Amplified response to phenobarbital promotion of hepatotumorigenesis in obese yellow Avy/A (C3H x VY) F-l hybrid mice

Mottled yellow A^vy/A and agouti ^A/a (C3H x VY) F-l hybrid malemice were fed untreated control diet or diet with a target doseof 500 p.p.m. sodium phenobarbital (PB) for 17–19 months.No differences in prevalence of hepatocellular adenomas or carcinomaswere found between untreated yellow and agouti mice. PB treatmentincreased prevalence of adenomas but decreased prevalence ofcarcinomas. No difference in enhancement of adenoma formationby PB was observed between yellow and agouti mice bearing singleadenomas. However, the proportion of PB-treated yellow micebearing multiple adenomas (66%) was much greater than the proportionof analogous agouti mice (18%). Fatty changes in the peri-portalarea of the liver and focal cytoplasmic vacuolization were inducedto a much greater extent in PB-treated yellow mice than amongtreated agoutis. PB increased the prevalence and severity offocal areas of chronic inflammation in the liver considerablymore in agouti than in yellow mice. The possible relation ofthis finding to the altered immune responses of obese yellowmice remains to be determined. The results of this study suggestthat the use of yellow A^vy/A and agouti A/a (C3H x VY) F-l hybridmice in carcinogenicity asays make make it possible to differentiatebetween weak and strong promoters as well as between promotersand complete carcinogens. Weak promoters should induce hepatocellularadenomas in yellow mice even if they fail to do so in agoutimice. Promoting substances which act similarly to PB may beidentified in this system by simultaneously increasing adenomaprevalence and decreasing carcinoma prevalence. Complete carcinogensshould increase carcinoma prevalence in the yellow mice evenat low dose levels.     

Comparison of the effects of acute and subacute treatment of phenobarbital in different strains of mice

A strain specificity has been demonstrated for the effect of subsequent administration of phenobarbital (PB), in which diethylnitrosamine (DENA)-initiated hepatocarcinogenesis was promoted in C3H mice, inhibited in B6C3F1 (C57BL x C3H) and not affected in C57BL mice. A correlation has been established between the ability of barbiturates and hydantoins to promote tumor formation and their ability to induce liver growth, hepatic DNA synthesis and mixed function oxidase activities. Therefore, we examined in these 3 strains of mice and in C3B6F1 (C3H x C57BL) mice the effect of PB administered in their drinking water for 4 days or 28 days. The liver weight to body weight ratio was increased by PB in all types of mice. Microsomal protein concentrations were increased in C57BL mice after 28 days of treatment, in C3H after both 4 days and 28 days and in B6C3F1 after 4 days of treatment. No effect upon microsomal protein content was observed in C3B6F1 mice. DNA content was increased in C3H mice, both in the 4-day and 28-day treatment groups, while the other strains showed either a decrease or no difference from control. DNA synthesis was elevated in all strains of mice after 4 days of treatment with PB, however, after 28 days of treatment there was either a much reduced increase (C57BL and C3B6F1) or no difference (C3H and B6C3F1) from controls. In all 4 types of mice after 4 and 28 days of treatment, PB increased the concentration of cytochrome P-450, the activity of aminopyrine-N-demethylase (AmDm) and 7-ethoxyresorufin-O-deethylase (ErDe) and the oxidation of testosterone (T). The oxidative metabolites of T were similar in the 4 types of mice.     

