Enhanced preneoplastic liver lesion development under 'selection pressure' conditions after administration of deoxycholic or lithocholic acid in the initiation phase in rats

The initiating potential of the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), for rat hepatocarcinogenesis was investigated using the development of hyperplastic nodules and/or glutathione S-transferase placental form (GST-P)-positive liver foci as the end point. Five week old male F344 rats were given either basal diet, or diets containing 0.5% DCA or 0.5% LCA for 3 weeks in conjunction with partial hepatectomy performed midway, followed by a selection regimen consisting of 2 weeks feeding of 0.02% 2-acetylaminofluorene diet and a single gastric intubation of carbon tetrachloride. The animals were then placed on either basal diet or a diet containing 0.05% phenobarbital (PB) for 52 weeks. Significantly higher numbers of hyperplastic liver nodules developed in the DCA-treated rats irrespective of PB promotion as compared with the respective control groups. No such increase was evident in the LCA-treated rats. In contrast, both DCA and LCA treatments enhanced the development of GST-P-positive liver foci with or without subsequent PB promotion. Only one hepatocellular carcinoma was diagnosed in a control group animal. The present data indicate that a short period of feeding of DCA and LCA in the initiation stage in conjunction with partial hepatectomy results in enhanced development of preneoplastic liver lesions under selection pressure conditions with or without subsequent PB promotion. They thus confirm and extend our previous finding of enhanced gamma-glutamyltranspeptidase-positive liver foci development in a short-term assay of DCA and LCA, and suggest that these secondary bile acids either possess possible initiating activity or some other priming effect for rat hepatocarcinogenesis.     

Dose-dependent effects of 2-acetylaminofluorene on hepatic foci development and cell proliferation in rats

Dose-dependent development of pre-neoplastic liver cell foci induced by 2-acetylaminofluorene (2-AAF) was investigated in relation to cell-proliferative activity. Male F344 rats were initially given a single i.p. injection of diethylnitrosamine (DEN, 200 mg/kg) and starting 2 weeks later received diets containing 2-AAF at dose levels of 150, 100, 60, 45, 35 or 30 p.p.m., 500 p.p.m. phenobarbital (PB) or basal diet as a control for 6 weeks. Two-thirds partial hepatectomy (PH) was performed at week 3. The rats were sequentially killed from weeks 0 to 16 and liver sections were analysed by double staining for both BrdU incorporation and glutathione S-transferase placental form (GST-P) expression. 2-AAF increased numbers and areas of GST-P positive (GST-P+) foci in a dose-dependent manner, especially after PH. Proliferation of hepatocytes, as indicated by BrdU labelling indices (LI), was higher in GST-P+ foci than in surrounding hepatocytes in all 2-AAF-treated groups, even after cessation of carcinogen administration. Proliferative response of hepatocytes to PH was delayed in rats treated with the highest dose of 2-AAF in both foci and in surrounding areas possibly due to the 2-AAF toxicity. In the PB treated group, the results were similar to those for the lower dose 2-AAF-treated groups. It is concluded that the development of GST-P+ foci and cell proliferation in GST-P+ foci are directly related to 2-AAF treatment in a dose-dependent manner and the present assay system is reliable for detection of carcinogenicity of chemicals even at low doses.     

In vitro and in vivo response of hepatocytes from hepatic nodules to the mitoinhibitory effects of phenobarbital

