Effect of coadministration of phenobarbital sodium on N-nitrosodiethylamine-induced gamma-glutamyltransferase-positive foci and hepatocellular carcinoma in rats

The effect of concurrent administration of phenobarbital on the hepatocarcinogenicity of N-nitrosodiethylamine (diethylnitrosamine; DENA) in rats was investigated by determination of the incidence of gamma-glutamyltransferase (gamma-glutamyltranspeptidase) (GGT)-positive foci and liver tumors. Male outbred Sprague-Dawley rats received either a weekly oral dose of DENA (0.08 mol/kg), phenobarbital sodium (500 ppm) in their drinking water, or DENA and phenobarbital sodium concurrently. After 16 weeks, only the animals treated concurrently with DENA and phenobarbital sodium had GGT-positive foci (3.65 foci/cm2). At 30 weeks, the group treated with DENA and phenobarbital sodium exhibited more foci (23.6 foci/cm2) compared to the group that received only DENA (3.08 foci/cm2). The average size of foci in both of the DENA-treated groups was the same. The tumors in the group that received DENA plus phenobarbital sodium showed a greater incidence of GGT activity compared to the tumors in the DENA group. Under the conditions of this study the incidence of GGT-positive foci did not predict the incidence of hepatocellular carcinomas.     

Stable phenotypic expression of glutathione S-transferase placental type and unstable phenotypic expression of gamma-glutamyltransferase in rat liver preneoplastic and neoplastic lesions

Using immunohistochemical demonstration of glutathione S-transferase placental type (GST-P) and histochemical demonstration of gamma-glutamyltransferase (gamma-GT), the long-term development of preneoplastic and neoplastic lesions was followed in rats over a 50-week period. Rats were given a single i.p. injection of 200 mg/kg body weight of diethylnitrosamine (DEN), and then 2 weeks later were administered 0.02% 2-acetylaminofluorene (2-AAF) (group 1), 0.05% phenobarbital (PB) (group 2), 2.0% butylated hydroxyanisole (BHA) (group 3) or no supplement (group 4) in their diet for 6 weeks, all rats being subjected to partial hepatectomy at week 3. Hepatocellular proliferated lesions were classified as foci, nodules and hepatocellular carcinomas. Development of foci, nodules and hepatocellular carcinomas was enhanced strongly by 2-AAF and weakly by PB, and inhibited by BHA. Almost all foci and nodules were GST-P positive, although 5-10% of the GST-P-positive foci were gamma-GT negative. The areas of GST-P-positive foci and nodules increased with time in all groups. In contrast, while the areas of gamma-GT-positive lesions also increased with time in groups 2-4, they decreased from week 12 in group 1. As the percentage gamma-GT-positive area in GST-P-positive foci significantly decreased with time in all groups, the rate of phenotypic reversion of gamma-GT in foci in group 1 was revealed to be larger than the focus growing rate, whereas that in groups 2-4 was smaller. Gamma-GT-negative and GST-P-positive micro-nodules of altered morphology appeared within gamma-GT- and GST-P-positive nodules in later stages. All hepatocellular carcinomas found in this experiment consisted of GST-P-positive cells. In contrast, 37% (13/35) of the hepatocellular carcinomas were negative for gamma-GT. The results indicate GST-P to be the most accurate marker enzyme for detection of initiated cells during liver carcinogenesis and gamma-GT to be more appropriate for indicating changes of phenotypic expression in each lesion type.     

Comparative tumor-promoting activities of phenobarbital, amobarbital, barbital sodium, and barbituric acid on livers and other organs of male F344/NCr rats following initiation with N-nitrosodiethylamine

