Decreased expression of Bcl-x protein during hepatocarcinogenesis induced exogenously and endogenously in rats.

Dysregulations of apoptosis have been widely recognized as important events in multi-stage carcinogenesis. Bcl-x, a member of the Bcl-2 family, is known to act as a regulator of apoptosis. The present study was conducted to assess the role of altered Bcl-x protein expression in exogenous and endogenous hepatocarcinogenesis in rats. In the short-term exogenous models, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal injection of diethylnitrosamine (DEN) at a dose of 200 mg / kg body weight, partially hepatectomized at the end of week 3, administered phenobarbital at a concentration of 0.05% from the end of week 2 for 6 weeks, and sacrificed. In the livers, glutathione S-transferase (GST-P)-positive, putative preneoplastic lesions were induced, and Bcl-x protein expression was decreased in 24.7% of such lesions. The incidence of GST-P-positive lesions with decreased Bcl-x increased depending on the size of the lesions; 18.9%, 32.4% and 86.5% in the lesions smaller than 0.03, between 0.03 and 0.3, and larger than 0.3 mm(2), respectively. In GST-P-positive lesions larger than 0.3 mm(2), both apoptosis induction and cell proliferation activity were enhanced when Bcl-x protein expression was decreased. In the long-term exogenous models, rats were given 10 mg / kg of DEN, partially hepatectomized 4 h after treatment, administered 0.5 mg / kg of colchicine at the end of days 1 and 3, subjected to a selection procedure, and sacrificed at the end of week 45. Hepatocellular carcinomas were induced with the decreased Bcl-x protein expression. In the endogenous model, rats were fed a choline-deficient, L-amino acid-defined diet for 16 or 80 weeks and sacrificed. Bcl-x protein expression was decreased both in GST-P-positive lesions and hepatocellular carcinoma. These results suggest that this decrease of Bcl-x protein might serve as an indicator of the advanced form of preneoplastic lesions, and that this decrease could also be associated with a potential to progress into carcinoma in both exogenous and endogenous hepatocarcinogenesis of rats.

CB6F1-rasH2 mouse: overview of available data.

This article presents data from short-term carcinogenicity studies of compounds tested in the CB6F1-rasH2 transgenic mouse as part of the International Life Sciences Institutes' (ILSI) Health and Environmental Sciences' (HESI) Alternative to Carcinogenicity Testing (ACT) project. Additionally, data from other studies that were not conducted as part of the ILSI program, but used comparable or slightly modified protocols, are included here. A significant number (3 of 4) of the genotoxic carcinogens tested were positive in the rasH2 mouse; the other compound was equivocally positive. The positive control, N-Methyl-N-nitrosurea (MNU), gave reproducible responses across all participating laboratories with tumors noted at multiple sites in the animal. The immunosuppressive human carcinogen. Cyclosporin A, was equivocal. Two hormones that are human tumorigens. Diethylstilbestrol and 17beta-Estradiol, gave positive and negative results, respectively. Of the twelve additional compounds tested that are classified as non-genotoxic rodent carcinogens and putative human non-carcinogens, only the two peroxisome proliferators (clofibrate and diethylhexylphthalate(DEHP)) produced a positive response (liver effects). The three non-genotoxic non-carcinogens that were tested also gave negative responses in the rasH2 model. This result provides confidence that the model is likely to have a low false-positive rate.

Immunohistochemical characterization of hepatoblastomas in B6C3F1 mice treated with diethylnitrosamine and sodium phenobarbital.