One of the proposed mechanisms by which phenobarbital (PB) promoteshepatocarcinogenesis in the rat is by differential mitoinhibition.However, our earlier studies indicated that PB inhibited DNAsynthesis in vitro in hepatocytes isolated from both surroundingnon-nodular liver and hepatic nodules promoted by orotic acid(OA). Since nodules generated by one promoter need not necessarilybe resistant to another promoter, the present study was undertakento determine whether foci/nodules promoted by PB itself areresistant to the mitoinhibitory effects of PB. Accordingly,rats were initiated with diethylnitrosamine (DENA, 200 mg/kgi.p) and promoted with PB (0.07% of PB as its sodium salt) intheir drinking water for 16 or 33 weeks. In vitro studies indicatedthat PB (3–5 mM) inhibited DNA synthesis induced by epidermalgrowth factor (EGF) in hepatocytes from surrounding non-nodularliver as well as from nodules promoted by PB for 33 weeks. Inanother experiment, initiated rats exposed to PB for 33 weekswere subjected to either two-thirds partial hepatectomy (PH)or sham hepatectomy. Hepatocytes were labelled with tritiatedthymidine in vivo for 48 h. Autoradiographic analysis indicatedthat in the presence of PB, the hepatocytes from both foci/nodulesand the surrounding non-nodular liver responded to PH to thesame extent. In addition, they both responded to PH less efficientlyas compared to the corresponding controls. Further, initiatedrats exposed to PB for 16 weeks when subjected to PH and killed4 weeks thereafter, the percentage area occupied by     

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.     

Lack of promoting effect of clonazepam on the development of N-nitrosodiethylamine-initiated hepatocellular tumors in mice is correlated with its inability to inhibit cell-to-cell communication in mouse hepatocytes

The tumor-promoting ability of clonazepam (CZP), a widely usedbenzodiazepine anticonvulsant, was investigated in an in vivomouse liver tumor promotion assay and an in vitro mouse hepatocyteintercellular communication assay. The development of preneoplastichepatocellular foci of cellular alteration and hepatocellularneoplasms was studied in male B6C3F1 mice initiated, at 5 weeksof age, with a single i.p. injection of N-nitrosodiethylamine(NDEA; 90 mg/kg body weight) in tricaprylin, followed by administrationof either phenobarbital (PB; 0.05%) or CZP (0.068% or 0.136%)in diet beginning 2 weeks after carcinogen injection and continuingto 60 weeks of age. Several mice from each group were killedafter 9, 21, 33 or 53 weeks on test diet, and portions of liverand other organs were fixed In formalin and examined histotogically.Unlike PB, CZP did not promote the development of preneoplastichepatocellular foci or neoplasms (adenomas and carcinomas) inNDEA-initiated mice. Following limited (2 weeks) dietary exposureat 0.15%, CZP was a potent inducer of hepatic P450IIBl-mediatedalkoxyresorufin O-dealkylase activities. In contrast, the degreeof induction in hepatic tissue from mice fed 0.136% CZP for53 weeks was markedly lower than that in mice fed 0.05% PB for53 weeks. In the in vitro assay, diazepam, a strong tumor promoterin mouse liver, significantly inhibited mouse hepatocyte gapjunctional intercellular communication, while CZP had no significanteffect on this parameter. Thus, CZP, a drug structurally relatedto diazepam, is inactive as a liver tumor promoter in mice.     

Effect of timing of partial hepatectomy on the induction of preneoplastic liver foci in rats given hepatocarcinogens

Beginning 2 weeks after an intraperitoneal injection of diethylnitrosamine (DEN), rats were fed diet containing either 2-acetylaminofluorene (2-AAF), 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB), alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane (alpha-HCH), or phenobarbital (PB) for 6 weeks and subjected to partial hepatectomy (PH) after various periods of chemical administration. The effect of timing of PH was most apparent in rats fed 2-AAF. The size of gamma-glutamyl transpeptidase positive foci was largest when PH was performed 1-3 weeks after the start of chemical feeding. Similar tendency was slightly observed with 3'-Me-DAB or alpha-HCH administration, but not with PB or basal diet feeding. Thus, the enhancing effect of PH on the induction of the preneoplastic liver foci was affected by the type of chemical and the timing of the operation.     

Development of resistance during the early stages of experimental liver carcinogenesis.

