The effect of the format of administration and the total dose of phenobarbital on altered hepatic foci following initiation in female rats with diethylnitrosamine

The effect of changing the format of administration as well as the total dose of the promoting agent phenobarbital (PB) on the development of altered hepatic foci (AHF) was determined in an initiation-promotion protocol with female rats fed the purified AIN-76 diet. Effects on the total number of AHF and the volume percentage of liver occupied by AHF were determined for four histochemical markers, the placental form of glutathione S-transferase, gamma-glutamyl-transpeptidase canalicular ATPase, and glucose-6-phosphatase after 16 and 60 weeks of promotion with varying doses and formats of PB, as well as for a further 16-week period in which no PB was administered. At the 16-week point, animals fed 0.1% PB continuously exhibited the largest number and volume percentage of AHF, whereas rats fed 0.1% PB for 4 days followed by 10 days of no PB with continuous repetition of this pattern during the 16-week treatment period exhibited no increase in the number of AHF over control and only a slight increase in volume percentage. Rats fed a continuous repetition of 0.2% PB for 2 days followed by 12 days of no PB exhibited an intermediate increase in the number of AHF as well as the volume percentage fraction after 16 weeks of this regimen. After 60 weeks of feeding PB by these three different formats, the numbers of AHF observed in these groups were equivalent and had increased above those seen after 16 weeks of feeding. The volume percentage occupied by the AHF in these three groups was also similar, although animals receiving 0.2% PB intermittently showed a significantly lower volume percentage than animals receiving 0.1% PB continuously for 60 weeks. When animals were maintained for an additional 16 weeks without PB feeding, the numbers of AHF decreased dramatically, much more so in animals fed PB intermittently, whereas the volume percentage fraction of AHF in livers of animals receiving 0.1% PB continuously for 60 weeks almost doubled. In contrast, the volume percentage fraction of AHF in livers of animals receiving PB intermittently for 60 weeks followed by 16 weeks of no PB was slightly less. Examination of the individual size classes of AHF showed little change in their distribution at 16 and 60 weeks, but after 16 weeks of PB withdrawal (76 weeks total time), the distribution of AHF in animals that had received 0.1% PB continuously for 60 weeks exhibited a decidedly greater shift to larger AHF than animals receiving PB intermittently for the 60-week period.(ABSTRACT TRUNCATED AT 400 WORDS)     

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,     

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.     

Induction of hepatic ornithine decarboxylase by hypolipidemic drugs with hepatic peroxisome proliferative activity

The present investigation was conducted to determine (1) ifhepatomegalic and mitogenic effects of selected hypolipidemicperoxisome proliferators are associated with an elevation ofhepatic ornithine decarboxylase (ODC) levels and (2) if inductionof ODC is a specific event associated with the development ofpreneoplastic hepatocellular foci and the eventual formationof hepatomas. Following a single i.p. dose of Wy-14, 643 (Wy),the hepatic ODC activity in rats rose sharply, reaching a peakat 8 h, and returned to the normal level by 24 h. Other peroxisomeproliferators tested (methyl clofenapate, BR-931 and nafenopin)also increased the hepatic ODC activities significantly 8 hafter i.p. administration of a single dose. Continuous dietaryadministration of Wy, whether or not these animals were previouslyinitiated with the carcinogen diethylnitros-amine (DEN), resultedin a sustained elevation of hepatic ODC activity. Although Wyexerted an enhancing effect on DEN-initiated tumorigenesis,there was no additional increase of ODC activity. There wasno correlation between the level of ODC induction and the presenceor absence of altered liver cell foci. These results suggestthat induction of ODC by peroxisome proliferators is not a specificevent associated with the development of preneoplastic fociand hepatocarcino-genesis, but is an event associated with thesustained maintenance of hepatomegaly and increased liver cellproliferation.     

Induction of foci of altered hepatocytes by a single injection of azaserine to rats.