Hepatoblastomas (HBs) were induced in B6C3F1 male mice by diethylnitrosamine (DEN) and sodium phenobarbital (PB). Six-week-old mice received a single intraperitoneal dose of DEN followed by a continuous treatment with PB in diet at a concentration of 0 (group 1) or 500 (group 2) ppm for 50 weeks. HBs were observed in 13 of 21 (62%) group 2 mice, with typical histologic features as reported previously, while no such tumors were observed in group 1. Seven of 13 (54%) HBs were found in and/or adjacent to hepatocellular adenomas (HCAs) or hepatocellular carcinomas (HCCs). Immunohistochemically, all HBs were positive for S-100 protein but negative for keratin, alpha-fetoprotein (AFP), albumin (ALB) and vimentin, while HCC cells occasionally reacted positively for AFP with a mosaic pattern. HCC and HCA cells were occasionally positive for ALB. Non-neoplastic hepatocytes and normal bile ducts were positively stained for ALB and keratin/S-100 protein, respectively. S-100 protein is known to be expressed in many mesenchymal tissues and neoplasms including neuroectodermal elements but negative in cells of the hepatic lineage. Thus, the present immunohistochemical results suggested that mesenchymal differentiation occurs in mouse HB cells as observed in human HBs, one of the most frequent infant liver tumors in humans. Although the susceptibility of mouse HBs to PB-promotion suggests a hepatocytic histogenesis, the present immunohistochemical results support the hypothesis that the mouse HB is derived from pluripotent endodermal stem-like cells.

Greater expression of transforming growth factor alpha and proliferating cell nuclear antigen staining in mouse hepatoblastomas than hepatocellular carcinomas induced by a diethylnitrosamine-sodium phenobarbital regimen.

Transforming growth factor alpha (TGF-alpha) is a potent stimulator of normal hepatocyte proliferation, considered to have relationship to the liver regeneration or carcinogenesis. In this study, we investigated immunohistochemically the association between expression of TGF-alpha and cell proliferation activity in mouse hepatoblastomas (HBs) and hepatocellular carcinomas (HCCs) induced in B6C3F1 mice by diethylnitrosamine and sodium phenobarbital. The TGF-alpha-positive rate in HBs (29.2%) was significantly higher than that in HCCs (12.7%). Likewise, the proliferating cell nuclear antigen-positive rate (22.2%) was higher than the HCC value (14.5%). On the individual data for both TGF-alpha and PCNA, most of the HBs showed higher positive rates than HCCs. In HBs, TGF-alpha was localized only in the nuclei, whereas some HCC cells stained positive both in their nuclei and cytoplasm (0.6%). These results suggest expression of TGF-alpha and its localization might be linked to cell proliferation and play a role in malignant progression of mouse HBs.

Peroxisome proliferator-activated receptor alpha (PPAR alpha) agonist, WY-14,643, increased transcription of myosin light chain-2 in cardiomyocytes.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by xenobiotics and natural fatty acids. To assess the potential physiological activity of PPAR ligands on cardiac muscular cells, the effects of PPAR alpha agonist, WY-14,643, on both rat hearts and a rat cardiomyocyte cell line (H9c2 cells) were investigated. Male F344 rats were fed a diet containing WY-14,643 at a concentration of 100 ppm for 26 weeks. Cardiac muscular hypertrophy was revealed by morphometric analysis in which the diameter of the muscular fibers in WY-14,643-treated rats was larger than those of control rats. Using H9c2 cells in vitro, the protein content per cell was increased in a dose-dependent manner with the treatment of WY-14,643. The transcription of myosin light chain-2 (MLC-2), a parameter of myocardial hypertrophy, was increased in H9c2 cells transfected with the rat MLC-2/luciferase fusion gene by WY-14,643 as well as other peroxisome proliferators, clofibrate and di(2-ethylhexyl) phthalate. In addition, accumulation of myosin light chain protein was confirmed in H9c2 cells treated with WY-14,643 at 10 micrograms/ml for 7 days or more by immunohistochemistry. These results suggest that PPAR alpha ligands have a potential to regulate MLC-2, which is a contractile protein in cardiomyocytes and may play a part of role in the pathogenesis of cardiac hypertrophy.

Immunohistochemical localization of transforming growth factor alpha in chemically induced rat hepatocellular carcinomas with reference to differentiation and proliferation.

