5%VE. Remodeling of hyperplastic nodules was not affected by thediet containing 0.72% VE given after initiation with DEN andselection for 12 weeks. The staining characteristics of GGTwere different between remodeling and persistent nodules, exceptfor those of the glutathione-S-transferase placental form (GST-P).The results obtained suggest that VE could prevent the veryearly events during hepatocarcinogenesis, the induction of phenotypicallyaltered foci, but could no longer affect the later stages, theevolution of foci into persistent nodules.     

Enhancing effect of co-administration of polychlorinated biphenyls and diethylnitrosamine on enzyme-altered islands induced by diethylnitrosamine in rat liver

The effect of co-adminstration of diethytnitrosamine (DEN) andClophen A 50, a commercial mixture of polychlorinated biphenyls(PCB), on pre-neoplastk enzyme-altered islands in livers offemale Sprague-Dawley rats was studied. The islands were identifiedby the loss of adenosine-5'triphos-phatase (ATPase), emergenceof gamma-glutamyltranspep-tidase (GGTase) and glycogen storageafter fasting. DEN was given p.o. (0.4 or 4 mg/kg body wt respectively)twice a week for 11 consecutive weeks. Clophen A 50 (1 or 5mg/kg body wt respectively) was given alternatively three timesa week for 11 weeks. Four groups of rats each received eitherDEN or PCBs in the respective doses. Control animals were treatedwith the vehicle or remained untreated. All animals were killedat week 12. In rats treated with 4 mg DEN/kg body wt 80 ATPase-defidentislands/cm2 were observed. Additional treatment with ClophenA 50 enhanced the island number 3-fold. Treatment with 0.4mg/kg body wt DEN induced 17 islands/cm². Additional applicationof Clophen A 50 enhanced the island number 3-fold. The totalisland area was enhanced to the same extent in both groups.The island incidence in PCB-treated rats and controls was belowI/cm² with all markers tested. The results indicate that PCBsmay exhibit a co-carcinogenic activity.     

The natural history and dose-response characteristics of enzyme-altered foci in rat liver following phenobarbital and diethylnitrosamine administration

Enzyme-altered foci (EAF) were induced in the Liver of femalerats by 70? partial hepatectomy (PH), followed by a single intragastricadministration of diethylnitrosamine (DEN) at a dose of 10 mg/kg.The stability and response of these foci to various doses ofthe hepatic promoting agent, phenobarbital (PB), were studied.The number of yglutamyltranspeptidasepositive (GGT⁺) EAF resultingfrom PH/DEN followed by PB (0.05%) administration for 1 week,2 weeks, 1 month, 2 months, 3 months or 4 months did not significantlychange when the administration of the promoting agent was followedby a 6-month period of a diet containing no PB. These data demonstratethe stability of the fwi induced by the PH/DEN/PB regimen andindicate that the increased number of foci resulting from PBpromotion in the absence of overt hepatic necrosis are not reversibleon removal of the promoting stimulus. Chronic administrationof dose levels of PB below 0.001% in the diet failed to demonstratean increase in the number of EAF over the number in the controlanimals not promoted with PB. A linear increase in the numberof EAF was observed when rats were chronically fed doses ofPB ranging between 0.001% and 0.05% in the diet, whereas dietconcentrations of PB > 0.05% did not result in any furtherincrease in the number of EM. The number of EAF resulting fromPH/DEN followed by 0.05% PB in the diet increased during thefirst 3–4 months of promotion. Thereafter, the numberof foci did not change despite the continued administrationof PB for as long as 8 months. These data suggest the presenceof an apparent threshold (no effect level) for promotion byPB and demonstrate the presence of a maximal response of EAFto this promoting agent after initiation by a single dose ofDEN.     

Sex-dependent promoting effect of polychlorinated biphenyls on enzyme-altered islands induced by diethylnitrosamine in rat liver