Tumor-promoting abilities of four barbiturates, phenobarbital [(PB) CAS: 50-06-6], amobarbital [(AB) CAS: 57-43-2], barbital sodium [(BB) CAS: 144-02-5], and barbituric acid [(BA) CAS: 67-52-7], on the development of neoplasms in livers and other organs of rats following initiation with N-nitrosodiethylamine [(DENA) CAS: 55-18-5] were compared. Four-week-old F344/NCr male rats were given a single ip injection of 75 mg DENA/kg body weight. Beginning 2 weeks later, they were given either tap water (group 1) or drinking water containing 500 ppm of PB (group 2), the sodium salt of BB (group 3), AB (group 4), or BA (group 5) for the remaining experimental period. Control groups (groups 6-10) received an ip injection of saline alone and 2 weeks later were given either tap water or drinking water containing barbiturates as listed above. Animals were sacrificed at either 52 weeks or 78 weeks. None of the barbiturates altered the growth and survival of animals. PB and BB increased liver weights and significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 weeks and hepatocellular foci, hepatocellular adenomas, and trabecular carcinomas at 78 weeks in DENA-treated rats. No such enhancing effects were observed with AB or BA. PB or BB did not significantly enhance the incidence of nonhepatic tumors at 52 weeks. However, at 78 weeks BB significantly enhanced the development of renal tubular adenomas and carcinomas, while PB enhanced the development of thyroid follicular cell neoplasms in DENA-treated rats. These results clearly showed that barbiturates exhibited structure-promoting activity relationships and that their promoting abilities were not restricted to liver alone. Substitution of both hydrogen atoms at the C-5 position of the pyrimidine ring by alkyl or aryl groups appears to be essential but not sufficient for tumor-promoting activity of barbiturates.     

N-nitrosodiethylamine-induced hepatocarcinogenesis in estuarine sheepshead minnow (Cyprinodon variegatus): neoplasms and related lesions compared with mammalian lesions

Groups of estuarine sheepshead minnows (Cyprinodon variegatus) were exposed to approximately 57 mg N-nitrosodiethylamine [(DENA) CAS: 55-18-5]/liter for 5-6 weeks. Exposure was stopped and the fish were then transferred to clean, flowing seawater. Induced liver lesions were studied in periodic samples of fish taken during the next 140 weeks of holding. Lesions found following exposure were early altered basophilic and eosinophilic foci, oval cell hyperplasia, clear cell foci, neoplastic nodules, hepatocellular carcinomas, cholangiolar carcinomas, possible pericytomas originating in liver, hemangiopericytomas, spongiosis hepatis, and cholangiofibrosis. The relative prevalence of these lesions was given. Most of these lesions morphologically were compared to their counterpart lesions in the rat. Certain lesions in our fish such as hepatocellular carcinomas, cholangiolar carcinomas, pericytomas, hemangiopericytomas, spongiosis hepatis, and cholangiofibrosis have apparent similar cellular origins and morphogenesis to those lesions in rats and perhaps in other mammals. Spongiosis hepatis in the sheepshead minnow apparently arises from perisinusoidal cells and may be a neoplasm of this cell type. The general similarity of response to DENA in sheepshead minnows and rats suggests that this fish has promise as a model subject for studying some hepatocarcinogens and as a sentinel organism for detecting hepatocarcinogens in contaminated coastal waters.     

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.     

Influences of different polychlorinated biphenyls on cytocidal, mitoinhibitory, and nodule-selecting activities of N-2-fluorenylacetamide in rat liver

The influences of different polychlorinated biphenyl (PCB) isomers and congeners on distinct hepatotoxic responses to the hepatocarcinogen N-2-fluorenylacetamide [(2-FAA) CAS: 53-96-3] were examined in F344 rats. Cytocidal toxicity of 2-FAA (25-400 microM), determined by lactate dehydrogenase release during 20 hours in primary monolayer cultures of isolated rat hepatocytes, was reduced by in vivo pretreatment with either phenobarbitone [(PB) CAS: 50-06-6] or 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), a PB-type PCB inducer. However, cytocidal toxicity of 2-FAA was substantially potentiated by either 3-methylcholanthrene [(MCA) CAS: 56-49-5] or 3,3',4,4'-tetrachlorobiphenyl [(TCBP) CAS: 32598-13-3], an MCA-type PCB. In the same cell culture assays, all four pretreatments similarly reduced cytocidal toxicity of N-hydroxy-N-2-fluorenylacetamide (0.32-32 microM; CAS: 53-95-2). By comparison, pretreatments with either the PB-type or MCA-type PCB's (50-200 mumol/kg) diminished mitoinhibitory toxicity of 2-FAA in vivo, as measured by hepatic regenerative growth and hepatocyte labeling indices 7 days after partial hepatectomy (PH) in rats given 3 consecutive daily doses of 2-FAA (20/mg/kg/day) before PH. This regimen of 2-FAA and PH promoted rapid selective growth of gamma-glutamyltranspeptidase-positive (gamma-GT+) nodules at 2 and 4 weeks after PH in rats previously given an initiating hepatocarcinogen, diethylnitrosamine [(DENA) CAS: 55-18-5]. However, various PCB's, including 2,2',4,4',5,5'-HCBP, 3,3',4,4'-TCBP, 2,2',4,4'-TCBP, 2,2',5,5'-TCBP, and the commercial mixture Aroclor 1254, each given as a single dose of 50 mumol/kg by gavage 10 days after DENA and 7 days before 2-FAA, all reduced the size of 2-FAA-selected gamma-GT+ nodules during the 4-week period after PH. These results indicate that, in spite of predictable inducer-specific opposite influences of different types of PCB's on cytocidal toxicity of 2-FAA, all PCB's similarly reduce nodule selection by 2-FAA in initiated livers. Reduced growth of 2-FAA-selected nodules correlated with the consistent ability of all PCB's to enhance regeneration of liver mass after 2-FAA and PH.     