The present study was designed to determine whether the resistant phenotype is acquired at the initiated cell stage itself or requires further exposure to a promoting regimen to express resistance. Male Fischer 344 rats were initiated with diethylnitrosamine (DENA) (200 mg/kg i.p.) and were subjected to either no further treatment or to the resistant hepatocyte (RH) model of liver tumor promotion. Six weeks later, the resistance of the focal lesions generated in these two groups to the mitoinhibitory effects of 2-acetylaminofluorene (2-AAF) was determined by subjecting the rats to two-thirds partial hepatectomy (PH) in the presence of a mitoinhibitory dose of 2-AAF (5 mg/kg i.p.) given at the time of PH. Labeling index was determined by administering multiple injections of [(3)H]thymidine. All rats were killed 48 h post-PH. While only a small percentage (23%) of the glutathione S-transferase-positive foci generated by DENA in the absence of an exogenous liver tumor promoting regimen were resistant to the mitoinhibitory effects of 2-AAF, a majority (85%) of the foci became resistant to 2-AAF following exposure to the RH model of liver tumor promotion. Further, initiated rats exposed to either 2-AAF or to CCl(4) alone, the two components of the RH model, resulted in 71% of the foci being resistant to the mitoinhibitory effects of 2-AAF. Similar patterns of results were obtained when the resistance of the foci to the mitoinhibitory effects of orotic acid, a liver tumor promoter and an inhibitor of DNA synthesis in normal hepatocytes, was monitored. These results suggest that the majority of initiated hepatocytes are not of resistant phenotype, however, they have acquired a unique ability to express resistance upon exposure to certain agents such as 2-AAF and CCl(4) or to a promoting regimen such as the RH model of liver tumor promotion.     

Influences of diethylnitrosamine on longevity of surrounding hepatocytes and progression of transplanted persistent nodules during phenobarbital promotion of hepatocarcinogenesis

The possibility that phenobarbital (PB) selectively promotes liver nodule development by decreasing survival of surrounding hepatocytes previously exposed to diethylnitrosamine (DENA) was evaluated. Livers of F-344 rats were labelled with [3H-methyl]-thymidine (3H-TdR) during developmental or regenerative growth. Neonatal rats given 3H-TdR between days 3 and 12 were subjected at 12 weeks of age to partial hepatectomy (PH) followed 24 hr later by DENA (10 mg/kg) or saline. Subsequent administration of PB (0.1% in drinking water) for 28 weeks reduced total liver label to 46 +/- 10% (saline group) or 40 +/- 4% (DENA group). Adult male rats initiated with DENA (200 mg/kg) and later labelled with 3H-TdR after PH also lost total liver label during 28 weeks' promotion with PB (0.05% in water) at rates similar to those exhibited by noninitiated rats given PB, and by DENA-treated or control rats not given PB. Large persistent (12 weeks) liver nodules generated by DENA in the Solt-Farber model were transplanted as small fragments into the spleens of syngeneic rats previously given 0, 100 or 200 mg/kg of DENA. Subsequent exposure to PB (0.05% in drinking water for 40 weeks) or Aroclor 1254 (6 X 300 mg/kg per month) promoted nodule and cancer development only in livers of DENA-initiated recipients. Surviving transplanted nodules remained as small microscopic clusters even after 40 weeks of promotion. However, PB increased transplant survival (50% vs. 21% in controls) whereas Aroclor reduced it to 8%. These findings indicate that promotion of liver nodules by PB occurs without enhanced mortality of surrounding hepatocytes previously damaged by DENA. They further suggest that promoters such as PB and PCBs do not directly influence the progression of established persistent nodules.     

Inhibitory Effect of Chlorophyllin on Diethylnitrosamine and Phenobarbital-induccd Hepatocarcinogenesis in Male F344 Rats

