Dose-response relationship of phenobarbital promotion of diethylnitrosamine initiated tumors in rat liver

The dose-response relationship was determined in rats for the enhancement by phenobarbital of diethylnitrosamine (DENA)-initiated neoplastic nodules and hepatocellular carcinomas. Male Sprague-Dawley rats received a single oral dose of either 80 mg/kg DENA or water. Seven days later, the animals were divided into groups that started to receive 0, 62.5, 125, 250, 500 or 1000 ppm sodium phenobarbital in the drinking water. Animals from each group were killed at 48 and 70 weeks after the DENA. No significant difference was observed in the low response of neoplastic nodules among the DENA-initiated groups. The incidence of DENA-initiated hepatocellular carcinoma was enhanced at 70 weeks by 250, 500 and 1000 ppm sodium phenobarbital but not by 62.5 or 125 ppm sodium phenobarbital. Equal enhancement of the incidence of hepatocellular carcinomas was obtained with 250, 500 and 1000 ppm sodium phenobarbital. In non-DENA-initiated rats, phenobarbital did not induce neoplastic nodules or hepatocellular carcinomas. Our results suggest that a daily dose of at least 250 ppm sodium phenobarbital is required in order for it to exert tumor promoting activity.     

Inhibitory potential of acetaminophen and o-, m-, p- aminophenols for development of gamma-glutamyltranspeptidase-positive liver cell foci in rats pretreated with diethylnitrosamine

The inhibitory potential of treatment with acetaminophen (AAP) and 3 different isometric forms of monoaminophenols (o-, m-, p-AP) for the development of gamma-glutamyltranspeptidase-positive (gamma-GT+) liver cell foci was examined in an in vivo short-term assay system. Rats were initially given a single intraperitoneal injection of diethylnitrosamine (DEN) and fed test compounds from week 2 until they were killed at week 6, all rats being subjected to two-thirds partial hepatectomy at week 3. All 4 compounds exerted obvious inhibition of the development of gamma-GT foci with both number and area (mm2) being reduced. AAP proved the most potent agent whereas dose-related inhibitory potential was observed within the 2 doses of p-AP treated.     

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.     

Age-dependent induction of preneoplastic liver cell foci by 2-acetylaminofluorene, phenobarbital and acetaminophen in F344 rats initially treated with diethylnitrosamine

Effects of age on the induction of glutathione S-transferase placental form (GST-P)-positive hepatic foci in rats were examined using a medium-term liver bioassay system (for carcinogens). F344 male rats aged 6, 26 and 46 weeks were initially given a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and, beginning 2 weeks later, received 0.02% 2-acetylaminofluorene (2-AAF), 0.05% phenobarbital (PB) or 1.3% acetaminophen (AAP) in the diet for 6 weeks. All animals were subjected to two-thirds hepatectomy 3 weeks after the DEN injection and were killed at week 8. Quantitative analysis of GST-P-positive foci revealed significantly (P less than 0.001) increased induction over control levels in terms of both numbers and areas for 2-AAF at all ages (6, 26 and 46 weeks), but especially in the 6-week-old case. In the PB- and AAP-treated groups, the respective enhancing and inhibitory influences were most pronounced in the animals aged 6 weeks, and were less marked in older rats. Thus, the response of F344 rats to the modifying effects of chemicals was age-dependent, the conclusion being drawn that young rats are more susceptible and therefore more appropriate for assessment of carcinogenic, promoting and inhibitory effects of chemicals.     

Enhancement of glutathione S-transferase placental-form positive liver cell foci development by microcystin-LR in aflatoxin B1-initiated rats