Hepatocellular carcinomas (HCCs) were induced in male Fischer 344 rats with dietary 3'-methyl-4-(dimethylamino)-azobenzene treatment and were classified into solid, glandular (well- or poorly differentiated), and trabecular types. Investigation of cell proliferation kinetics and immunohistochemical localization of transforming growth factor alpha (TGF-alpha) demonstrated all solid (n = 24) and poorly differentiated glandular type (n = 6) HCCs to have TGF-alpha-positive nuclei. Nuclear staining of TGF-alpha was also observed in 13 of 28 (46%) trabecular-type HCCs, whereas 12 (43%) exhibited cytoplasmic staining, and 3 (11%) were negative. As for well-differentiated glandular HCCs, 7 of 20 (35%) were positively stained in their nucleus, another 7 (35%) demonstrated antibody binding in the cytoplasm, and 6 (30%) were negative. The order for growth rate evaluated by bromodeoxyuridine (BrdU) labeling was solid (38.22%), poorly differentiated glandular (26.82%), trabecular (7.98%), and well-differentiated glandular (2.57%) types. For trabecular HCCs with nuclear, cytoplasmic, or negative TGF reactions, values were 13.39% (n = 13), 3.61% (n = 12), and 2.01% (n = 3), respectively. Likewise, BrdU-labeling indices for the counterpart groups of well-differentiated glandular type HCCs were 4.53, 1.91, and 1.29%, respectively. The results indicate that TGF-alpha expression might be linked to histopathological differentiation and cell proliferation in rat HCCs.

Glomerulonephritis with fibrillary deposition in a transgenic mouse carrying the human prototype c-Ha-ras gene (rasH2 mouse).

Glomerulonephritis was observed in a 34-week-old transgenic CB6F1 mouse carrying the human prototype c-Ha-ras gene (rasH2 mouse) from a medium-term carcinogenicity study of N-methyl-N-nitrosourea (MNU). Lesions were characterized by severe diffuse enlargement and prominent hyalinization of glomeruli. The hyaline material was positive for periodic acid-Schiff but negative for amyloid by the Congo red method. Immunohistochemically, affected glomeruli were positive for polyclonal anti-mouse IgG. Ultrastructurally, there were characteristic subendothelial and mesangial deposits composed of fibrils showing a fingerprint pattern. Lamellae were 7.5-14.3 nm in diameter and formed multilayered structures. In addition to the renal lesions, a lymphoma was observed in the thymus, with metastasis to the spleen and some lymph nodes. However, there was no glomerulonephritis in 32 other mice bearing thymic lymphomas and in more than 40 males and females given MNU in the same study. Thus, the lesions in this mouse may have been spontaneous. Glomerulonephritis was not found in more than 120 other male and female rasH2 mice in our facility. This is the first report of glomerulonephritis in a rasH2 mouse, a promising candidate for medium-term carcinogenicity risk assessment.

Establishment and characterization of a cell line from a chemically-induced mouse hepatoblastoma.

We established a cell line (MHB-2) from a hepatoblastoma (HB) induced by diethylnitrosamine (DEN) and sodium phenobarbital (PB) in male B6C3F1 mice and examined the biological characteristics of MHB-2. MHB-2 cells grew as monolayers in culture and showed a spindle or polygonal shape. Immunohistochemically, the original tumor cells and MHB-2 cells were negative for keratin, alpha-fetoprotein and albumin. Electron microscopically, MHB-2 cells had irregular-shaped nuclei with prominent nucleoli, abundant free ribosomes, myelinosomes, desmosomes and surface microvilli. Growth of this cell line was significantly accelerated by hepatocyte growth factor (HGF) and expression of its receptor c-met was confirmed by the reverse transcription-polymerase chain reaction (RT-PCR). MHB-2, however, was not found to be tumorigenic when transplanted into the subcutaneous tissue of syngeneic, nude or scid mice. To our knowledge, this is the first report on the establishment of a cell line derived from a mouse HB. MHB-2 would be useful for further studies to clarify the biological characteristics of mouse HB.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 25). Effects of 2- and 4- week repeated-dosing of dibromoacetic acid.