The promoting effect of Clophen A 50, a commercial mixture ofpolychlorinated biphenyis (PCBs) on preneoplastic islands, initiatedby diethylnitrosamine (DEN), was studied in male and femaleSprague-Dawley rats. The islands were identified histochemicallyby loss of adenosine-5'-triphosphatase (ATPase) and/or emergenceof gamma-glutamyltranspeptidase (GGTase). Treatment with 12x 8 mg DEN/kg body wt./day initiated a similar number and totalarea of islands in males and females. Additional weekly applicationof Clophen A 50 (50 or 100 mg/kg body week, for 7 weeks) enhancedthe number of ATPase-deficient islands 3-fold in males and 9-foldin females. The total area was increased 4-fold in males and15-fold in females. Number and area of GGTasepositive islandswere similarly enhanced. The emergence of a small number ofislands after application of Clophen A 50 alone may indicatea weak carcinogenic potency. PCB treatment caused an increasein liver weight, which amounted to 55% in males and 20% in femalescompared to controls. This increase is partly due to cell hypertrophy,as indicated by determination of cell size. The mitogenic activityof Clophen A 50 was evaluated by measurement of the mitoticindex of unaltered hepatocytes at 24, 48 h, and 7 days afterapplication of a single dose (100/mg/kg body wt.) of ClophenA 50. The mitotic index in control animals of both sexes was0.3%, and was enhanced 8-fold in males, 24 h after PCB treatment.In females only a slight, non-significant increase was observed.The results indicate that the sex-dependent promoting effectof Clophen A 50 is independent from its mitogenic action.     

Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver

Aroclor 1254 is a complex mixture of polychlorinated biphenyls (PCB) that upon prolonged administration has been reported to produce hepatic tumors in mice and rats. The ability of Aroclor 1254 to promote enzyme-altered foci was determined in an initiation/promotion bioassay in rat liver. Initiation was accomplished in rats that received a 2/3 partial hepatectomy followed in 24 h by diethylnitrosamine (DENA). Aroclor 1254 was administered to each rat 7, 28 and 49 days after the DENA and some of the rats were killed 21 days after each dose of Aroclor. The liver of rats that received Aroclor 1254 on either day 7 or on day 7 and 28 contained an increased incidence of gamma-glutamyltranspeptidase (GGTase)-positive foci compared to partial hepatectomized and DENA treated rats given tricaprylin (the solvent for Aroclor 1254). Therefore, Aroclor 1254 was demonstrated to enhance the appearance of enzyme-altered foci after only a single oral dose.     

Inhibitory effect of nafenopin upon the development of diethylnitrosamine-induced enzyme-altered foci within the rat liver

The effect of nafenopin and phenobarbitone upon the distributionof -glutamyltranspeptidase activity and epoxide hydrolase antigenicsites in the liver and upon the development of enzyme-alteredfoci during hepatocarcinogenesis have been compared. Phenobarbitoneinduced -glutamyltranspeptidase activity in perilobular hepatocytes.Nafenopin did not alter the distribution of this enzyme. Bothcompounds appeared to induce epoxide hydrolase; phenobarbitoneincreased the enzyme content of centrilobular cells, whilstnafenopin altered immunostaining mainly in portal regions. Hepaticlesions were induced by treating one day-old rats with diethylnitrosamine.Phenobarbitone and nafenopin were then administered in the dietupon weaning. Animals were killed after either 2, 4 or 8 weeksfeeding and liver sections were stained for the two enzymes.Only sections from nitrosamine-treated animals contained enzyme-alteredfoci. In general, -glutamyltranspeptidase-containing foci stainedalso for epoxide hydrolase; but many hydrolasepositive focidid not stain for -glutamyltranspeptidase activity. Phenobarbitonetreatment stimulated the formation of enzymealtered foci. Thiseffect was more marked in male animals. Nafenopin treatmentsuppressed the development of foci at all time points, suchthat less hepatic lesions were seen than in animals which receivedonly diethylnitrosamine. The results cast doubt upon the generalityof -glutamyltranspeptidase as a marker for preneoplastic lesionswithin the liver.     

Lack of acetylaminofluorene-DNA adduct formation in enzyme-altered foci of rat liver