The objective of this study was to elucidate whether microcystin-LR(MC-LR), a hepatotoxic blue-green algal toxin in drinking water,is carcinogenic or possesses the ability to modulate aflatoxinB₁ (AFB₁)-induced hepatocarcinogenicity. In a medium-term liverbioassay, male Fischer 344 rats were given a single i.p. injectionof diethylnitrosamine (DEN, 200 mg/kg) followed by an i.p. injectionof MC-LR for 6 weeks after 2 weeks of DEN treatment. To studythe synergism between AFB₁ and MC-LR, DEN-treated rats weregiven an i.p. injection of AFB₁ (0.5 mg/kg) dissolved in dimethylsulfoxide (DMSO) followed by MC-LR at 2 weeks after the treatment.In a separate experiment, the rats were first given AFB₁ (0.5mg/kg) and 2 weeks later an i.p. injection of 1 or 10 µg/kgof MC-LR twice a week for 6 weeks. Most rats were subjectedto a two-thirds partial hepatectomy (PH) at week 3 and werekilled under anesthesia at week 8. Liver sections were analyzedfor glutathione S-transferase placental form (GST-P) expression,and subjected to histopathological examination for phenotypicalteration of hepatocellular foci. In rats that did not receiveDEN, MC-LR did not cause a significant increase in the numbersof GST-P-positive foci, whereas AFB₁ induced a slight increasein GST-P-positive foci development. In rats given DEN, MC-LRenhanced the expression of GST-P-positive foci, as did AFB₁but no synergism was observed. Histopathological analysis revealedthat the area of eosinophilic foci, a biomarker for preneoplasticliver lesion, markedly increased because of MC-LR. In rats givenAFB₁ as an initiator, treatment with MC-LR resulted in a synergisticincrease in the development of GST-P-positive foci. These resultssuggest that the hepatocarcinogenicities of MC-LR and AFB₁ canbe predicted in experimental animals with a medium-term bioassay.Furthermore, tumor promoting activity of MC-LR was demonstratedin rats treated with AFB₁.     

Failure of glutathione to prevent liver cancer development in rats initiated with diethylnitrosamine in the resistant hepatocyte model

This study was undertaken to observe whether the administrationof reduced glutathione intragastrically to male Fischer 344rats during the precancerous steps of liver carcinogenesis hasany protective effect on the development of hepatocellular carcinoma.Hepatocyte nodules were induced in the liver with a single initiatingdose of diethylnitrosamine followed by selection of resistanthepatocytes to generate nodules by a two week exposure to dietary2-acetylamino-fluorene coupled with partial hepatectomy. Animalshad hepatocyte (‘hyperplastic’) nodules when examinedby iaparotomy at three months. At that time, the animals weredivided into two groups. One received daily intragastric giuta-thionefor 8 months while the other received no further treatment.An additional control group received only the selecting (promoting)regimen with no initiator or glutathione. At 12 months, theanimals receiving the initiator and promoter regimen had a 65%incidence of hepatocellular carcinoma and those receiving glutathionein addition bad a 71% incidence. Under these experimental conditions,the long term administration of glutathione appears to haveno observable influence on liver cancer development in thismodel.     

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.     

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.     

Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3F1 mice

Potentially preneoplastic hepatocellular hyperplastic foci andhepatocellular neoplasms were studied in weanling male B6C3F1mice that received a single i.p. injection (80 mg/kg) of diethylnitrosamine(DEN) at 4 weeks of age, followed by oral administration ofphenobarbital (PB) or di(2-ethylhexyl)- phthalate (DEHP) thatbegan 2 weeks after DEN injection and continued for up to 6months. PB was administered in drinking water at 500 p.p.m.and DEHP in the feed at 3000, 6000 or 12 000 p.p.m. Groups ofmice were sacrificed at 2, 4 and 6 months after DEN exposure;formalin-fixed liver samples were evaluated histologically.Hepatocellular neoplasms and foci of hyperplasia were quantifiedwith the aid of an image analysis computer. Few foci were seenat 2, 4 or 6 months in mice exposed to DEN, PB or DEHP alone,while numerous foci and neoplasms were seen in mice given DEHPor PB after DEN. Area-perimeter measurements for each hepatocellularfocus or neoplasm transection revealed that foci and neoplasmsin PB-exposed mice increased both in size (area and volume)and in number throughout the study. In DEHP-exposed mice thepattern of response was different in that the numbers of focidid not increase between 4 and 6 months, but the foci increasedin mean diameter and volume throughout the experiment. Fociand tumors appeared earlier in mice given higher dietary levelsof DEHP than in those given lower doses. By the end of the studythe number of foci per unit volume of liver was similar in micegiven any dose of DEHP, but their volume was dose-related. Hepatocellularfoci and neoplasms in PB-exposed mice were composed predominantlyof eosinophilic hepatocytes, while in DEHP exposed mice, basophilicfoci and neoplasms predominated; the latter were more malignantin appearance than neoplasms in PB-exposed mice. At 6 months,the neoplasms in high dose DEHP-exposed mice were significantlylarger than those in PB exposed mice. Histochemistry, however,revealed similarities between lesions in mice exposed to PBor DEHP. PB given continuously for 6 months revealed no initiatingactivity of DEHP given once by gavage and followed by PB indrinking water. Both morphology and biology of hepatocellularfoci and neoplasms, which develop in mice after a single exposureto a carcinogen with initiating activity, thus depend, in part,on the subsequent promoting agent. More than one process oftumor promotion, as characterized by a specific sequence ofmorphologic and biochemical changes, is possible for the mousehepatocyle.     