Tumorigenic responses to lindane in mice: potentiation by a dominant mutation

Obese mottled yellow Avy/a, lean pseudoagouti Avy/a and lean black a/a (YS X VY) F-1 hybrid female mice were fed diet containing 160 p.p.m. lindane (gamma-hexachlorocyclohexane) for 6, 12, 18 or 24 months. Clara cell hyperplasia was present in a majority of the mice after six months of lindane ingestion; however, more yellow mice (77%) than pseudoagouti (50%) or black (56%) mice had developed this lesion. Continued ingestion of lindane increased the incidence of Clara cell hyperplasia and resulted in similar prevalences in the three phenotypes. Lung tumors associated with lindane ingestion for 24 months were found only in yellow (19%) and pseudoagouti (14%) mice but not in the black mice. Prevalences of hepatocellular adenomas and carcinomas were very low (less than 10%) in untreated pseudoagouti and black mice. Lindane ingestion for 24 months resulted in an hepatocellular adenoma prevalence of 12% in pseudoagouti mice and 3% in black mice; comparable hepatocellular carcinoma prevalences were 5% and 1%. Among yellow mice fed lindane diet for 24 months, adenoma prevalence was 35% (9% among untreated controls) but carcinoma prevalence was only 17% (13% among controls). The tumorigenic responses evoked by lindane feeding in the lean pseudoagouti Avy/a mice but not in the black a/a mice indicate, for the first time, that the Avy gene itself, in the absence of obesity, sensitizes cells to transformation. The greater prevalence of hepatocellular adenomas in obese yellow Avy/a than in lean pseudoagouti Avy/a mice implicates obesity-associated factors in tumor promotion. Similarly, the increased prevalence of hepatocellular carcinomas in untreated obese yellow Avy/a mice, as compared to lean pseudoagouti mice, implicates obesity-associated factor as favoring histiotypic progression of liver tumors. Thus, the Avy gene not only sensitizes cells to respond to tumorigenic stimuli but also, by the induction of obesity, enhances promotion and progression of transformed cells.     

Manifestation of hyperplastic alveolar nodules and mammary tumors in "viable yellow" and non-yellow mice.

The time course of appearance of hyperplastic alveolar nodule(s) (HAN) and mammary tumor(s) (MT) was determined in untreated virgin "viable yellow" (Avy/A) and non-yellow (A/a) (C3H/HeNIcrWf X VY/Wf)F1 female mice. The first HAN was detected in a yellow female at age 16 weeks, the first period in which mice were killed. HAN were not found in the non-yellow mice until they were 19 weeks old. The incidence of HAN increased to 92% among yellow females and to 75% among non-yellow females by 36 weeks of age. MT were first observed at age 22 weeks in yellow mice and at 28 weeks in non-yellow mice. The incidence of MT at 36 weeks was 75% among yellow mice and 22% among non-yellow mice. Type B adenocarcinoma was the predominant class of MT found in the yellow mice. The time courses of appearance and incidence of HAN and MT in the non-yellow F1 mice were similar to those observed in inbred C3H female mice. MT first appeared in each population when the incidence of HAN bearers had reached 40--45% regardless of age, body weight, or number of HAN per HAN bearer. Apparently, the phenotypic effects of the Avy gene primarily stimulated the multiplication of nodule-transformed cells to form HAN and thus indirectly enhanced MT formation.     

Wide spectrum detection of precarcinogens in short-term bioassays by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes

The use of Aroclor 1254 to induce S9 liver fractions is a standard method for conducting short-term genotoxicity assays. An alternative induction procedure, using beta-naphthoflavone (beta-NF), as a safe (non-carcinogenic) substitute for polychlorinated biphenyls, combined with sodium phenobarbital (PB), was found to be equally effective. The aim of this work is to realize a novel schedule of induction for the preparation of metabolizing systems containing a wider spectrum of induced cytochrome P450s. Five inducers of different 'classes' such as PB (class IIB P450s), beta-NF (IA), isosafrol (IA2), ethanol (IIE1) and pregnenolone 16 alpha-carbonitrile (IIIA) were injected daily both separately (to achieve maximal monooxygenase induction) in male and female mice. Induction was monitored using specific P450-linked activities. In the optimal schedule for complete induction, the various monooxygenases were greater (2- to 4-fold) than those achieved by the classical schedule. More than a 14-fold increase of total P450 and 3.3-fold increase of NADPH-cytochrome (P450) c-reductase activity, over those uninduced, account for the above increase. For example, there was a marked increase in the deethylation of ethoxyresorufin (37-fold) compared to the uninduced mice that was considerably higher than classical induction (8-fold over uninduced). On the contrary, phase II reactions i.e. epoxide hydrolase, glutathione S-transferase, glutathione S-epoxide transferase and UDP-glucuronosyl transferase, examined to compare the phase I/phase II ratios in the traditional and proposed procedures, were increased to a lesser extent (2-fold over uninduced). No significant sex differences were seen. Five precarcinogens specifically metabolized by each of the induced P450s elicited a higher mutagenicity response in the presence of superinduced fractions with respect to the classical one, when tested on Salmonella typhimurium (cyclophosphamide, benzo[alpha]pyrene, 2-naphthylamine and dimethylnitrosamine) or Saccharomyces cerevisiae D7 strain (diethylstilbestrol). These novel metabolizing biosystems, with an enhanced spectrum of induced P450s and oxidative/post-oxidative reaction rates, are recommended for detecting unknown xenobiotics in genotoxicity studies.     