Modifying effects of chlorophyllin (CHL) on the diethylnitrosamine (DEN)-phenobarbital (PB) hepatocarcinogenesis model were examined in rats. Five-week-old male F344 rats were divided into 8 groups. Groups 1 through 5 were given i.p. injections of DEN (100 mg/kg body weight) once a week for 3 weeks beginning one week after the start of the experiment, while groups 6 through 8 received vehicle treatment. Groups 1, 2, 3 and 7 received drinking water with 500 ppm PB from one week after the end of carcinogen or vehicle treatment. CHL-containing diet (2000 ppm) was given to group 2 during the initiation phase and to groups 3 and 5 during the promotion and the post-initiation phase, respectively. Group 6 was given the experimental diet alone throughout the experiment (24 weeks). Liver neoplasms were present in DEN-treated groups and PB treatment promoted liver tumorigenesis. The incidences of adenoma in groups 2 and 3 were significantly smaller than in group 1 (P<0.05 and P<0.02), although the reductions in the incidences of liver cell cancer were not significant. The average numbers of liver neoplasms/rat in group 2 were significantly smaller than in group 1 (P<0.05-P<0.005), Glutathione S -transferase placental form-positive foci were also significantly decreased by CHL treatment (P<0.05 and P<0.001). DEN and PB exposure increased liver ornithine decarboxylase activity and this increase was significantly inhibited by feeding of CHL during the initiation phase (P<0.001). These results suggest that CHL is a chemopreventive agent for liver neoplasia.     

Molecular profiling of genes up-regulated during promotion by phenobarbital treatment in a medium-term rat liver bioassay

In search of genes that are steadily up-regulated during the promotion stage in carcinogenesis, suppression PCR subtractive hybridization and following northern blot screening were performed using a phenobarbital (PB)-promotion model based on a medium-term liver bioassay. Two weeks after a single injection of diethylnitrosamine (DEN; 200 mg/kg body wt, i.p.), rats were given 600 p.p.m. PB in the drinking water for up to 64 weeks. For comparison, animals fed 1 p.p.m. ethinylestradiol (EE) or 3000 p.p.m. butylated hydroxytoluene (BHT) in the diet at promotion stage were also included. Rats were subjected to partial hepatectomy (PH) at week 3. In addition, dose-dependence of PB at week 8 of promotion and responsiveness to representative non-genotoxic carcinogens without DEN initiation were examined. Fragments of a total of 67 different genes were isolated from the up-regulated gene population in the liver at day 10 of PB treatment by subtracting from basal expression of DEN + PH alone. Using northern blot screening for signal-detectable 48 genes, 16 genes showed up-regulation in the livers at week 8 of promotion, common to the PB and EE treatments with the levels being three times or more than the basal expression of unpromoted liver. The majority of these genes were also up-regulated at week 8 by BHT treatment, and were also constitutively expressed in the DEN(-), PH(-) untreated rat livers. Among the up-regulated genes common to the PB and EE promotion, and not responding to the non-genotoxic carcinogens in uninitiated liver, the following six genes showed overexpression in PB-promoted hepatocellular carcinomas at week 64, with the levels three times or more than untreated rat liver: ubiquitously expressed mammalian ABC half transporter, apolipoprotein A4, nuclear receptor binding factor-2, CD81, hypothetical protein (HSPC014) and one unidentified gene. These genes might be candidates for biomarkers in screening of non-genotoxic hepatocarcinogens by analysis in two-stage carcinogenesis models.     

Suppressive Effect of Irsogladine Maleate on Diethylnitrosamine-initiated and Phenobarbital-promoted Hepatocarcinogenesis in Male F344 Rats

Modifying effects of irsogladine maleate (IRG) on diethylnitrosamine (DEN)-induced hepatocarcinogenesis were examined in male F344 rats. Six-week-old rats were divided into 8 groups. Groups 1 through 4 were given a single i.p. injection of DEN (200 mg/kg body weight) at the start of the experiment, whereas groups 5 through 8 received a single i.p. injection of saline as the vehicle treatment. Groups 1 and 8 were kept on the basal diet and distilled water throughout the experiment (36 weeks). Groups 2 and 7 were exposed to 500 ppm phenobarbital (PB) in the drinking water, starting one week after the carcinogen or vehicle treatment. Groups 3 and 5 were fed the diet mixed with 125 ppm IRG from one week after DEN or vehicle treatment. Groups 4 and 6 were given 125 ppm IRG-containing diet and drinking water with 500 ppm PB after the carcinogen or vehicle treatment. Liver neoplasms developed in groups 1 (1/15 rats, 7%) and 2 (14/14 rats, 100%). However, no liver tumors were found in rats of groups 3 through 8. Incidence and average number of liver neoplasms in group 4 (0/14 rats, 0%) were less than those in group 2 ( P <0.0001). The number of glutathione S -transferase placental form (GST-P)-positive liver cell foci in group 3 or 4 was significantly smaller than that in the appropriate control ( P <0.01, P <0.001, respectively). The average and unit areas of these foci in group 4 were also significantly smaller than those in group 2 ( P <0.001, P <0.05, respectively). These results suggest that IRG could be a chemopreventive agent for rat 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.     