Differences between the promoting activities of the peroxisome proliferator WY-14,643 and phenobarbital in rat liver

In order to characterize the promoting activity of the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (WY-14,463), male F344 rats which received a single 150-mg/kg dose of diethylnitrosamine (DEN) were fed 0.1% WY-14,643 or 0.05% phenobarbital in the diet for 11, 22, or 54 wk. WY-14,643 promoted the development of ATPase-deficient foci but not GGTase-positive or G6Pase-deficient foci, in contrast to phenobarbital which promoted development of foci detected by all three markers. The mode of promotion of ATPase-deficient foci by WY-14,643 was distinctly different from that of phenobarbital. WY-14,643 primarily increased mean volume of foci at 11 and 22 wk, while phenobarbital primarily increased the numerical density of foci at these time points. At 54 wk, the yield of hepatic neoplasms per liver was higher in rats fed WY-14,643 than in rats fed phenobarbital. To evaluate the possibility that the promotional activity of WY-14,643 was more effective at a later stage in hepatocarcinogenesis, rats receiving a dose of DEN and then phenobarbital in the diet for 11 wk were changed to a diet containing WY-14,643 for an additional 11 or 43 wk. However, WY-14,643 feeding from wk 11 to 22 caused a reduction in volume density of ATPase-deficient foci relative to the volume density of foci at 11 wk. In addition, feeding WY-14,643 from wk 11 to 54 caused similar yields of hepatic neoplasms whether or not phenobarbital was fed for the initial 11 wk. WY-14,643 induced hepatic peroxisome proliferation as indicated by palmitoyl CoA oxidase activity regardless of prior treatment with DEN and/or phenobarbital. The yield of neoplasms in rats not receiving DEN was greater in rats fed WY-14,643 for wk 11 to 54 than in rats fed WY-14,643 for wk 1 to 54. In summary, the peroxisome proliferator WY-14,643 was a more efficient promoter of hepatocarcinogenesis in DEN-initiated rats than phenobarbital. The promotional activity of WY-14,643, when evaluated by stereological analysis and by changing promoters, is distinct from that of phenobarbital, perhaps suggesting different cellular and/or molecular mechanisms of promotion. Understanding the role of promotion by WY-14,643 and other peroxisome proliferators may be important in understanding the mechanism of their hepatocarcinogenicity.     

Inhibition by d-limonene of experimental hepatocarcinogenesis in Sprague-Dawley rats does not involve p21(ras) plasma membrane association.

The effects of d-limonene on hepatocarcinogenesis induced by N-nitrosomorpholine (NNM) and on membrane-associated p21(ras) and labeling and apoptotic indices of the liver were investigated in male Sprague-Dawley rats. Rats were given drinking water containing NNM for 8 weeks, and from the beginning of experimental week 9, they received chow pellets containing 1% or 2% limonene. The preneoplastic and neoplastic liver lesions (cellular alteration foci, neoplastic nodules and hepatocellular carcinomas), and hepatic foci staining positive for glutathione-S-transferase, placental type (GST-P) were examined microscopically and histochemically. At week 16, quantitative histologic analysis showed that oral administration of 1% or 2% limonene resulted in significant reductions in the number and mean area of GST-P-positive hepatic foci and the number of cellular alteration foci, neoplastic nodules and hepatocellular carcinomas. Limonene, at both doses, also caused significant decreases in the labeling indices and significant increases in the apoptotic indices of cellular alteration foci, neoplastic nodules, hepatocellular carcinomas and adjacent liver. However, limonene, at both doses, had no significant influence on the production of membrane-associated p21(ras) in the visible liver white nodules. These findings indicate that limonene inhibits hepatocarcinogenesis and suggest that this effect may be clearly related to its effect in inhibiting cell proliferation and in enhancing apoptosis, but not through ras oncoprotein plasma membrane association.     