The quantitative analysis and stability of histochemical markers of altered hepatic foci in rat liver following initiation by diethylnitrosamine administration and promotion with phenobarbital

The stability and response of histochemical phenotypes of altered hepatic foci (AHF) were studied both in the presence and following the withdrawal of 0.05% phenobarbital (PB) treatment in rats previously given a single dose of diethylnitrosamine (DEN) 20-24 h following partial hepatectomy (PH). AHF were scored by their expression of three biochemical markers: gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase and glucose-6-phosphatase (G6P). AHF demonstrated significant heterogeneity with respect to the marker alterations. The use of three markers in the present study confirmed the findings of our earlier study, which showed the maximal response of GGT+ AHF to PB administration following PH/DEN initiation and the stability of GGT+/AHF induced by the PH/DEN/PB regimen after the withdrawal of PB. In the regimen employed, the GGT marker alone scored the great majority of the AHF detected by all three markers. The frequency distribution of histochemical phenotypes remained relatively constant in AHF during continuous PB administration and in AHF promoted by PB followed by a 6-month period of feeding a diet containing no PB. These findings suggest that individual AHF remain phenotypically stable throughout the PB promotion phase, i.e., do not progress from one phenotype to another. In every marker class, the mean volume of AHF increased during continuous PB administration. These data illustrate the enhancing effect of PB on the growth of the AHF. The size of AHF continued to increase following the withdrawal of PB in the 3-month PB treatment group, but not in the animals treated for 4 months. A mechanism that may account for the differences in these two treatment groups is discussed.     

Expression of c-myc in altered hepatic foci induced in rats by various single doses of diethylnitrosamine and promotion by 0.05% phenobarbital

Among the proto-oncogenes examined by northern blot analysis, c-myc, c-Ha-ras, c-fos, and c-raf-1 have been reported to be activated in rat liver cell carcinomas. However, there are relatively few reports on proto-oncogene expression in altered hepatic foci (a) early during hepatocarcinogenesis in the rat. In this study, diethylnitrosamine (a) at doses ranging from 10 to 200 mg/kg was used to initiate and phenobarbital (0.05%) to promote AHF in rats. AHF were detected by the presence of the marker enzymes glutathione s-transferase, placental form (GST-P); γ-glutamyltranspeptidase (a); glucose-6-phosphatase (G6Pase); and canalicular ad-enosine triphosphatase (a). Proto-oncogene expression in individual AHF was investigated by in situ hybridization (a). ISH for the mRNAs of c-Ha-ras, c-fos, and c-raf-1 revealed little or no expression in AHF. However, the levels of c-myc mRNA were increased in about 10% of the AHF initiated by the highest dose of DEN (200 mg/kg). Thus, altered expression of proto-oncogenes was not seen in AHF initiated by nonnecrogenic doses of DEN and promoted by phenobarbital. However, at the necrogenic dose of 200 mg/kg DEN, c-myc expression was found mostly in AHF in which abnormal expression of GST-P, GGT, G6Pase, and ATPase was also present, indicating that c-myc expression is correlated with phenotypically greater complexity of the AHF, a characteristic of malignant hepatic neoplasms in the rat. © 1995 Wiley- Liss, Inc.     