Formation of the N-(deoxyguanosin-8-yl)-aminofluorene adductwas studied in enzyme-altered foci induced by four differentliver carcinogenesis models. Foci were detected and scored forenzyme phenotype by a computer-aided image overlay technique.Localization of the enzymes -glutamyl transpeptidase, canalicularATPase and glucose-6-phosphatase was performed by enzyme histochemistry,allowing identification of foci of seven different phenotypes.Patterns of foci obtained by image overlay were compared toin situ 2-acetylaminofluorene-DNA adduct distribution obtainedby immunofluorescence. Foci were induced by the following models:(1) chronic feeding of 0.02% 2-acetylaminofluorene (2-AAF) for8 weeks; (2) intubation of diethylnitrosamine (DEN) (10 mg/kg)24 h after a 70% partial hepatectomy (PH), followed 8 weekslater by a diet containing 0.05% phenobar-bital for 9 months;(3) intubation of DEN (10 mg/kg) 24 h after PH, followed bya diet containing 0.01% ciprofibrate for 5 months, and afteran additional 4 months a diet containing 0.05% phenobarbitalfor 2 months; (4) maintenance for 7.5, 16.5 or 19.5 months aftertransplantation of DEN/2-AAF/PH (‘Solt-Farber’ protocol)donor liver cells into host rats receiving a brief 2-AAF/PHselective regimen then no further treatment until sacrifice.To test the capacity of both foci and morphologically normallivers to form DNA adducts, the animals in models 2–4received a diet containing 0.02% 2-AAF for 5 or 6 days beforesacrifice. In all of the enzyme-altered foci identified in models1–3 there were no DNA adducts visible by immunofluorescence.Scattered groups of positive cells were occasionally seen inthe otherwise dark foci induced by model 4. For technical reasonssome enzyme-altered foci were not identifiable on the fluorescence-stainedslides. In liver serial sections from rats in models 1–4,there were 75, 304, 125 and 68 enzyme-altered foci of sevendifferent phenotypes which were identified as AF-DNA negative.In models 1 and 4 there were some additional adduct-negativefoci not associated with any of the seven identified focus phenotypes.These studies demonstrate that loss of the ability to form DNAadducts in hepatic enzyme-altered foci is a common and veryearly biochemical adaptation to xenobiotic exposure in differenthepatocarcinogenesis models. This adaptation also is retainedby the majority of foci in later stages of hepatocarcinogenesis.     

Application of quantitative stereology to the evaluation of enzyme-altered foci in rat liver

The mathematical science of quantitative stereology has established relationships for the quantitation of elements in three-dimensional space from observations on two-dimensional planes. This report describes the utilization and importance of such mathematical relationships for the quantitative analysis of focal hepatic lesions in terms relative to the volume of the liver. Three examples are utilized to demonstrate the utility of such calculations in the three-dimensional quantitation of hepatic focal lesions. The first is that of a computer-simulated experiment based on defined hypothetical situations. The simulations demonstrate the applicability of the computations described in this report to the evaluation of two-dimensional data from typical animal experiments. The other two examples are taken from actual experiments and involve the transplantation of hepatic cell populations into the liver suitably prepared hosts and the quantitation of altered foci produced by initiation with diethylnitrosamine-partial hepatectomy followed by promotion with phenobarbital. The quantitation of altered foci by means of a two-dimensional analysis (simple enumeration of focal intersections/area of tissue section) is proportional to the quantitation of foci per volume of liver provided that the mean diameter of the foci for each treatment is sufficiently uniform, as exemplified in the text by the transplantation experiment. When such mean diameters are unequal as in the diethylnitrosamine-phenobarbital experiment described herein, quantitation from three-dimensional analysis gives significantly different results as compared with enumeration of focal intersections on two-dimensional areas. These studies clearly demonstrate that the frequency and size of foci intersections viewed on two-dimensional tissue sections do not necessarily reflect the number of size of foci in the three-dimensional tissue. Only by quantitating the number and size of the foci in relation to the three-dimensional volume of the tissue can one determine the validity of the proportionality of data from two-dimensional measurements to the total number of foci per volume of tissue. Such a conclusion has important implications for quantitative studies on hepatocarcinogenesis as well as for the enumeration of premalignant lesions which occur during the natural history of carcinogenesis in any solid tissue.     

Comparison of three rat liver foci bioassays--incidence of preneoplastic foci initiated by diethylnitrosamine

Three rat liver foci bioassays have been compared with respect to their sensitivity by the histochemical demonstration of preneoplastic foci, and by the biochemical determination of alterations in enzyme activities of serum indicating hepatotoxicity. We studied the initiation/promotion schedules according to Oesterle and Deml (A), and according to Pereira (B, Broad Spectrum Protocol), and the initiation/selection protocol according to Tatematsu et al. (C), with diethylnitrosamine (DEN), given as a single initiating dose of 10 and 30 mg/kg body wt respectively. With all schedules Sprague-Dawley rats, either females, 3 weeks old (A), or males, 6 weeks old (B, C) were used. For promotion polychlorinated biphenyls (A) or phenobarbital (B) were administered. Selection was performed with 2-acetylaminofluorene (C). The rats in schemes (B) and (C) underwent partial hepatectomy one day prior to initiation. The number and total area of foci deficient in adenosine-5'-triphosphatase (ATPase) and positive in gamma-glutamyltranspeptidase (GGTase) was evaluated. In the complete schedule with 30 mg of DEN in system (A) foci incidence exceeded that of the other systems by about 7-fold (ATPase) and 2-fold (GGTase) respectively. The lower dose of DEN and all control experiments resulted in a respective lower foci yield. With scheme (C), but not with schemes (A) and (B), e.g. serum fructose-1.6-bisphosphatase and alkaline phosphatase were increased, suggesting liver cell damage. Thus tested with DEN, scheme (A) is most sensitive and causes a low impairment of animals' welfare.     