To assess the sensitivity of rats to a testicular toxicant, 2- and 4-week repeated-dose studies using dibromoacetic acid (DBAA) were performed. Four groups of 6- or 8-week old male SD rats were given DBAA at a daily dosage of 0, 5, 50 or 250 mg/kg. The highest dose was given for 2 weeks, and the others for 2 and 4 weeks. There were no effects on body or testicular weights in any of the DBAA-treated groups. However, the mean absolute epididymal weight in the 250 mg/kg group was significantly lower than that of the control group. Histopathologically, atypical residual bodies (ARBs) and retention of Step 19 spermatids were evident with this high dose. In the same group, ARBs in the epididymal ducts and narrowing of these lumina were also observed. Retention of Step 19 spermatids was similarly apparent in the testes of animals given 50 mg/kg for 2 or 4 weeks, and in one animal given 5 mg/kg for 4 weeks. Based on these data, DBAA testicular toxicity is histopathologically detectable within 2-weeks of repeated dosing at an appropriate dose.

Effects of carbon tetrachloride administration on initiation of liver cell foci by the non-hepatocarcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P).

In a development trial for an initiation bioassay system, cell proliferation kinetics after partial hepatectomy (PH) or CCl4 administration (1 ml/kg b.w., i.g.) and the effect of administration time after PH or CCl4 treatment on liver cell foci induction by the direct and indirect non-hepatocarcinogens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and benzo(a)pyrene (B(a)P) were investigated. Male F344 rats were killed 12, 18, 24, 36, 48, 72 or 96 h after PH or CCl4 treatment and liver cell proliferation was examined with the bromodeoxiuridine (BrdU) labeling method. Appreciable increase in the BrdU labeling index was observed 18-36 h after PH with a peak at 24 h, and 18-72 h following treatment with CCl4 with a peak at 48 h. MNNG (80 mg/kg i.g.) or B(a)P (100 mg/kg i.g.) were administered to 7-week-old male F344 rats at various times after PH or CCl4 treatment and lesion induction was assessed using the resistant hepatocyte model. MNNG caused significant numbers of glutathione S-transferase placental form (GST-P)-positive liver cell foci in rats when given 12-36 h after PH, with a peak at 24 h. In contrast, the numbers of foci induced by B(a)P were maximal with exposure at 12 h after PH. In the CCl4 study, both MNNG and B(a)P induced significant increase in GST-P-positive liver cell foci when given 12-72 h after CCl4 treatment, with a peak at 48 h, the results being directly in line with the changes in BrdU labeling. From these findings, it is concluded that initiation assay protocols with a CCl4 proliferative stimulus to hepatocytes may prolong the appropriate administration period for effective detection of the initiation potential of both direct and indirect carcinogens targeting sites other than the liver.

Sequential observation of micrometastasis formation by bacterial lacZ gene-tagged Lewis lung carcinoma cells.

Sequential events in micrometastasis formation including entry into the blood circulation and arrest, extravasation and initial growth in the lung was investigated using bacterial lacZ gene-tagged Lewis lung carcinoma cells (4A1-1). Micrometastases in the lung could thereby be specifically detected at the single cell level by X-Gal staining. After intravenous injection, X-Gal positive tumor cells appeared to extravasate within hours, but most cells then degenerated or died in the alveolar space by 2-3 days postinjection. A decreased BrdU labeling index to a negligible level at 2 days postinjection and reduction of X-Gal positive foci to a basal level (less than 0.1% of injected cells) by 4 days are in line with rapid clearance of tumor cells from the lung. The size and BrdU labeling indices of the persisting X-Gal positive foci, however, started to increase from 4 days postinjection. Type IV collagen immunostaining demonstrated loss of pre-existing basement membranes with growth of micrometastases: When 4A1-1 cells were inoculated subcutaneously, lung micrometastases from resulting tumors were detected as single or small numbers of X-Gal positive cells at 2 weeks postinjection. Progressive development of micrometastasis to macroscopic metastasis was noted by 4-5 weeks postinjection. The results indicate that micrometastasis formation by Lewis lung carcinoma cells involves a sequence of events starting with rapid extravasation after arrest in the lung within 1 day, followed by death of most cells at 2-3 days and subsequent new growth and expansion of persisting tumor cells from 4 days postinjection.