Pathogenesis of dimethylnitrosamine-induced hepatocellular cancer in hamster liver and lack of enhancement by phenobarbital

The early stages of dimethylnitrosamine (CAS: 62-75-9)-induced liver cancer and the effect of administration of phenobarbital (CAS: 50-06-6) after dimethylnitrosamine were studied in Syrian golden hamsters. A single ip injection of 6 mg dimethylnitrosamine/kg (body wt) induced hepatocellular altered foci by 6 months. Most foci were composed mainly of large clear glycogen-containing cells, while smaller numbers were formed by cells with abnormally acidophilic or basophilic cytoplasms. Biliary cysts, but not biliary neoplasms, also occurred. A few neoplastic (hyperplastic) nodules and hepatocellular carcinomas developed by 12 months. gamma-Glutamyltranspeptidase activity was not present in any of the hepatocellular lesions, but foci of all types, neoplastic nodules, and carcinomas were characterized by a lack of iron accumulation. Phenobarbital at 0.05% in the diet for up to 12 months did not increase the number of lesions of any type, indicating a lack of promoting effect under these conditions.     

Early stages of N-2-fluorenylacetamide-induced hepatocarcinogenesis in male and female rats and effect of gonadectomy on liver neoplastic conversion and neoplastic development

The early stages of N-2-fluorenylacetamide [(2-FAA) CAS: 59-96-3]-induced liver carcinogenesis in inbred F344 male and female rats and the effect of gonadectomy on liver carcinogen biotransformation capability and hepatocarcinogenesis were studied. Feeding of 2-FAA induced more altered hepatocellular foci characterized by exclusion of cellular iron and gamma-glutamyl-transferase activity in male rats than in female rats. At 6-22 weeks after cessation of carcinogen exposure, only males developed liver neoplastic nodules. Liver cytochrome P450, aryl hydrocarbon hydroxylase, and uridine-5'-diphosphateglucuronosyltransferase activity toward p-nitrophenol, but not phenolphthalein, were greater in males than in females. Gonadectomy of males reduced the activities that were greater than in females, whereas none was significantly altered by gonadectomy of females. Gonadectomy of male rats prior to 2-FAA feeding suppressed the induction of both altered foci and neoplastic nodules, whereas in female rats gonadectomy prior to 2-FAA feeding enhanced the induction of foci. Gonadectomy of males after administration of 2-FAA slightly enhanced the persistence of foci at 6 and 12 weeks after removal of carcinogen, but it did not affect their persistence by 22 weeks post carcinogen or the incidence of neoplastic nodules. However, only the males that were gonadectomized after receiving 2-FAA developed hepatocellular carcinomas at 22 weeks. Gonadectomy of females after receiving 2-FAA did not affect the persistence of foci, and no liver neoplasms developed. Thus gonadectomy of male rats, which reduced liver carcinogen metabolism, when done before carcinogen feeding had the greatest effect on hepatocarcinogenesis.     

Profound postinitiation enhancement by short-term severe methionine, choline, vitamin B12, and folate deficiency of hepatocarcinogenesis in F344 rats given a single low-dose diethylnitrosamine injection

The potential promoting and/or complete carcinogenic activity of a methyl group-deficient (MD) diet lacking methionine, choline, vitamin B12, and folate on liver tumor induction in weanling male F344/NCr rats was examined. Each of 50 rats per group received one injection 20 mg diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine]/kg body weight at 4 weeks of age, and then each was maintained on a methyl group-adequate (MA) diet for 52 weeks (groups 2 and 5) or on an MD diet for 15 weeks followed by the MA diet for 37 weeks (group 4). Controls received injections of saline and were maintained on the same two respective diet regimens (groups 1 and 3, respectively). Histologic results from sacrifices at 6, 10, 15, 22, 39, and 52 weeks revealed early development of foci of eosinophilic gamma-glutamyltransferase (GGT)-positive hepatocytes by week 6 in DENA-MD diet-treated rats, with subsequent development of a diffuse hyperplasia of hepatocytes, oval cell proliferation, cholangiofibrosis, nodular cirrhosis, and neoplastic nodule (NN) formation and, at 52 weeks, hepatocellular carcinomas (HCC) in 13 of 15 rats. Similar but significantly fewer lesions were observed at slightly later sacrifice times in the livers of saline-MD diet-treated rats, with development of NN in 5 of 12 rats and an HCC in 1 of 12 rats at 52 weeks. DENA-treated rats on MA diets developed relatively few GGT-positive foci, and none developed any neoplastic lesions. Except for basophilic foci, areas and foci of cellular alteration containing glycogen-rich hepatocytes frequently exhibited diminished uptake of injected iron and decreased glucose-6-phosphatase and ATPase contents focally or throughout. This study indicates that a relatively brief exposure of both untreated and DENA-treated weanling rats to a severely MD diet produces classical preneoplastic and neoplastic lesions in their livers.     