Chlorobenzilate-induced effects on enzyme-altered foci in rat liver and intercellular communication in rat liver WB-F344 epithelial cells

The mouse liver carcinogen chlorobenzilate (CB), a DDT-related pesticide, was investigated for enhancement of enzyme altered foci incidence in partially hepatectomized, diethyl-nitrosamine-initiated rats. In this in vivo experiment, CB administered per os (25 or 100 mg/kg per day for 10 weeks) enhanced foci incidence at the high dose level. In order to study potential mechanisms involved, CB was investigated for inhibition of gap-junctional intercellular communication in rat liver epithelial WB-F344 cells and Chinese hamster V79 cells in vitro. CB abolished dye transfer in WB-F344 cells and inhibited metabolic cooperation in V79 cells. Two CB metabolites were unable to induce such tumor promotion related effects. The results of this investigation provide support for the involvement of an epigenetic, tumor promoting mechanism in CB-induced liver tumors in laboratory animals.     

Inhibitory effect of dietary iron deficiency on the induction of putative preneoplastic foci in rat liver initiated with diethylnitrosamine and promoted by phenobarbital.

The effects of dietary iron deficiency on induction of putative preneoplastic, gamma-glutamyltransferase (GGT)-positive hepatocyte focal lesions in the liver of rats treated with diethylnitrosamine (DEN) followed by phenobarbital (PB) were investigated. Male Fischer 344 rats of 4 weeks old were placed on an iron deficient (ID) diet containing less than 5 p.p.m. of iron or an iron supplemented (IS) diet containing 180 p.p.m. of iron throughout experimental period of 12 weeks. Both groups of rats were administered 200 mg kg-1 body weight of DEN by a single intraperitoneal injection at Week 4 followed by PB mixed into each diet at a concentration of 0.05% from Week 6 to the final sacrifice at Week 12 when induction of GGT-positive foci was quantitatively analysed. On the ID and IS diets, respective numbers of GGT-positive foci were 6.3 and 14.2 cm-2. The sizes of foci were not altered by the iron content of the diet. The present results indicate that iron plays a role in the development of preneoplastic foci in the livers of rats initiated with DEN and promoted by PB especially in the initiation phase.     

Application of quantitative stereology to the evaluation of phenotypically heterogeneous enzyme-altered foci in the rat liver

Quantitative stereologic relationships are applied in this report to the evaluation of F344 rat liver foci where the tissue sections exhibit congruent enzyme-altered areas of the several different phenotypes as well as enzyme-altered areas within a larger area of another enzyme alteration, that is, a "focus within a focus.' Quantitation of both the numbers and volume occupied by each of the phenotypes of the enzyme-altered foci was accomplished by the unique logic described in this report. The application of this logic to four representative experimental protocols with the use of three phenotypic markers demonstrated all possible congruent phenotypes as well as a small number of "foci within foci.' The variance of the quantitation of the experimental data was shown to depend on the number of focal transections identified in the sections, the number of sections examined, and the distribution of phenotypic alterations among foci.     

The effect of the dose of diethylnitrosamine on the initiation of altered hepatic foci in neonatal female rats

The dose—response characteristics of initiation of hepatocarcinogenesisby diethylnitrosamine (DEN) was investigated in the neonatalfemale rat by means of the quantitative stereologic estimationof altered hepatic foci (AHF) expressing multiple markers. At5 days of age, female Sprague—Dawley rats were given asingle i.p. dose of DEN (0.1–30 mg/kg body wt) or thevehicle (trioctanoin). The semisynthetic AIN-76A diet was providedto half of the rats in each treatment group, while the remainderreceived this diet containing 500 mg phenobarbital (PB)/kg for8 months from weaning until the animals were killed. To ascertainmore exactly the dose—response relationship for initiationby DEN, the number, volume percentage and phenotypes of theresulting AHF were determined by quantitative stereologicalanalysis on serial sections of frozen tissue, each stained forone of four markers of preneoplasia. A linear relationship wasobserved between the dose of DEN (0–30 mg/kg) and thenumber and volume percentage of AHF detected, with each singlemarker or the total number of AHF detected when the placentalisozyme of glutathione S transferase,     