Promotion of malignant 'embryonal' liver tumors by phenobarbital: increased incidence and shortened latency of hepatoblastomas in (DBA/2 x C57BL/6)F1 mice initiated with N-nitrosodiethylamine

In order to analyze the genetics of susceptibility to promotion of hepatocarcinogenesis in DBA/2NCr and C57BL/6NCr mice by phenobarbital (PB), reciprocal F1 hybrid male mice were given 90 mg N-nitrosodiethylamine (NDEA)/kg body weight, i.p. at 5 weeks of age followed by 0.05% PB in drinking water. Hepatocellular adenomas and carcinomas were comparably increased in incidence and multiplicity in both reciprocal hybrids over mice given NDEA alone. Eight of 10 D2B6F1 progeny of DBA/2NCr females mated with C57BL/6NCr males, but only 1/10 B6D2F1 mice (progeny of C57BL/6NCr females mated with DBA/2NCr males) given PB after NDEA initiation developed single or multiple hepatoblastomas within 42 weeks. These small-celled, intensely basophilic tumors were characteristically hemorrhagic and highly malignant. Hepatoblastomas were mostly found within or adjacent to hepatocellular tumors. No hepatoblastomas were seen in either hybrid given NDEA alone. PB consistently enhanced development of malignant hepatoblastomas, as well as promoted hepatocarcinogenesis in D2B6F1 males, but did not elicit hepatoblastoma development in B6D2F1 males that were genetically identical to D2B6F1 males except for the reverse origin of their X and Y chromosomes.     

Modulation of mRNAs P450 and hepatocarcinogenesis promotion

The adaptive response of the liver to phenobarbital is characterized by a strong cell hypertrophy and coordinate induction of specific P450 forms (IIB1, 2; IIC7, IIIA1). The pattern of active mRNA is significantly changed, demonstrating the establishment of PB phenotype. Employed as a promoting agent in experimental hepatocarcinogenesis, PB triggers a significantly different, uncoordinated response. Only P450 IIB1 is positively modulated while P450 IIC7 mRNA becomes repressed. Mechanisms underlying the differential P450 adaptative response to PB in the initiated versus non-initiated liver are discussed in the light of both the importance of epigenetic events and the possible role of P450 mono-oxygenases in hepatocarcinogenic promotion by PB.     

Susceptible and resistant subgroups in genetically identical populations: response of mouse liver neoplasia and body weight to phenobarbital

Following 17 – 19 months of feeding 500 p.p.m. sodiumphenobarbital (PB) in the diet to yellow A^vy/A and agouti A/a(C3H x VY) F₁ hybrid male mice, two subgroups differing in responsivenessto PB with respect to promotion of hepato-cellular adenomasand body weight gain were observed within each genotype. Inuntreated mice of both genotypes, the presence of an adenomaat necropsy was associated with decreased body weight gain duringthis study. However, PB treatment inverted this association.In treated mice the presence of an adenoma at necropsy was precededby a greater increase in body weight during the study than whenno tumor was present. This increase in average body weight gainwas more pronounced among the yellow mice (44%) than among theagouti mice (21%). Among yellow mice PB treatment had no effecton body weight gain unless an adenoma was present at necropsy.However, in those yellow mice in which an adenoma was found,body weight was greater than in untreated yellow controls throughoutthe study beginning at week 27. The mean body weight curve oftreated yellow mice bearing one adenoma was slightly higherthan that of treated yellow mice in which no adenoma was found.The mean body weight curve of treated yellow mice bearing multipleadenomas was significantly higher than those of yellow micewith no or only one adenoma.     

P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats

Male F344/NCr rats, 6 wk old, were fed 500 ppm of phenobarbital (PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or 5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufin O-dealkylase and aminopyrine N-demethylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective. To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 rats were given a single i.p. injection of 75 mg N-nitrosodiethylamine/kg body weight. Beginning 2 wk later, they were placed either on normal diet or diet containing 500 ppm of PB or equimolar doses of EPH or EEH for the remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificed at either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk and hepatocellular carcinomas at 78 wk in N-nitrosodiethylamine-initiated rats. Neither the incidence of hepatocellular neoplasms nor the number and size of hepatocellular foci was significantly increased by EEH. At 78 wk, both PB and EPH enhanced the development of thyroid follicular cell neoplasms in N-nitrosodiethylamine-initiated rats while no such enhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticonvulsant, like the structurally similar PB, promoted hepatocellular and thyroid follicular cell carcinogenesis and induced the PB-inducible form(s) of cytochrome P-450 (P-450b) in rats. In contrast, EEH unlike barbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochrome P-450 in rats.     