The induction of foci of altered, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes by azaserine was investigated. After injection of a single dose of azaserine, many foci developed in male Wistar rats fed a choline-devoid (CD) diet containing acetylaminofluorene (AAF), but only a few in rats fed a choline-supplemented (CS) diet containing AAF. Similar results were obtained in rats fed a plain CD diet or a plain CS diet and injected with a single dose of azaserine after a partial hepatectomy. These findings indicate that azaserine is an effective initiator of liver carcinogenesis in rats, and that a CD diet acts as a strong promoter of the evolution initiated liver cells to foci of altered, GGT-positive hepatocytes.     

Long-term effects of hypolipidemic peroxisome proliferator administration on hepatic hydrogen peroxide metabolism in rats

The effects of prolonged dietary administration of peroxisome proliferators, such as clofibrate, bezafibrate and di(2-ethylhexyl)phthalate (DEHP), on hepatic hydrogen peroxide (H2O2) level and on hepatic activities of the enzymes relating to H2O2 metabolism were examined. Male rats were treated for 79 weeks with the above three peroxisome proliferators. The activities of the peroxisomal beta-oxidation and catalase were increased 8- to 20-fold and 2- to 3-fold, respectively, after 2 or 4 weeks of treatment with these peroxisome proliferators. However at 79 weeks the peroxisomal beta-oxidation activity was 3-8 times that of control. The level of catalase activity was kept at approximately 2-fold even after prolonged treatment of peroxisome proliferators. Although the activities of glutathione peroxidase (GSH-Px) and glutathione S-transferase (GST) were decreased 50-60% at 4-12 weeks by the treatment with peroxisome proliferators, from 20 to 79 weeks those activities approached control levels in the case of clofibrate and bezafibrate but not DEHP-fed rats; GSH-Px and GST activities were kept at approximately 40% those of control. However hepatic capacities of H2O2-degrading enzymes, catalase and GSH-Px, apparently exceeded the H2O2-generating levels obtained on the basis of peroxisomal beta-oxidation activities in the livers of control and treated rats throughout the experimental period. The hepatic H2O2 levels increased only slightly but this increase did not correspond to changes in peroxisomal beta-oxidation. Our results suggest that a large part of H2O2 produced by peroxisomal beta-oxidation could be rapidly scavenged by catalase and GSH-Px in the liver of rats treated with peroxisome proliferators.     

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.     

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to mitochondrial alterations

Recent studies have indicated a lack of correlation betweenhepatic 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels and thecarcinogenicity of peroxisome proliferators (PP) and suggestedthat DNA in intact hepatic nuclei may be insensitive to increasesin 8-OHdG resulting from PP exposure. The possibility that PP-inducedelevations in acyl CoA oxidase (ACO) activity might result inoxidative damage to mitochondrial DNA (mtDNA) was thereforeinvestigated by feeding male F344 rats the hepatocarcinogenicPP Wy-14,643 (Wy, 0.1% in the diet) for 3, 6, 11, or 22 weeks,or clofibric acid (CA, 0.5% in the diet) for 22 weeks. Followingthe respective PP exposures, hepatic peroxisomal acyl CoA oxidaseactivity was determined and DNA isolated from either mitochondriaor unfractionated liver homogenates and analysed for the presenceof 8-OHdG. PP treatment caused an increase in ACO activity (10-to 15-fold) at all time points examined and an increase of 8-OHdG(1.5- to 2-fold) in DNA isolated from unfractionated liver homogenatesfollowing PP treatment for 11 or 22 weeks. No increase of 8-OHdGin mtDNA was detected. However, quantitation of a PCR amplifiedregion from the D-loop of mtDNA demonstrated a 2- to 3-foldincrease in the relative amount of mtDNA in DNA isolated fromunfractionated liver homogenates following 3, 11, and 22 weeksexposure to Wy or CA (22 weeks only). In addition, a slightincrease in the mitochondrial volume density (1.4-fold) wasobserved in electron micrographs of liver samples from ratsexposed to Wy for 22 weeks. These results (i) demonstrate thatPP treatment, at levels which cause an increase in ACO activity,does not cause oxidative damage to mtDNA, and (ii) suggest thatone reason for the observed increase of 8-OHdG in DNA from unfractionatedliver homogenates may be an increase in the amount of mtDNApresent in these samples. Furthermore, these studies provideadditional evidence against a role of oxidative DNA damage,measured as 8-OHdG, in PP-induced rodent hepatocarcinogenesisand suggest that alterations in mitochondria or other effectsmay be more pertinent to PP-related carcinogenesis.     