N-Nitroso-N-methylurea initiation in multiple tissues for organ-specific tumor promotion in rats by phenobarbital

Effects of phenobarbital [(PB) CAS: 50-06-6], a systemic tumor promoter, on carcinogenesis initiated by the broad-spectrum carcinogen N-nitroso-N-methylurea [(NMU) CAS: 684-93-5] were investigated in F344/NCr rats. Single and divided doses of NMU were evaluated for this purpose in 4-week-old rats of both sexes. Rats received iv injections of either 0.2 mmol NMU/kg (body wt) once or 0.05 mmol NMU/kg (body wt) for 4 weeks (1 injection/wk), followed by or concurrently with PB (0.05% in drinking water) that was continued until the termination of the experiment. Half the rats were killed at 52 weeks and survivors at 80 weeks. At 52 weeks, PB given subsequent to NMU or concurrently with divided doses of NMU significantly enhanced the incidence of thyroid follicular tumors only in male rats. This sex difference in thyroid tumorigenesis was somewhat less pronounced in animals killed at 80 weeks. Only 1 liver cell adenoma occurred in males and none in females given NMU alone. PB given concurrently with divided doses of NMU enhanced the yield of hepatocellular foci/cm2 but had no significant effect on hepatic tumor development. Subsequent exposure to PB, however, significantly promoted hepatocarcinogenesis in rats of both sexes given NMU in divided doses; 50% of males and 40% of females given NMU (0.05 mmol/kg, administered four times) followed by PB had hepatocellular adenomas and carcinomas by 80 weeks. PB did not affect the incidence of any other kind of neoplasms seen in NMU-initiated or control rats. These lesions included squamous cell neoplasms of the skin, oropharynx, and forestomach; nonsquamous epithelial tumors of mammary gland, pituitary body, intestinal mucosa, and urinary bladder; tumors of the central and peripheral nervous system; and mesenchymal tumors of the kidney. A sequence of multiple low doses of NMU appeared to be a convenient and useful systemic, multitissue, tumor-initiation regimen for systematic investigation of organ-specific tumor promotion in rats.     

Possible tumor development from double positive foci for TGF-alpha and GST-P observed in early stages on rat hepatocarcinogenesis

Expression of TGF-alpha during promotion of neoplastic development from GST-P-positive foci in rat chemical hepatocarcinogenesis was investigated. One-hundred male F344 rats were given a single intraperitoneal injection of DEN (200 mg/kg bodyweight) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2 weeks from the start, PB at doses of 0 or 500 p.p.m. was fed to the rats for 46 weeks. Groups of 10 rats were killed at weeks 4, 8, 16, 32, 48 and their livers were immunohistochemically examined for expression of GST-P and TGF-alpha. TGF-alpha-positive foci and single positive cells were observed from week 4, partially overlapping with GST-P-positive foci but being much fewer. Numbers of TGF-alpha-positive lesions did not increase from weeks 4-48, but their areas showed increment at weeks 32 and 48, especially with PB administration. Almost all of the tumors observed at weeks 16, 32 and 48 were positive for TGF-alpha (98%). In addition, epidermal growth factor receptor overexpression was observed in most TGF-alpha-positive lesions (foci and tumors). The proliferating cell nuclear antigen labeling index in double positive foci for GST-P and TGF-alpha was significantly higher than that in TGF-alpha-negative foci. In conclusion, TGF-alpha may be closely related with promotion from altered foci to neoplasms in rat hepatocarcinogenesis. Our data suggest that double positive foci for GST-P and TGF-alpha in the early stages of rat hepatocarcinogenesis may develop into tumors with promotion.     