UDP-GT involvement in the enhancement of cell proliferation in thyroid follicular cell proliferative lesions in rats treated with thiourea and vitamin A.

The mechanisms underlying enhanced cell proliferation in thyroid proliferative lesions of rats simultaneously treated with large amounts of vitamin A (VA) and thiourea (TU) were investigated. Male F344 animals were initiated with N-bis(2-hydroxypropyl)nitrosamine (2800 mg/kg body weight, single s.c. injection). Starting 1 week later, groups received water containing 0.2% TU (TU group), diet containing 0.1% VA (VA group), both 0.2% TU and 0.1% VA (TU + VA group) or tap water/basal diet without supplement (control group) for 10 weeks. The serum levels of triiodothyronine (T3) and thyroxine (T4) were decreased and the thyroid stimulating hormone (TSH) levels were elevated in the TU and TU + VA groups, with the degree of change being significantly greater in the combined treatment group. The induction of P450 isoenzymes by TU was not enhanced by VA supplementation, but uridine diphosphate glucuronosyltransferase (UDP-GT) activity in the liver was significantly increased in the TU + VA group compared to the TU group. Thyroid weights were increased in both the TU and TU + VA groups, this being more pronounced with VA supplementation. Thyroid follicular cell hyperplasias and neoplasias were induced to similar extents in both TU treated groups, but their cell proliferation appeared to be increased by the VA supplementation. The results of the present study suggest that enhanced cell proliferation is due to increased TSH stimulation, resulting from the decrease in serum T3/T4 levels brought about by induction of liver UDP-GT activity with the combined action of TU + VA as well as inhibition by TU of thyroid hormone synthesis in the thyroid.

Dose-dependent promoting effects of catechol on glandular stomach carcinogenesis in BALB/c mice initiated with N-methyl-N-nitrosourea.

The effects of catechol administration in the diet on stomach carcinogenesis in mice after initiation with N-methyl-N-nitrosourea (MNU) in the drinking water were investigated in a development trial for a new experimental protocol. Male 6-week-old BALB/c mice were given MNU in the drinking water intermittently for a total of three one-week periods, with one-week intervals, at the concentration of 120 ppm (groups 1 and 2). Groups 3 and 4 served as non initiated controls. From week 7, groups 1 and 3 were divided into three subgroups and the mice were fed on diet containing 0.05% (groups 1a and 3a), 0.2% (groups 1b and 3b), 08% (groups 1c and 3c) or 0% (groups 2 and 4) catechol for 29 weeks. At week 20, appreciably enhanced development of pepsinogen 1-altered pyloric glands was noted in all catechol-treated groups, in a partially dose-dependent manner (12.8 +/- 12.5, 13.8 +/- 11.7, and 24.0 +/- 12.7/100 pyloric glands respectively, for groups 1, 2 and 3). The incidences of adenomas (groups 1, 2 and 3) were also increased. At week 35, dose-dependent induction of adenocarcinomas in groups 1 (3/19), 2 (3/19) and 3 (14/20) was evident. In addition, the depth of invasion of the adenocarcinomas was enhanced by catechol in a dose-dependent manner, though the histological type was not influenced. Thus, the administration of catechol in the diet strongly enhanced the preneoplastic and neoplastic lesions in mouse glandular stomach induced by MNU in the drinking water, in a dose-dependent manner.

Decreased connexin32 and a characteristic enzyme phenotype in clofibrate-induced preneoplastic lesions not shared with spontaneously occurring lesions in the rat liver.

