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.     

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.     

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).     

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.     

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.     

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.     

Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci

Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet.     

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.     

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.     

Potential initiating and promoting activities of diacetyl and glyoxal in rat stomach mucosa

The potential initiating and promoting activities in the rat glandular stomach of the dicarbonyl compounds diacetyl (DA) and glyoxal (G), which are found in various heated foods, were studied. Administration of DA at doses of 300 to 1500 mg/kg body weight and of G at doses of 150 to 400 mg/kg body weight by gastric intubation to male F344 rats induced up to 100-fold increase in ornithine decarboxylase activity (formation of 195 pmol CO2/30 min/mg protein by DA and 302 pmol CO2/30 min/mg protein by G) with maxima after 16 hr. These treatments also induced a more than 10-fold increase in DNA synthesis (incorporation of 11,400 dpm of [3H]dThd/microgram DNA by DA and 15,100 dpm of [3H]dThd/microgram DNA by G) with maxima after 16 hr, and induced apparent unscheduled DNA synthesis in the pyloric mucosa of the stomach within 3 hr after administration. These results suggest that DA and G have potential tumor-promoting activities and may also have initiating activities in carcinogenesis in the glandular stomach.     

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.     

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.     

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.     

Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver

The effect of a low dose of preadministered diethylnitrosamine(DEN) on the induction of enzyme-altered foci in the liversof male full-grown Fischer 344 rats was studied. As a pretreatment,DEN at a dose of 10 mg/kg body wt was injected i.p. At varioustimes after DEN pretreatment a complete initiation, consistingof administration of the same dose of DEN by the same routein rats subjected to partial hepatectomy (PH), was performed,followed by application of selection pressure. Enzyme-alteredfoci stained with -glutamyltrans-peptidase (-GTP) and glutathioneS-transferase placental form (GST-P) were then assayed. Decreasesin the numbers and areas of foci in the rats which receivedsaline + PH 14 or 28 days after DEN pretreatment were observedin comparison with rats which received saline + PH immediatelyafter DEN. On the other hand, the numbers and areas of fociwere not decreased in rats which received the complete initiation,consisting of DEN + PH, at various times after DEN pretreatmentwhen compared with rats which received these at the same timeas the DEN pretreatment. This persistent effect of DEN pretreatmenton the complete initiation lasted up to 182 days after the timeof DEN pretreatment. In this experiment, GST-P was found tobe a more sensitive marker for the detection of putative preneoplasticliver-cell foci than -GTP.     

Comparative tumor initiating activities of N-nitrosomethylbenzylamine and N-nitrosodimethylamine in rat liver

The tumor initiating activities of nitrosomethylbenzylamine(NMBzA), an esophageal carcinogen, and nitrosodimethylamine(NDMA), a hepatocarcinogen, were compared in rat liver usingmodifications of the initiation assays of Tsuda et al. (CancerRes., 40, 1157, 1980) and Pitot et al. (Nature, 271, 456, 1978).Equimolar doses of NMBzA or NDMA (33.5 µmol/kg) were injectedi.p. into male Sprague-Dawley rats 18 h after partial hepatectomy.Following selection, foci of preneoplastic hepatocytes weredetected by histochemical staining for / glutamyltranspeptidase.Nitrosomethylamylamine (NMAmA), 115 µmol/kg), also anesophageal carcinogen, was tested in the assay of Tsuda et al.,and nitrosodiethylamine (NDEA, 33.5 µmol/kg), a hepaticand esophageal carcinogen, was tested in the assay of Pitotet al. Both NDMA and NDEA induced significant increases in thenumber of preneoplastic foci above background. In contrast,neither NMBzA nor NMAmA increased the number of foci above background.Microsomes from regenerating liver had a lower capacity to metabolizeboth NDMA and NMBzA compared to microsomes from intact liver,but the decrease in activity was similar for both compounds.Neither NDMA nor NMBzA significantly inhibited the first waveof DNA synthesis in vivo in the regenerating liver. The resultsdemonstrate that in contrast to NDMA and NDEA, NMBzA and NMAmAlack tumor initiating activity in the liver.     

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.     

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.     

Further evidence that mitogen-induced cell proliferation does not support the formation of enzyme-altered islands in rat liver by carcinogens

Our earlier studies have revealed that direct hyperplasia induced by liver mitogens such as lead nitrate, ethylene dibromide, nafenopin and cyproterone acetate, unlike compensatory cell proliferation induced by partial hepatectomy and CCl4, does not support the formation of enzyme-altered islands induced by chemical carcinogens in the liver. In the previous studies carcinogens were given at the peak of DNA synthesis induced by the liver mitogens. If the mitogens have altered the sensitive phase of the hepatocyte to the carcinogenic attack, administering the carcinogen at one time point following the mitogenic stimulus might have missed the sensitive phase. In order to overcome this possibility in the present study male Wistar rats weighing 200-250 g were given N-methyl-N-nitrosourea (MNU; 60 mg/kg, i.p.) at three points representing G1, S and G2/M phases of the cell cycle following different types of liver cell proliferative stimuli. In another experiment MNU (60 mg/kg, i.p.) and diethylnitrosamine (15 mg/kg, i.p.) were given prior to the administration of proliferative stimuli. The initiated hepatocytes were also assayed following promotion by two different promoting regimens, namely phenobarbital and the resistant-hepatocyte model. Further, the initiated hepatocytes were monitored not only by using the appearance of islands of enzyme-altered hepatocytes but also using the incidence of hepatocellular carcinoma. The results of this study clearly revealed that irrespective of the protocol used, only the compensatory liver cell proliferation but not the mitogen-induced direct hyperplasia supported the formation and the growth of enzyme-altered islands in the liver induced by chemical carcinogens.     

Prevention of fibrosis reduces enzyme-altered lesions in the rat liver

A choline-deficient L-amino acid-defined (CDAA) diet led tothe development of liver cirrhosis in 100% of male Wistar ratsafter 16 weeks. In contrast, an ordinary (semipurified) choline-deficient(CD) diet led to the development of liver cirrhosis in only33.3%. After 16 weeks, the liver hydroxyproline content, whichreflects the amount of collagen, increased to a level more thanfour times higher in rats fed a CDAA diet than in rats fed acholinesupplemented L-amino acid-defined (CSAA) diet Concurrentadministration of a prolyl 4-hydroxylase inhibitor, 2,4-pyridinedicarboxylic acid bis(2-methoxyethyl amide) (HOE 077), to ratsfed a CDAA diet reduced this increase in liver hydroxyprolinecontent in a dose-dependent manner for doses up to 200 p.p.m.Microscopically, reduction in the hydroxyproline content ofthe liver resulted in a reduced number of pseudolobuli and thinnerfibrous septa. HOE 077 showed no effect on liver hydroxyprolinecontent in rats fed a CSAA diet The administration of a CDAAdiet for 16 weeks led to a substantial induction of GSTP-positivelesions in the liver. The concurrent administration of HOE 077reduced the number, average diameter and percent area of GSTP-positivelesions in a dose-dependent manner, in parallel with the reductionin hydroxyproline content These data suggest that inhibitionof fibrosis may limit the development of subsequent neoplasms.