Modulation of altered hepatic foci induction by diallyl sulphide in Wistar rats.

Diallyl sulphide (DAS) is a sulphur-containing volatile compound present in garlic (Allium sativum). It has been shown to inhibit a number of chemically induced forms of cancer in experimental animals. The present study demonstrates the inhibitory effect of DAS on the development of diethylnitrosamine (DEN) initiated and 2-acetyl-aminofluorene (2-AAF) promoted preneoplastic altered hepatic foci (AHF) in Wistar rats. AHF were scored and analysed by quantitative stereology using the Image Analysis system from frozen liver sections stained for biological markers, namely glutathione S-transferase, placental form (GST-P), gamma-glutamyl transpeptidase (GGT), adenosine triphosphatase (ATPase), glucose-6-phosphatase (G6 Pase) and alkaline phosphatase (AlkPase). DAS-supplemented rats were found to restore the near-normal levels of enzymes GST-P and GGT when exposed to DEN and 2-AAF. DAS administration following DEN and 2-AAF exposure led to the restoration of enzymic activity of ATPase, G6 Pase and AlkPase, as evident by number and area of the foci. These findings suggest the protective role of DAS in rat hepatocarcinogenesis, by suppressing DEN- and 2-AAF-induced AHF development.     

Elevated 8-hydroxydeoxyguanosine in hepatic DNA of rats following exposure to peroxisome proliferators: relationship to carcinogenesis and nuclear localization

Increased oxidative DNA damage due to increased peroxisomalgeneration of H₂O₂ is a potential mechanism in the carcinogenicityof chemical peroxisome proliferators (PP) in rodent liver. Inorder to determine the relationship between carcinogencity andperoxisome-dependent DNA damage, levels of DNA base oxidationwere examined by comparing 8-hydroxydeoxyguanosine (8-OHdG)in DNA from unfractionated liver of male F344 rats followingdietary exposure to PP [WY-14, 643, 0.1% or 0.005%; di(2-ethylhexyl)phthalate(DEHP), 1.2%; clofibric acid, 0.5%] or phenobarbital (0.05%).Exposure-related increases in 8-OHdG were not observed at 3or 11 weeks for any of the compounds fed. At 22 weeks, 8-OHdGwas similarly elevated (2–3x) by WY-14, 643 (0.1% and0.005%) and clofibric acid (0.5%). These equivalent increasesin 8-OHdG in DNA from unfractionated liver did not parallelthe divergent carcinogencity of these different dietary exposuresin the present or previous studies. The potential oxidationof nuclear DNA was examined by comparing levels of 8-OHdG inDNA isolated from purified liver nuclei and unfractionated liver.Elevated levels of 8-OHdG were not detected in DNA isolatedfrom nuclear fractions of livers from rats fed clofibric acidfor 22 weeks, indicating the dependence of PP-induced oxidativeDNA damage on extranuclear components of samples for DNA isolation.The absence of a quantitative relationship between PP-inducedcarcinogenicity and oxidative DNA base damage (as 8-OHdG), andthe failure to localize this oxidative damage to nuclear DNA,suggest two possible conclusions: (1) quantitation of 8-OHdG,a specific and sensitive indicator of oxidative DNA damage,does not accurately reflect the potential peroxisomal H₂O₂-dependentDNA damage and carcinogenicity of PP exposure in rodents; (2)other hepatic responses may be more critical features of themechanism of PP carcinogenicity.     