Two different types of focal preneoplastic lesions, tentatively named Type I and II lesions, were recognized in the liver of rats chronically treated with clofibrate for 104 weeks. Type I lesions were characterized by mostly negative glucose-6-phosphate dehydrogenase (G6PD) activity (6 out of 10, 60%) and positive expression of succinate dehydrogenase (10 out of 10, 100%), in addition to the previously documented complete lack of expression of glutathione S-transferase, placental form (GST-P) and gamma-glutamyl transpeptidase (GGT). Furthermore, most importantly, Type I lesions exhibited a clear decrease in immunohistochemically demonstrated connexin32 (Cx32) spot counts on their hepatocyte membranes, similarly to nitrosamine-induced lesions. In contrast, Type II lesions, mostly small in size and positively expressing GST-P and/or GGT and G6PD, similarly to their previously reported nitrosamine-induced counterparts, did not exhibit a significant decrease in Cx32 count. In addition, spontaneously occurring lesions, again sharing the same enzyme phenotype, did not show a decrease in Cx32. The results indicate that: (i) a clear distinction between the two lesions, with Type I being involved in clofibrate-induced tumors and Type II being more likely to be spontaneous in nature; (ii) a decrease in Cx32 is closely linked to lesion development and possibly stage of progression, irrespective of the enzyme phenotype and the applied carcinogen; (iii) the unaltered condition of Cx32 may suggest a slow growing or non-progressive nature.

Modifying influence of swine-serum-induced liver fibrosis on development of preneoplastic lesions in rat liver.

Modifying effects of fibrosis or a cirrhotic state, caused by treatment with swine serum (SS), on the induction of preneoplastic focal lesions were assessed in a rat medium-term liver bioassay model for the detection of environmental carcinogens, in which the test compound is administered during the promotion phase after initiation with diethylnitrosamine. In experiment I, repeated intraperitoneal administration of SS concomitantly with the hepatopromoting agent deoxycholic acid (DCA) or phenobarbital (PB) resulted in a cirrhotic state and a significant increase in the number or size of preneoplastic glutathione S-transferase placental form (GST-P)-positive liver cell foci as compared to the corresponding DCA or PB alone groups. In experiment II, SS was given prior to commencement of the same medium-term bioassay system, in which a known hepatopromoting agent, DCA, 17-alpha-ethynylestradiol, or 2-acetylaminofluorene, was applied. In this case, the liver did not show obvious cirrhotic change and, rather than any enhancement, slight inhibition of promotion occurred. The results indicate that a coexisting, but not a pre-existing, cirrhotic condition acts to increase growth pressure on GST-P+ preneoplastic foci, and suggest that concomitant administration of SS with the promoting agent could be applied to improve the sensitivity of the assay protocol.

Clofibrate-induced neoplastic development in the rat liver is associated with decreased connexin 32 expression but not with a co-ordinated shift in expression of marker enzymes.

Altered enzyme phenotype and expression of connexin 32 (Cx32), a gap junction protein were studied during the development of rat liver tumors induced by the non-genotoxic carcinogen, clofibrate. (1) In contrast to previous findings for nitrosamine-induced lesions, preneoplastic enzyme-altered foci (EAF) and neoplastic nodules (NN) lacked any clear association with degree of altered enzyme expression because of an almost complete negativity for GST-P and GGT. (2) Immunohistochemically demonstrated Cx32 spots on the hepatocyte membranes showed a clear decrease in clofibrate-induced lesions. (3) Naturally occurring EAF demonstrating GST-P and/or GGT positivity did not show a significant decrease of Cx32 counts suggesting a reversible nature. Therefore, the Cx32 decrease appears closely linked to progression of hepatocarcinogenesis irrespective of the enzyme phenotype of neoplastic focal lesions and the carcinogens used for their induction.

Modification of BHA forestomach carcinogenesis in rats: inhibition by diethylmaleate or indomethacin and enhancement by a retinoid.