The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital

The ability of the dietary methyl donors methionine and choline to inhibit the carcinogenic and tumor-promoting effects of phenobarbital (PB) in the livers of male weanling C3H mice was examined. The mice were fed a commercial rodent diet with or without 0.05% PB. Thirty animals from each set received the diet with either: (1) no dietary supplementation, (2) an additional 1.0% choline chloride, (3) 1.5% DL-methionine or (4) both 1.5% DL-methionine and 1.0% choline chloride. Additional groups of 30 animals with the same eight dietary and PB-treatment regimens described above were given a single initiating dose of 150 mg diethylnitrosamine (DENA)/kg body wt dissolved in saline, or the saline solution only, 1 week prior to the start of PB feeding. The 16 treatment groups were fed their respective diets for 12 months. Statistical trend analysis showed that increasing levels of supplemental methyl donors gave highly significant protection in PB-treated mice (P less than 0.01). The incidence of liver carcinomas in the four dietary groups not receiving PB or DENA varied from 0 to 7%. The PB-treated animals not receiving an initiating dose of DENA developed hepatocellular carcinomas (HCCs) at incidences of 79% in group 1 animals, 74% in group 2 animals, 60% in group 3 animals, and 31% in group 2 animals respectively. Thus, incidence of HCCs in group 4 was significantly lower than in groups 1, 2 or 3 (P less than 0.01). However, the total incidence of liver tumors (adenomas plus carcinomas) was about the same in all DENA or PB-treated groups. Thus, dietary supplementation with methyl donors increased the proportion of animals bearing liver adenomas as their most advanced hepatic lesion in PB-treated mice. In DENA-treated mice fed PB, dietary supplementation with methionine and choline protected against the formation of liver carcinomas (P less than 0.02); however, methionine and choline had no significant effect on liver tumor formation in mice fed the PB-free diets. Methionine and choline supplementation gave significant protection against HCC metastases in the lungs of the tumor-bearing mice in groups initiated with DENA followed by PB promotion. These results support the hypothesis that PB exerts it tumorigenic activity in mice at least in part through a physiological insufficiency of labile methyl groups.     

Lack of Promoting Effects of Phenobarbital at Low Dose on Diethyl-nitrosamine-induced Hepatocarcinogenesis in TGF-a Transgenic Mice

Phenobarbital (PB), a rodent non-genotoxic carcinogen, showed hormesis, biphasic effects on rat livercarcinogenesis. To test the hypothesis that the hormesis earlier observed for PB induced hepatocarcinogenesis mightalso exist in the TGF-α transgenic mice model, one which is highly susceptible to carcinogenesis, the carcinogenic orpromotion effects of a wide range of phenobarbital (PB) concentrations were investigated. Two weeks after a singlei.p. dose of 5 mg /kg bw of diethylnitrosamine (DEN) to 15 day old mice, animals were treated with diet containingPB at doses of 0, 2, 15 or 500 ppm. The incidence and multiplicity of tumors, including hepatocellular adenomas andcarcinomas, were significantly increased by the high dose of PB, but no significant difference among the groupsreceiving 2 and 15 ppm for liver tumors when compared to DEN alone group. The proliferating cell nuclear antigenindices for liver tumors and surrounding hepatocytes in high dose PB treated mice were significantly increased, butno change was noted at the lower doses. The total cytochrome P450 content in the liver was also elevated by 500 ppmof PB, while hepatic 8-OHdG levels demonstrated no significant change. In conclusion, PB at high dose enhancesDEN-induced hepatocarcinogenesis in TGF-α transgenic mice, but low doses lack any significant effects. One possiblemechanism of phenobarbital carcinogenicity might be influenced by cytochrome P450 system exhibiting a strongpromoting activity for liver of mice.     