Evidence of two separate mechanisms for the decrease in aryl sulfotransferase activity in rat liver during early stages of 2-acetylaminofluorene–induced hepatocarcinogenesis

Enzymatic and immunohistochemical experiments were conducted to evaluate the mechanistic basis for the downregulation of the important detoxication/bioactivation enzyme aryl sulfotransferase IV (AST IV) during 2- acetylaminofluorene (2AAF)–induced hepatocarcinogenesis. To distinguish between possible genotoxic and cytotoxic actions of 2AAF, three different dietary protocols were used in these experiments: group 1 received 2AAF for 12 wk, group 2 received 2AAF for 3 or 6 wk and then a control diet lacking xenobiotics for 3 or 6 wk, and group 3 received 2AAF for 3 or 6 wk and then phenobarbital for 3 or 6 wk. When hepatic AST IV activity was assessed, N-hydroxy-2AAF sulfotransferase activity was found to decrease 80–90% in response to 2AAF feeding, but activity recovered to essentially normal levels in the livers of rats subsequently placed on either control diets or diets with phenobarbital, suggesting a reversible cytotoxic mechanism for loss of AST IV activity. However, when liver sections from the rats were evaluated immunohistochemically, two distinct patterns were detected for the downregulation of AST IV expression was observed throughout the liver and among most but not all newly developed nodules. In tissue sections from rats initially fed 2AAF and then placed on a control diet (group 2) or a diet with phenobarbital (group 3), the nodules continued to show low levels of AST IV expression, while expression in the areas surrounding nodules returned to the normal, high levels. In addition, among those rats fed 2AAF for just 3 wk and then control diet or diet containing phenobarbital for 6 wk, only rats fed phenobarbital developed altered foci that stained weakly for AST IV expression. These results show that there were two kinds of 2AAF-mediated decrease in hepatic AST IV activity: a general overall loss of AST IV expression dependent on administration of 2AAF and reversible upon removal of 2AAF from the diet and a loss of AST IV expression among newly developed liver foci and nodules that persisted in the absence of 2AAF administration and appeared to be a property of 2AAF-induced subpopulations of cells. These patterns may correspond, respectively, to cytotoxic and genotoxic mechanisms of 2AAF action. © 1994 Wiley-Liss, Inc.     

Immunohistochemical detection of c-Ha-ras oncogene p21 product in pre-neoplastic and neoplastic lesions during hepatocarcinogenesis in rats

An immunohistochemical study of c-Ha-ras expression was performed on preneoplastic and neoplastic stages of diethylnitrosamine (DENA)-induced hepatocarcinogenesis in rats, using an antibody raised against a peptide sequence of the Ha-ras p21 product. Moderate to high immunostaining intensity was observed in the following hepatocytic lesions: (1) hepatocellular carcinomas (14/14) and associated neoplastic nodules (8/8) and foci of phenotypic alterations (35/40) (after 13-20 months of treatment); (2) neoplastic nodules (6/6) and associated foci (42/50) (after 5-9 months); (3) foci (10/10) (after 2 months); and (4) small, slowly growing foci (26/40) found 9 months after treatment with DENA without prior partial hepatectomy, resulting in low number of nodules and no tumor even after 15 months. No c-Ha-ras p21 was detected immunohistochemically in normal nor in regenerating rat liver. Our results indicate that increased Ha-ras expression is an early and stable event in liver lesions associated with hepatocarcinogenesis. They also imply that increased Ha-ras expression is insufficient (if at all implicated) for inducing fully malignant hepatocyte transformation. It might be indicative of cell populations at an increased transformation risk.     