The long-term effects of butylated hydroxyanisole (BHA), in combination with various other chemicals on the development of forestomach lesions in rats were investigated. BHA is a synthetic antioxidant, and the other agents included the glutathione-depleting agent diethylmaleate (DEM), the anti-inflammatory drugs indomethacin (IM), dexamethazone (DEX), 6-aminocaproic acetate (6-ACA) and FOY (gabexate mesilate), and the vitamin all-trans-retinol acetate (RA). Concurrent treatment with BHA (1% in diet) and DEM, IM, DEX or FOY for 52 weeks inhibited development of forestomach epithelial hyperplasia as compared to BHA alone, while simultaneous treatment with RA enhanced hyperplastic development. However, the inhibition by DEX or FOY was only partial and in the DEX case, in particular, might have been due to weight loss. Since the most effective inhibitory influence on BHA-induced forestomach lesions exerted in this 1-year experiment was by DEM, a further 2-year experiment was conducted to confirm whether DEM actually can exert inhibitory effects on BHA (2% in diet)-induced forestomach carcinogenesis. The results demonstrated that induction of forestomach hyperplasias and papillomas by BHA was significantly reduced by combination treatment with DEM. Both multiplicity and incidence of forestomach papillomas were significantly decreased, while squamous cell carcinoma development showed a tendency for decrease only.

Modifying effects of various chemicals on tumor development in a rat wide-spectrum organ carcinogenesis model.

The efficacy of a wide-spectrum organ carcinogenesis model for detection of modification potential of exogenous agents was investigated in F344 male rats. Groups of animals were sequentially injected with N-bis(2-hydroxypropyl)nitrosamine (1000 mg/kg body weight, i.p., in saline, twice in week 1), N-ethyl-N-hydroxyethylnitrosamine (1500 mg/kg body weight, i.g., in distilled water, twice in week 2) and 3,2'-dimethyl-4-aminobiphenyl (75 mg/kg body weight, s.c., in corn oil, twice in week 3) for wide-spectrum initiation of target organs and then given one of 10 test chemicals, comprising 6 hepatocarcinogens and 4 non-hepatocarcinogens, for 12 weeks. All 10 chemicals exerted modifying effects in their respective target organs. Enhancing influence could be detected in the liver and urinary bladder with 2-acetylaminofluorene, ethionine, and 3'-methyl-4-dimethylaminoazobenzene; in the liver and thyroid with 4,4'-diaminodiphenylmethane and phenobarbital; in the esophagus and urinary bladder with N-butyl-N-(4-hydroxybutyl)nitrosamine; in the forestomach and urinary bladder with butylated hydroxyanisole; in the liver with 7,12-dimethylbenz[a]anthracene and in the liver and lung with 3-methylcholanthrene. Inhibitory effects on development of glutathione S-transferase placental form-positive liver cell foci were observed with clofibrate. The results indicate that the present model can be reliably utilized as a whole body medium-term bioassay system for assessment of environmental cancer modifiers.

Effects of phenolic antioxidants in low dose combination on forestomach carcinogenesis in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine.

The combined effects of low doses of promoters or carcinogens on two-stage forestomach carcinogenesis were examined in rats pretreated with N-methyl-N'-nitro-N-nitrosoguanidine. Groups of 15 rats were given a single 150 mg/kg body weight intragastric dose of N-methyl-N'-nitro-N-nitrosoguanidine. Starting 1 week later they were fed a diet containing low doses of known forestomach promoters/carcinogens (0.5% caffeic acid, 0.2% catechol, 0.5% butylated hydroxyanisole, or 0.25% 2-tert-butyl-4-methylphenol), alone or in combination, or basal diet without antioxidant supplement for 35 weeks. Histopathological examination revealed the incidences of forestomach squamous cell carcinomas in animals treated with N-methyl-N'-nitro-N-nitrosoguanidine followed by caffeic acid, catechol, butylated hydroxyanisole, 2-tert-butyl-4-methylphenol, and basal diet to be 27, 20, 13, 13, and 7%, respectively, whereas the incidence increased to 80% by the combined treatment with these four chemicals. The present results thus show that although the low doses of individual promoters/carcinogens did not have significant promoting activity, their combination exerted a strong enhancing influence on rat forestomach carcinogenesis. The findings indicate the importance of summation and synergism at a low dose for agents present in the human environment.