Sex-differentiated and growth-hormone-regulated mitoinhibition in rat liver during treatment with 2-acetylaminofluorene and partial hepatectomy in the resistant hepatocyte model

The previously observed sex difference in the growth rate of enzyme-altered foci (male greater than female) in rats treated according to the resistant hepatocyte model (RH model) occurs during selection/promotion of diethylnitrosamine-initiated cells with dietary 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH). The secretory pattern of growth hormone (GH) is sex dependent in the adult rat and is a major determinant for sex-differentiated liver functions. In the present study the capacity for liver regeneration following PH in the RH model was studied in rats of both sexes, in castrated males and in males receiving GH infusion, both treatments leading to a feminine pattern of GH secretion. Mitoinhibition during treatment with 2-AAF was shown to be more pronounced in liver from male than from female rats, both in initiated and non-initiated animals. Castration of male rats and continuous infusion of GH to males during the selection period, previously shown to decrease the focal growth rate towards that in female rats, 'feminized' also the degree of mitoinhibition. Autoradiography of sections from animals receiving continuous infusion of [3H]thymidine for 1 week, starting at the time of PH, demonstrated a lower labeling index in surrounding liver from male rats treated in the RH model than in surrounding liver from female rats. Males treated with continuous infusion of human GH during 2-AAF/PH treatment had an intermediary labeling index in the surrounding tissue. No differences in labeling index in enzyme-altered foci was observed between males, females or males plus hGH. These findings support the concept that sex-differentiated promotion in the RH model is exerted by selective mitoinhibition and that this feature is regulated by GH.     

Anti-carcinogenic action of phenobarbital given simultaneously with diethylnitrosamine in the rat

The present work has been planned in order to elucidate the effect of phenobarbital (PB: 15 mg per rat of ingested dose) on carcinogenesis when it is administered simultaneously with diethylnitrosamine (DEN: 10 mg/kg/day). Wistar rats (180 g) were treated by DEN alone or by DEN + PB during 2, 4 and 6 weeks according to our schedule for hepatocarcinogenesis. After the end of the treatment, the number and the size of induced PAS positive preneoplastic foci was significantly reduced when PB was given simultaneously with DEN for 4 and 6 weeks. The mitotic inhibition and the production of micronuclei normally observed after partial hepatectomy in DEN treated rats were also significantly decreased in DEN + PB treated rats. When the treatment last only 2 weeks, the presence of PB did not change significantly the last parameters. In DEN + PB treated rats, the survival was prolonged and the tumor incidence decreased as compared with the results obtained by DEN alone. It is concluded that PB, which promotes carcinogenesis when administered after the DEN treatment, reduces the carcinogen effect when given simultaneously with DEN. This ‘anti-carcinogen’ effect acts on the initiation as well as on the promotion of the precancerous lesions. Biochemical investigations are in progress to obtain more information about this ‘paradoxical’ PB effect.     

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.     

Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver.