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.     

Induction of hepatic tumors by diethylstilbestrol alone or in synergism with n-nitrosobutylurea in castrated male WF rats

Inbred male WF rats were castrated at 40 days of age and divided into 5 groups. Group I was given no further treatment. Groups III, IV, and V received pellet implants of 5.0 mg diethylstilbestrol (DES) concurrently with castration. At 50-55 days of age, groups II, IV, and V were given drinking water containing 5.0 mg N-nitrosobutylurea (NBU) per day for 30 days (subthreshold dose). At the termination of NBU treatment, group V further received daily sc injections of 2-bromoergocryptine (CB-154; 0.4 mg/100 g body wt) four times a week throughout the experiment. None of castrated rats or rats castrated and treated with NBU alone developed hepatic tumors (HT) and pituitary tumors (PT). Incidences of HT and PT in groups III, IV, and V were 4/9 (44%) and 7/9 (78%), 15/17 (88%) and 12/17 (71%), and 17/20 (85%) and 4/20 (20%), respectively. The treatment of DES alone resulted in the concurrent development of HT and PT in castrated male rats (group III), and further NBU treatment significantly increased the incidence of HT (group IV). CB-154 treatment did not change the incidence of HT, the number of HT per rat, and the liver weight, although it significantly reduced the incidence of PT, the pituitary weight, and the serum prolactin level in castrated male rats given DES and NBU (group V). These results indicate that DES itself had a direct carcinogenic effect on the liver; this effect was not mediated by prolactin, and NBU increased the effect of DES in this process.     

Effects of separate and combined treatments with gamma radiation and diethylnitrosamine in neonatal rats on the induction of altered hepatocyte foci and hepatic tumors

To characterize the effects of combined treatments with gammaradiation and diethylnitrosamine (DEN) on the induction of histochemicallydetectable altered hepatocyte foci and hepatic tumors, we assessedthe yields of these lesions in the livers of 150-day-old ratsthat had been treated neonatally with a single dose of gammaradiation (75 red, whole body) and i.p.-injected DEN (0.15 µmol/gbody wt), either separately or in combination. The combinedtreatments involved the administration of the two stimuli inboth possible sequences, with the interval between treatmentsset at 1 h. The focus population was examined for two histochemicalmarkers (elevated gamma-glutamyl transpeptidase [GGT(+)J andiron exclusion [FE(–)], giving rise to three detectablefocus phenotypes, i.e. GGT(+) foci, FE(–) foci, and GGT(+),FE(–) foci. Frequencies of the three phenotypes were quantitatedthrough the use of serial frozen sectioning techniques and computer-assistedimage analysis. GGT(+) focus induction was synergistkally enhancedby the combined treatment irrespective of the order in whichthe two stimuli were administered; the remaining two phenotypesdid not show such enhancement. The magnitude of the GGT(+) focusresponse was significantly greater when the treatment sequencewas gamma DEN as opposed to DEN gamma. Tumor yields in ratsreceiving combined gamma-DEN treatment were similar to thosein rats receiving the DEN alone, irrespective of the gamma-DENtreatment sequence. These results suggest that (i) phenotypicallydistinguishable lesions, including foci with different histochemicalmarker patterns and tumors, originate from specific types ofdamage at different genetic loci and are developmentally independent;and (ii) the expression of the GGT(+) marker per se in alteredhepatocyte foci is not a reliable index of incipient hepaticneoplasia.     