Influence of dietary fat and selenium in initiation and promotion of aflatoxin B1-induced preneoplastic foci in rat liver

Aflatoxin B₁-induced hepatic -glutamyl transpeptidase-positivefoci were quantified in rats fed different levels of fat andselenium during either initiation or early promotion. Male Sprague-Dawleyrats were divided into 12 groups. One of six experimental dietswere fed to groups 1–6 prior to and during aflatoxin B₁exposure (initiation, weeks 1–4.5) and to groups 7–12during weeks 4.5–15 (promotion). The six experimentaldiets contained 2 or 20% corn oil, each with either <0.02,0.15 or 2.5 (or 1.9) p.p.m. selenium. When not fed the experimentaldiets, rats were fed a modified MN 76A diet. In groups 1–6,0.03% phenobarbital was added as a promoter to the AIN-76A diet.Individual and interactive effects of selenium and fat weredependent on the stage of carcinogenesis. High dietary fat fedwith either <0.02 or 0.15 p.p.m. selenium during initiationresulted in a significant increase in the number and size offoci when compared with low fat groups. In rats fed 20% fatand 2.5 p.p.m. selenium during initiation, preneoplastic developmentwas reduced below all low fat groups. In contrast, seleniumstatus but not dietary fat level influenced focal formationduring promotion. Rats fed <0.02 p.p.m. selenium had a significantlygreater percentage of liver section occupied by foci than ratsfed either 0.15 or 1.9 p.p.m. seleniwn. Feeding 1.9 p.p.m. seleniumduring promotion did not afford greater protection above the0.15 p.p.m. level. Hepatic glutathione peroxidase activity atweek 15 was significantly diminished in animals fed <0.02p.p.m. selenium during promotion. Feeding 1.9 p.p.m. seleniumwhen compared with 0.15 p.p.m. did not result in a consistentincrease in enzyme activity. Although differences were observedin growth due to dietary treatment, there were no significantcorrelations between preneoplastic foci and body weight, foodconsumption or food effidency. These findings indicate an interactionbetween dietary fat and selenium during initiation, but notduring early promotion. Furthermore, dietary selenium and fatmay function by different mechan isms at different stages ofcarcinogenesis.     

Inhibitory effect of vanadium on rat liver carcinogenesis initiated with diethylnitrosamine and promoted by phenobarbital.

The chemoprotective effect of vanadium, a dietary micronutrient, against chemically induced hepatocarcinogenesis in rats was investigated. Initiation was performed by a single intraperitoneal injection of diethylnitrosamine (DENA; 200 mg kg-1) followed by promotion with phenobarbital (0.05%) in the diet. Supplementary vanadium (0.5 p.p.m.) in the drinking water was provided ad libitum throughout the experiment, before the initiation or during the promotion period. At the end of the study (20 weeks), vanadium supplementation throughout the experiment reduced the incidence (P < 0.01), total number and multiplicity (P < 0.001) and altered the size distribution of visible persistent nodules (PNs) as compared with DENA control animals. Mean nodular volume (P < 0.05) and nodular volume as a percentage of liver volume (P < 0.01) were also attenuated following long-term vanadium treatment. It also caused a large decrease in the number (P < 0.001) and surface area (P < 0.01) of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci and in the labelling index (P < 0.001) of focal cells, coupled with increased (P < 0.01) remodelling. The activity of GGT, measured quantitatively, was found to be significantly less in the PNs (P < 0.001) and non-nodular surrounding parenchyma (P < 0.01) of vanadium-supplemented rats. The anticarcinogenic effect of vanadium was also reflected in the histopathological analysis of liver sections that showed a well-maintained hepatocellular architecture as compared with DENA control. Similar results were observed when vanadium was given only before the initiation. However, supplementation of vanadium during the promotion period did not result in significant alterations of these parameters. Our results, thus, strongly suggest that vanadium may have a unique anti-tumour potential which is primarily exerted on the initiation phase and only secondarily on the promotion stage.     