The effects of tea polyphenols and tea pigments on rat liver precancerous lesions and some cell cycle regulators were studied. A modified Solt-Farber model in rats was established by multiple low-dosage of N-nitrosodiethylamine (NDEA) i.p. injections, followed by i.p. CCl(4) injection and partial hepatectomy. Sixty male Wistar rats were randomly divided into four groups: positive control group, two tea-treated groups, and negative control group. Rats in tea-treated groups were given tea polyphenols (0.1%) and tea pigments (0.1%) in drinking fluid during the whole experiment. The number and area of glutathione S-transferase P (GST-P)-positive foci in the rat liver were used as biomarkers of precancerous liver lesions. Western blotting assay was carried out to detect the expression of cyclin D1, CDK4, and P21(WAF1/CIP1) on whole liver extract. At the end of the experiment (56 days), the number and area of GST-P-positive foci in liver increased significantly in carcinogen-administered positive control group, whereas no GST-P-positive foci were found in the negative control group in which animals did not receive carcinogen exposure. The number and area of GST-P-positive foci in tea-treated, carcinogen-exposed groups were significantly reduced as compared with the positive control group. It was also found that the expression of P21(WAF1/CIP1) was significantly induced and the expression of cyclin D1 and CDK4 was significantly inhibited in tea-treated groups. These results suggest that tea polyphenols and tea pigments are effective in preventing the precancerous liver lesions in rats, and modulation of cell cycle by regulating cell cycle regulators may be a possible mechanism.     

Incorporation of bromodeoxyuridine in glutathione S-transferase-positive hepatocytes during rat multistage hepatocarcinogenesis

Cell proliferation is pivotal to all stages of the carcinogenesisprocess and is one of the primary characteristics of the promotionstage of cancer development. Both a two-stage model of initiationand promotion for analysis of early preneoplasia and a three-stageinitiation-promotion-progression model of hepatocarcinogenesiswere used to address the effect of the liver tumor-promotingagent phenobarbital (PB) on hepatic cellular proliferation.Male rats were subjected to a 70% partial hepatectomy and 10mg diethyInitrosamine (DEN)/kg or the solvent alone and wereadministered PB for 4–8 months. Analysis of bromodeoxyuridine(BrdU) incorporation (1 h pulse) in liver within (focal) andnot with in (non-focal) altered hepatic foci (AHF) demonstrateda labeling index in AHF of 2% in DEN-initiated rats; the non-focallabeling index of placental glutathione S-transferase expressinghepatocytes was 0.3–0.6%. The focal labeling index wasconstant over the 8 month period of promotion. Inasmuch as onecharacteristic of promotion is the reversibility of the inducedeffects on clonal expansion of initiated cells, groups of ratsinitially promoted with PB were maintained in the absence ofcontinued promotion for 4 or 8 months prior to being killed.Assessment of the focal labeling index after cessation of PBtreatment indicated a drop in the index from 2.3% to 0.7%. Whena progressor agent, ethylnitrosourea, was given at the timePB was discontinued for 4 or 8 months, a significant changein focal labeling index was not observed relative to the indexin AHF when the animals were killed immediately after 8 monthsof PB promotion. Thus, cell proliferation plays an integralrole in both the promotion and progression stages of multistagerat hepatocarcinogenesis and is influenced by administrationof promoting and progressor agents.     

Compared promoting potential of D-galactosamine, carbon tetrachloride and partial hepatectomy in rapid induction of preneoplastic liver lesions in the rat

Effectiveness of two different chemically induced stimuli for hepatocellular proliferation was compared with regard to that of commonly used partial hepatectomy (PH), for the purpose of developing short-term protocol for the assay of promoting agents of hepatocarcinogenesis. Enhancing effect of D-galactosamine (DGA) and carbon tetrachloride (CCl4) given during the promotion procedure by 2-acetylaminofluorene (2-AAF) was compared along with PH in rats initiated by diethylnitrosamine (DEN), using preneoplastic glutathione S-transferase positive (GST-P+) hepatocyte foci as an end-point marker lesion. The number of GST-P+ foci per cm2 was largest in the group given CCl4 followed by DGA, no treatment (2-AAF alone) and PH. In contrast, the area (mm2) per cm2 and mean diameter of the focus were largest in the PH group then DGA followed by CCl4 and no treatment. The results indicate that the number of GST-P+ foci were not clearly affected by 3 different treatments whereas area and size of foci which represented the result of promoting effect were clearly influenced by those treatments, indicating they caused differential proliferation of initiated cells. In this respect, even though PH is the most potent procedure, DGA is also efficient and preferred to CCl4 for the non-surgical enhancing method.