Characterization of histochemically detectable altered hepatocyte foci and their relationship to hepatic tumorigenesis in rats treated once with diethylnitrosamine or benzo(a)pyrene within one day after birth

A new experimental system was used to examine the stages of chemically induced hepatic neoplasia in the rat. The treatment protocol involved the i.p. injection of a single dose of carcinogen [diethylnitrosamine or benzo(a)pyrene] into male and female rats within 1 day after birth, followed by dietary exposure to promoter (0.05% dietary phenobarbital) beginning at weaning. Rats were killed at intervals, and their livers were examined for tumors and for histochemically detectable foci of altered hepatocytes using six histochemical markers. Through serial frozen-sectioning techniques and computer-assisted image analysis, foci containing between one and six markers were identified, and their average diameters were calculated. The same complement of histochemical tests was applied to the primary hepatic tumors observed in this study. The principal findings were the following. (a) Both the diethylnitrosamine and benzo(a)pyrene treatments were tumorigenic and produced foci with similar phenotypic properties (numbers and identities of histochemical markers). (b) Foci relative growth rates and growth capacities (ranges of possible growth rates) were directly related to foci phenotypic complexity levels (numbers of markers per focus). (c) Individual foci were phenotypically stable; i.e., they neither gained nor lost markers. (d) A substantial fraction of the tumors observed in this study had fewer markers than the most complex foci. On the basis of these observations, we suggest that foci emerge as the result of a specific set of cellular changes solely inducible by carcinogenic stimuli, but the foci do not evolve through progressively more deviated forms into tumors. Instead, we postulate that tumorigenesis involves a separate transformation event that may occur in a susceptible fraction of foci subsequent to their induction or, alternatively, may occur at the time of exposure to carcinogen, in parallel with the carcinogen-mediated events leading to focus formation.     

Effect of the peroxisome proliferators ciprofibrate and perfluorodecanoic acid on hepatic cell proliferation and toxicity in Sprague--Dawley rats

The objective of this study was to compare the effects of perfluorodecanoicacid (PFDA) and ciprofibrate on the induction of hepatic toxicityand on hepatocellular proliferation in rats. In the first study,rats were first subjected to partial hepatectomy and then injectedwith [³H]thymidine (20 µCi/injection) at 23, 24, 25, 47,48 and 49 h afterwards. After a 2 week recovery period, ratswere injected with one of four levels of PFDA (03, 1.0, 3.0or 10 mg/kg/injection) in four i.p. doses every 14 days, orwere fed 0.01% or 0.003% ciprofibrate. Six days after the lastPFDA injection and three days before the animals were killed,an osmotic minipump containing 20 mg/ml 5-bromo-2’-deoxyuridine(BrdU) was implanted s.c for the measurement of DNA synthesis.Peroxisomal fatty acyl-CoA oxidase activity was significantlyenhanced in both PFDA and ciprofibrate-treated groups in a dose-dependentmanner. Hepatotoxicity, measured as the loss of [³H]thymldinefrom hepatic DNA, was not significantly affected by any of thetreatments. Hepatic DNA synthesis was significantly increasedonly in rats receiving the highest dose of PFDA. In order todetermine the time course of ciprofibrate- and PFDA-inducedcell proliferation, we conducted another study with more timepoints. Rats were fed 0.01% ciprofibrate or were injected every14 days with 3 or 10 mg PFDA/kg body weight for 10 days, 24days, 6 weeks, 26 weeks or 54 weeks. Cell proliferation wasquantified as in the first study. Ciprofibrate increased cellproliferation at the early but not the later time points, whereasPFDA increased cell proliferation at most times throughout thestudy. This study demonstrates that PFDA and ciprofibrate donot selectively induce hepatic toxicity and that their effectson cell proliferation do not correlate with their carcinogenicor promoting activities.     