Induction of gamma-glutamyltransferase-positive and nonspecific esterase-positive foci in rat liver by partial hepatectomy following single injection of N-nitrosodiethylamine

The effect of partial hepatectomy (PH) on alteration of liver foci induced by N-nitrosodiethylamine (DENA) was studied in inbred F344 male rats. As early as 2 weeks after PH was performed 6 hours after an injection of 100 mg DENA/kg, gamma-glutamyltransferase-positive hepatocellular foci were induced, whereas DENA alone induced no foci until 12 weeks after PH. The focus counts of the group with PH performed 6 hours after an injection of 100 mg DENA/kg were consistently greater than those of a group with PH performed at 24 hours following DENA injection. At 3 and 6 weeks after PH was done at 12 weeks following treatment with 100 or 200 mg DENA/kg, the focus count was significantly increased compared with that in nonhepatectomized groups. The results indicate that increased liver cell proliferation resulting from PH enhances the conversion of persisting DNA damage to a permanent alteration in DNA. The effect at 12 weeks after exposure supports the concept that DNA damage in hepatocytes is highly persistent.     

An electron microscope study of hepatocellular changes in the rat during chronic treatment with acetamide. Parenchyma, foci and neoplasms

Male Leeds strain rats were fed a diet containing 5.0% by weightof acetamide (AA), for up to 35 weeks, inducing a high incidenceof hepatic cell neoplasms. The sequential morphological changesinduced by AA in the hepatic parenchymal cells were studiedby electron microscopy and were compared with those observedin foci of cellular alteration, neoplastic nodules and hepatocellularcarcinomata. The observed fine structural changes includingearly glycogen depletion, dispersal and dislocation of the roughendoplasmic reticulum (ER), smooth ER proliferation and nuclearirregularity and nucleolar abnormalities. Glycogenosis developedin some cells during treatment and was characteristic of the‘clear cell’ foci, as well as being common in theneoplasms. The most consistent alteration, observed in parenchyma]cells and the cells of foci, nodules and carcinomas, was thatinvolving the rough ER. This persisted after withdrawal of AAfrom the diet, whereas many other changes were transient, andmay represent a change in differentiation associated with neoplasticinduction.     

Effect of phenobarbital on the development of liver tumors in juvenile and adult mice

The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal's age at the start of exposure.     

Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses

The hepatocarcinogenicity of aflatoxin B1 (AFB1) was compared in male Syrian golden hamsters and inbred F344 rats. AFB1 was administered by gavage 5 days/week for 6 weeks at doses of 1 and 2 mg/kg body weight/day to rats and hamsters, respectively; rate did not survive beyond 3 weeks with doses of 2 mg/kg/day. After 6 weeks the animals either received no further treatment or were given 0.1% phenobarbital sodium in the drinking water. ATPase-deficient foci of hepatic parenchymal cells, neoplastic nodules, and hepatocellular carcinomas were observed in liver sections from AFB1-treated rats killed at 6, 14, or 23 weeks; they were not seen in sections from AFB1-treated hamsters killed at 6, 14, or 46 weeks. Each of the 50 AFB1-treated rats developed hepatocellular carcinomas by 46 weeks; many also had cholangiocarcinomas and mixed hepatocellular-cholangiocellular carcinomas. Hepatocellular carcinomas were found in only 2 of 49 AFB1-treated hamsters by 78 weeks. At this time cholangiocarcinomas were found in 15 hamsters, and microscopic cholangiomas were seen in all of the livers. Compared to the rat, the hamster was resistant to the hepatotoxic and hepatocellular carcinogenic effects of AFB1.     

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.     

Promotion mechanism of phenobarbital and partial hepatectomy in DENA hepatocarcinogenesis cell kinetics effect

Diethylnitrosamine (DENA, 10 mg kg-1 per day) was fed to rats for 2, 4 and 6 weeks. One week after the cessation of DENA, animals were submitted either to partial hepatectomy or to phenobarbital administration. Partial hepatectomy did not promote neoplastic transformation, except after a 6-week DENA treatment. A minimum of phenobarbital was required to reach a significant promoting effect in DENA carcinogenesis. A too-limited treatment was ineffectual but could be compensated for by prolonged DENA administration. The phenobarbital treatment became unnecessary when neoplastic nodules were present. Phenobarbital continuously given after the carcinogen administration promoted neoplastic transformation even after a subcarcinogenic DENA treatment (2 weeks). It accelerated the pathological evolution and increased the tumour incidence. In these conditions, phenobarbital increased the proliferation advantage of preneoplastic cells over normal cells. In the different experimental modalities, the promoting effect was associated with the induction of chronic cell proliferation, the inhibition of the rapid response to the 2/3 partial hepatectomy and the mitotic circadian rhythm normally present during liver regeneration. It is concluded that the promotion mechanism could consist in disturbing the mitotic control in order to maintain, for a long time, a chronic low level of cell proliferation permitting the selective growth of preneoplastic cells and their subsequent transformation.     

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.