Effect of phenobarbital on diethylnitrosamine and dimethylnitrosamine induced hepatocellular tumors in male B6C3F1 mice

The effect of the type of carcinogen initiator on the ability of phenobarbital (PB) to promote hepatic tumor formation in 15-day-old initiated male B6C3F1 mice was evaluated. Fifteen-day-old male B6C3F1 mice were divided into 6 groups of 10 mice each. Groups 1 and 2 received a single intraperitoneal (i.p.) injection of diethylnitrosamine (DENA) (5 micrograms/body wt). Groups 3 and 4 received a single i.p. injection of diethylnitrosamine (DENA) (5 micrograms/g body wt). Groups 3 and 4 received a single i.p. injection of dimethylnitrosamine (DMNA) (5 micrograms/g body wt). Groups 5 and 6 received a single i.p. injection of saline. At weaning (28 days of age), mice in groups 2, 4 and 6 received PB (500 mg/ml) in their drinking water. Mice in groups 1, 3 and 5 received deionized drinking water. Drinking water treatment continued for 24 weeks at which time mice were sampled. At sampling, mice were examined for hepatic tumors by histology. Mice in groups 5 (no treatment) and 6 (PB only) did not exhibit hepatic tumors. Groups 2 (DENA + PB) displayed a decrease in hepatic adenomas from that of group 1 (DENA only), confirming previous observations. Treatment with DMNA and PB (group 4), however, resulted in a significant increase in both hepatic adenoma incidence and number over that of DMNA only (group 3) treated mice. The promoted adenomas appeared to be predominantly eosinophilic in appearance. The type of initiator therefore appears important in determining if 15-day-old initiated male B6C3F1 mice respond to the promotion effects of PB.     

Comparison in C3H and C3B6F1 mice of the sensitivity to diethylnitrosamine-initiation and phenobarbital-promotion to the extent of cell proliferation

The ability of diethylnitrosamine (DENA) to initiate and phenobarbitalto promote altered-hepatocyte foci and hepatocellular carcinomasin C3H and C3B6F1 (C3H x C57BL/6) mice was compared to the extentof cell proliferation. Fifteen day old female mice receivedDENA (16 mg/kg, i.p.) or the vehicle control followed at weaningwith 500 p.p.m. sodium phenobarbital in their drinking wateruntil killed at 161 days of age. Six days prior to death eachmouse had implanted an osmatic minipump containing 30 mg/mlbromodeoxyuridine in order to measure cell proliferation asthe labeling index of bromodeoxyuridine incorporation into newlyreplicated DNA. C3H mice were more susceptible to DENA thanC3B6F1 mice, 54.1 versus 0.57 adenomas/mouse respectively, andphenobarbital increased the yield of altered-hepatocyte fociin C3H mice. Hepatocellular adenomas had a greater bromodeoxyuridinelabeling index than altered-hepatocyte foci, which was greaterthan non-involved/ normal hepatocytes. In DENA-initiated C3Hand C3B6F1 mice, phenobarbital increased the labeling indexin eosinophilic foci, while decreasing the labeling index innormal/non-involved hepatocytes with/without DENA initiation.However, the level of cell proliferation in normal/non-involvedhepatocytes, foci and adenomas was lower in C3H mice than inC3B6F1 mice. Thus, the level of cell proliferation did not correlateto the higher yield of foci and tumors in C3H mice. The resultsare consistent with the strain sensitivity to DENA-initiationand to phenobarbital-promotion correlating to the ratio of thelabeling index in the foci to the labeling index in non-involvedhepatocytes. An increase in this labeling index ratio for cellproliferation indicates that the foci are selectively stimulatedto proliferate resulting in a greater expansion of the populationof precancerous cells in the liver.     

Phenobarbital promotion in diethylnitrosamine-initiated infant B6C3F1 mice: influence of gender