Effects of calcium glucarate on the promotion of diethylnitrosamine-initiated altered hepatic foci in rats

Calcium glucarate (CGT), an inhibitor of beta-glucuronidase, is a potent inhibitor of chemically-induced tumors when administered orally. The present study was undertaken to determine the effects of CGT on the promotion of hepatocarcinogenesis by phenobarbital following initiation with diethylnitrosamine (DENA). Partially hepatectomized, DENA-initiated female Sprague-Dawley rats, previously maintained only on chow diet for 2 months, were supplemented with either 0.05% phenobarbital alone or 0.05% phenobarbital plus 4% dietary CGT, for varying time intervals up to 6 months. Histopathologic evaluation of the liver sections showed that CGT significantly delayed the development of altered hepatic foci (AHF). By the seventh month post-initiation, however, the frequency and severity of changes seen in the livers of experimental animals approximated those of the controls.     

Tumor promotion by the peroxisome proliferator nafenopin involving a specific subtype of altered foci in rat liver

The involvement of tumor promotion in the hepatocarcinogenic action of peroxisome proliferators has not been generally accepted. We studied the effect of nafenopin (NAF) as a model compound in a two-stage initiation-promotion protocol. Carcinogenesis was initiated by a single dose of aflatoxin B1 (AFB1) in female (AFB1, 5 mg/kg) and male (AFB1, 2 mg/kg) Wistar rats. After recovery NAF was fed via the diet, providing a daily dose of 100 mg/kg body weight. Phenobarbital (PB) (50 mg/kg body weight) was fed to female rats as a positive control. The following results were obtained. (a) At weeks 40, 55, 59, and 70, significantly more and larger liver tumors were present in AFB1-NAF-treated rats than in rats receiving either compound alone, and the effect of the combined treatment was clearly more than additive, in three independent experiments including both sexes. This suggests tumor promotion by NAF. Male rats responded more strongly than females. Similarly, PB enhanced the yield of liver tumors. Histologically, tumors were hepatocellular adenoma or carcinoma. In group AFB1-PB the majority consisted of eosinophilic and glycogenstoring cells. However, adenoma and carcinoma of groups AFB1-NAF and O-NAF consisted of weakly basophilic cells. (b) Phenotypically altered foci were evaluated in hematoxylin- and eosin-stained liver sections from the female rats. NAF treatment after AFB1 had little effect on number and size of eosinophilic-clear cell foci and decreased the number of trigroid foci. However, it led to a dramatic increase (20-fold after 70 weeks of NAF treatment) in number and size of foci of a special phenotype that was extremely rare after AFB1 alone and virtually absent in group AFB1-PB. Hepatocytes in these foci are characterized by weak diffuse basophilia and some eosinophilia, similar to the phenotype in adenoma and carcinoma, and by absence of gamma-glutamyltranspeptidase (GGT) expression. Based on these findings, we propose the hypothesis that NAF promotes the development of liver tumors via a mechanism involving amplification of a specific subtype of altered hepatic foci.     

Induction of foci of altered, gamma-glutamyltranspeptidase-positive hepatocytes in carcinogen-treated rats fed a choline-deficient diet.

A series of experiments was performed to investigate whether, after exposure of rats to a chemical hepatocarcinogen, feeding a choline-deficient (CD) diet would promote the proliferation of initiated liver cells, and their evolution to foci of altered γ-glutamyltranspeptidase (GGT)-positive hepatocytes, without subjecting the animals to further experimental manipulations.     

Expression of a 65 kda oncofetal phosphoprotein in the altered hepatic foci of rats fed 2-acetylaminofluorene followed by phenobarbital

The altered hepatic foci (AHF) developed in livers of Sprague-Dawley male rats,by acetylaminofluorene/phenobarbital protocol, were analyzed on paraffin sections for gamma-glutamyl-transpeptidase, glutathione S-transferase P and 65 kDa oncofetal protein (p65). 92% percent of the foci were GST-P-positive, 89% of those were also positive for GGT but only 10-12% were positive for p65. Comparison of tissues from different sources such as fetal and normal liver, foci, nodules, surrounding parenchyma and hepatocellular carcinomas revealed the presence of p65 protein in all tissues except normal rat liver and liver parenchyma surrounding the focal lesions.