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.     

Modulation of aflatoxin B1-induced glutathione S-transferase placental form positive hepatic foci by pretreatment of rats with phenobarbital and buthionine sulfoximine

Induction of glutathione S-transferase placental form (GST-P)positive hepatic foci has been examined by immunohisto-chemicalanalysis in young male Fischer rats 3 weeks after a single i.p.injection of aflatoxin B₁ (AFB₁). Pretreatment of rats withL-buthionine sulfoximine (BSO), a GSH depleter, at a dose of4 mmol/kg body wt 4 and 2 h before 1.0 mg AFB₁ treatment enhancedboth the number of AFB₁-induced hepatic foci and the area occupiedby these foci by     

A mechanism of inhibition of aflatoxin B1-DNA binding in the liver by phenobarbital pretreatment of rats

The effect of phenobarbital (PB) pretreatment of rats on both hepatic aflatoxin B1 (AFB1)-DNA binding and AFB1-glutathione (AFB1-SG) conjugation have been examined in studies in vivo and in vitro. Male Sprague-Dawley rats fed a commercial diet with 0.1% PB in their drinking water for 1 week had total wet liver weight and microsomal protein content about 27% and 38% higher, respectively, than controls. Hepatic cytochrome P-450 content, microsomal cytochrome P-450 mediated AFB1 binding to exogenous DNA and formation of hydroxy metabolites of AFB1 were also about threefold higher in PB-treated rats and cytosolic reduced glutathione S-transferase activities were about doubled. Microsome-mediated AFB1-DNA binding, when examined at 2 microM and 10 microM levels of AFB1, was inhibited two-to threefold more by cytosols of treated rats whereas AFB1-SG conjugation was two- to threefold higher by cytosols of treated rats. In reconstitution experiments with 2 microM AFB1, with intact nuclei serving as a source of endogenous DNA, addition of microsomes from either group generated a large amount of AFB1-DNA binding (68-105 pmol) and a smaller amount of AFB1-SG conjugate (12-21 pmol). The presence of cytosol from the controls reduced AFB1-DNA binding to a much lesser extent than the cytosol from the treated group whereas AFB1-SG conjugation was much higher with the cytosol from the treated group. These results are in agreement with the studies in vivo. In isolated hepatocytes at 33 nM, 2 microM and 10 microM AFB1 levels, AFB1-DNA binding was decreased 50 to 70% by prior PB-treatment whereas AFB1-SG conjugation was two- to threefold higher in treated compared to control hepatocytes. In hepatocytes, addition of 1 mM diethylmaleate increased DNA binding two- to threefold with a corresponding decrease in AFB1-SG conjugation. Addition of 1 mM styrene oxide caused 5- to 10-fold increases in AFB1-DNA binding at levels of AFB1 of 33 nM and 2 microM; but at 10 microM AFB1, increases in AFB1-DNA binding were two- to threefold. In intact rats, PB treatment reduced hepatic AFB1-DNA binding to 30% of controls with concomitant increase in biliary excretion of AFB1-SG conjugate. It appears that the induced cytosolic GSH S-transferases after PB treatment of rats plays a significant role in inhibiting hepatic AFB1-DNA binding and hepatocarcinogenesis presumably by inactivation of the reactive AFB1-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Enhancing effect of oxymetholone, an anabolic steroid, on development of liver cell foci in rats initiated with N-diethylnitrosamine

The promoting potential of oxymetholone (OXM) administration on development of liver cell foci was investigated in male F344 rats previously treated with N-diethylnitrosamine (DEN). One week after a single injection of DEN (100 mg/kg, i.p.), rats were given OXM at a dietary level of 0.2% for the first 4 weeks and then at a concentration of 0.1% for an additional 35 weeks. All rats were killed at week 40 for histopathological and immunohistopathological examination of liver tissue. The numbers and areas of both clear cell and glutathione S-transferase placental form (GST-P) positive foci were significantly increased in the group treated with DEN and OXM as compared with the respective values for the DEN alone group. The results thus suggested that OXM possesses promoting potential for rat liver carcinogenesis.