Phenobarbital (PB) promotes hepatic tumorigenesis when chronicallyadministered to male B6C3F1 mice after initiation with diethylnitrosamine(DENA) at 30 days of age. In contrast, when male B6C3F1 micewere initiated with DENA at 15 days of age, an inhibition ofhepatic tumorigenesis occurred. The present study was undertakento evaluate the Influence of gender on the inhibiting abilityof PB in the 15 day old DENA-initiated B6C3F1 mouse. Mice wereinjected with either DENA (5 µg/g) or saline at 15 daysof age. At weaning mice were given either PB (500 p.p.m.) containingdrinking water or deionized drinking water for 24 weeks. Malemice treated with DENA and PB demonstrated a significant decreasein the number of hepatocellular adenomas compared to males receivingDENA only. In contrast, females exposed to DENA and PB exhibitedan enhancement of hepatic adenoma number compared to those receivingonly DENA. In an additional experiment, individual preneoplastkfoci from male and female B6C3F1 mice initiated with DENA at15 days of age were examined for their responsiveness to theniitogenic stimuli of PB. Mice were exposed to either PB-containingor PB-free drinking water for 7 days. In non-PB treated malesand females, preneoplastk hepatocytes demonstrated higher ratesof DNA synthetic labelling compared to normal hepatocytes withno gender difference noted. Males exposed to PB exhibited increasedlevels of DNA synthesis in normal cells but not in preneoplastkfoci. Females treated with PB, however, demonstrated significantincreases in DNA synthesis in both preneoplastk and normal hepatocytescompared to non-PB treated females and PB-treated males. Thesefindings suggest that in male mice initiated with DENA at 15days of age, the preneoplastk foci are refractory to the proliferativeeffects of PB which may account for the observed inhibitionof hepatic tumorigenesis by PB in this mouse strain.     

Neoplastic and nonneoplastic lesions in aging (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice.

Neoplastic and nonneoplastic lesions in untreated (C57BL/6N x C3H/HeN)F1 (B6C3F1) mice used as controls in carcinogenesis tests were tabulated and evaluated. The most common neoplasms in 2,543 male mice were hepatocellular adenomas and carcinomas. In 2,522 female mice, common tumors were lymphomas, leukemias, pulmonary adenomas and carcinomas, hepatocellular adenomas and carcinomas, and pituitary adenomas. The risk of developing most neoplasms increased with the age of the mouse. Hepatocellular carcinomas metastasized in 12% of the animals with these tumors. Other than lymphomas and leukemias, few other tumors metastasized. Nonneoplastic lesions included cystic hyperplasia of the uterus, nephritis, ovarian and uterine cysts, inflammatory lesions of the lung, mineralization in the brain, and focal hyperplasias in several tissues. The focal hyperplasias in lung and pituitary, adrenal, and thyroid glands were suggestive of the early stages of neoplasia. Comparative aspects of lesions in aging mice and their interpretation in carcinogenesis tests are discussed.     

Tumor-promoting and hepatocarcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in DBA/2NCr and C57BL/6NCr mice and an apparent promoting effect on nasal cavity tumors but not on hepatocellular tumors in F344/NCr rats initiated with N-nitrosodiethylamine.

The tumor-promoting and carcinogenic effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) in the liver and in other organs were quantified and compared to those of phenobarbital (PB) in two inbred strains of mice (C57BL/6NCr, DBA/2NCr) and in F344/NCr rats initiated at 5 weeks of age with N-nitrosodiethylamine (NDEA; 90 mg/kg in mice, 75 mg/kg in rats). Two weeks later animals were placed on a regimen of TCPOBOP once every 2 weeks (administered i.p. or i.g.) or on a diet containing 500 p.p.m. PB as a positive control for the duration of the experiment. Mice were administered TCPOBOP (3.0 mg/kg/dose) for 30 weeks followed by control diet, while rats were given the TCPOBOP regimen (3.0 or 30 mg/kg/dose) for the full 78 weeks of the experiment. TCPOBOP was a complete carcinogen and an extremely potent promoter in both strains of mice, particularly the DBA strain in which NDEA followed by TCPOBOP (i.p.) resulted in death of all the animals within 30 weeks from multiple hepatocellular tumors. TCPOBOP alone induced 100% tumor incidence in DBA mice within 60 weeks. In addition, in both strains of mice, a high proportion of those animals with liver tumors had metastases to the lungs. In contrast, TCPOBOP was ineffective as a liver tumor promoter in F344 rats at even 10 times the dose administered to mice. Interestingly however, TCPOBOP, when given subsequent to NDEA, caused a significant increase in nasal cavity tumors in F344 rats. PB was an effective liver tumor promoter in male DBA mice and male F344 rats, but was relatively ineffective as a promoter in C57 mice. When tumor-promoting activity and induction of cytochrome P450 IIB1 were compared, good agreement between these two parameters was observed. PB was an effective inducer of P450 IIB1 in the rats and in both strains of mice and a potent liver tumor promoter in both DBA mice and F344 rats, whereas TCPOBOP was a potent inducer and tumor promoter in both strains of mice but was negligibly effective as either an inducer or a promoter in F344 rats at even 10-fold higher dosage.     

Significance of strain and sex differences in the development of 252Cf neutron-induced liver tumors in mice.