Vinyl acetate, a structural analog of vinyl carbamate, fails to induce enzyme-altered foci in rat liver

The development of hepatic enzyme-altered foci (ATPase, GGTase)was investigated after dosing vinyl acetate (200 and 400 mg/kgper day, orally) to newborn rats for 3 weeks, with or withoutsubsequent promotion by phenobarbital. Whereas the structurallyrelated compounds vinyl carbamate and vinyl chloride induceenzyme-altered foci under comparable experimental conditions,no foci were observed in vinyl acetatetreated animals at theage of 14 weeks. This is consistent with investigations on metabolismand pharmacokinetics of vinyl acetate which show that this compound,after entering the organism, is immediately split by blood esterasesand thus may not be available for epoxidation to an ultimatelycarcinogenic metabolite.     

The rat liver foci bioassay: II. Investigations on the dose-dependent induction of ATPase-deficient foci by vinyl chloride at very low doses

In order to study the dose-dependence of the genotoxic effectof vinyl chloride (VC) hepatocellular ATPase-deflcient fociwere evaluated after subchronic exposure of newborn rats. Wistarrats were exposed from day 1 after birth over 10 weeks to 10,40, 70, 150, 500 and 2000 p.p.m. VC (8 h/day; 5 days/week).One week after cessation of exposure hepatic ATPase-deficientfoci were quantitated. For a subsequent investigation lowerdose range groups of female and male Wistar and Sprague-Dawleyrats were exposed (8 h/day; 5 days/week) to 2.5, 5, 10, 20,40 and 80 p.p.m. VC. Exposure started at day 3 of life and lastedfor 3 weeks. After cessation of exposure the animals were maintainedfor 10 weeks without further treatment until ATPase-deflcientfoci were quantitated. Both sets of experiments revealed a straightlinear relationship between the dose of VC and the % foci areainduced. Within the dose range investigated, no obvious thresholdfor the induction of pre-neoplastic foci by VC was observed.     

Cell proliferation and promotion of rat liver carcinogenesis: different effect of hepatic regeneration and mitogen induced hyperplasia on the development of enzyme-altered foci

A series of experiments was performed to investigate the effectof different types of cell proliferation on the developmentof enzyme-altered preneoplastic hepatic foci in male Wistarrats. Animals were given a single dose of diethylnitrosamine(100 mg/kg body weight). After a 2-week recovery period livercell proliferation was repeatedly induced by four or eight necrogenicdoses of carbon tetrachloride (compensatory cell proliferation),or by four or eight treatments with three different liver mitogens,namely lead nitrate, ethylene dibromide and nafenopin (directhyperplasia). The carcinogen altered hepatocytes were monitoredas --glutamyltransferase positive or adenosine triphosphatasenegative foci. The results indicate that compensatory cell proliferationinduced by both four and eight carbon tetrachloride treatmentsenhanced the growth of diethylnitrosamine-initlated hepatocytesto enzyme- altered foci. On the contrary, repeated waves ofcell prolifera tion induced by liver mitogens did not resultin any significant number of enzyme-altered foci.     

Strain and species differences in the induction of ATPase-deficient hepatic foci by diethylnitrosamine

Diethylnitrosamine (5 mg/kg) was intraperitoneally administered to rats, mice, guinea pigs and tupaias daily for 3 weeks. After 12 weeks of cessation, focal areas of ATPase-deficiency of hepatocytes appeared only in rats and, to a much lesser extent, in guinea pigs. Female rats were more sensitive than males and Wistar rats were less sensitive than Sprague-Dawley rats. These data show that quantitation of ATPase-deficient foci as a determinant of hepatocarcinogenicity is mainly restricted to the rat species.     

Quantitative analysis of enzyme-altered foci in rat hepatocarcinogenesis experiments--I. Single agent regimen

Considerable recent attention has focused on the quantitativeanalysis of enzyme-altered foci in rodent hepatocarcinogenesisexperiments. These foci are believed to represent clones premalignantcells. A method is presented for the quantitative analysis ofthese foci that takes into account both the total number offocal transections observed in each liver crosssection and thesize distribution of these transections. The method, which hasa natural interpretation within the framework of a two-mutationmodel for carcinogenesis, yields estimates of rates of initiationand of growth rates of enzyme-altered foci as functions of doseof the agent under consideration. Definitions of initiationand promotion potencies are proposed. The method is illustratedby application to an experiment in which rats were administeredN-nitroso morpholine at various concentrations in their drinkingwater.