Mouse liver tumors occurring in C3H/HeN, C57BL/6N and C3B6F1 hybrid (C3H x C57BL) were studied following 252Cf fission neutron irradiation. Three strains of mice of both sexes (about 30 mice/group) were irradiated once with 252Cf at doses of 0, 12.5, 50 and 200 cGy. The groups were observed for 13 months after irradiation. The incidence of liver tumors in the non-irradiated controls was 0% in both sexes of C57BL/6N, 11.7% in males and 0% in females of C3B6F1 and 39.5% in males and 11.4% in females of C3H/HeN mice. In the four strains of mice thus far studied, including B6C3F1 hybrid (C57BL x C3H) which was previously studied, 252Cf irradiation has increased the tumor incidence dose-dependently in males and in females, but less effectively in females. The mean number and size of liver tumors were clearly correlated with tumor incidence. The incidence was always highest in C3H/HeN mice of both sexes, followed by B6C3F1, C3B6F1 and C57BL/6N mice. The influence of sex hormones was studied in B6C3F1 mice of both sexes after 200 cGy of 252Cf irradiation. In males, the incidence of liver tumors was significantly decreased from 55.2% to 23.3% and 25.9% after orchidectomy, and in females it was slightly decreased from 27.6% to 14.8% and 18.8% after ovariectomy. Supplementation of testosterone in orchidectomized mice did not restore the occurrence of liver tumors.     

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.     

Promoting effects of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene in mouse hepatocarcinogenesis

The effects of phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene(TCPOBOP) on liver hyperplasia, induction of microsomal enzymeactivities, and two-stage hepatocarcinogenesis were evaluatedin B6C3F1 female mice. For 4 weeks four groups of mice receivedPB (500 p.p.m. in the drinking water), TCPOBOP (3 mg/kg i.p.once every week), PB together with TCPOBOP or corn oil vehiclei.p. TCPOBOP induced liver hyperplasia and hypertrophy and increasedp-nitroanisole-O-demethylase and aminopyrine-N-demethylase morethan PB. Neither chemical changed UDPG-transferase activity.The association of PB and TCPOBOP gave the same effects as TCPOBOPalone. Other four groups of mice were treated with N-nitroso-N-diethylamineat 7 days of age and then, starting from 8 weeks of age, receivedthe above specified weekly treatments for 20 weeks and werethen sacrificed. Hepatocellular nodules > 150 µm werefound in all animals of all groups. Due to increased size ofthe liver compared to controls, the number of nodules/cm³ decreasedafter PB and TCPOBOP treatments given alone or together; howeverthe mean volume of nodules and the percentage of liver volumeoccupied by nodules increased after TCPOBOP but not after BPtreatment, and the association of PB and TCPOBOP was even moreeffective than TCPOBOP alone. Hepatocellular adenomas >2.4mm in diameter were observed in 5 of 10 TCPOBOP-treated mice(total of 11 nodules) and in 5 of 11 mice that received PB plusTCPOBOP (total of 15 nodules). Hepatocellular carcinomas wereseen in one mouse treated with PB and in three mice given PBand TCPOBOP.     

Inhibition by phenobarbital and lack of effect of amobarbital on the development of liver tumors induced by N-nitrosodiethylamine in juvenile B6C3F1 mice

The effects of phenobarbital (PB) and amobarbital (AB) on the rate of development of hepatocarcinogenesis induced by N-nitrosodiethylamine (DEN) were studied in mice. Groups of 40 B6C3F1 male mice were injected i.p. at 15 days of age with 5 micrograms DEN/g body wt. Beginning at 4 weeks of age, DEN treated groups were given either normal drinking water or water containing either 0.05% PB or AB for up to 36 weeks. DEN alone induced multiple focal hepatic lesions including hepatocellular foci, hepatocellular adenomas and trabecular carcinomas. Subsequent exposure to PB had a suppressing effect on DEN-induced hepatocarcinogenesis. Hepatocellular foci in PB-exposed mice were significantly smaller in size (area) and fewer in number throughout the study. Also, PB treatment either prolonged the latency period or significantly slowed the rate at which hepatocellular tumors developed in these mice. No such effects were seen in AB-exposed mice; AB neither inhibited nor promoted the development of focal hepatic lesions in DEN-pretreated mice. Possible mechanisms responsible for the inhibition of DEN-induced hepatocarcinogenesis include the feminizing effects of perinatal administration of PB.