Shuji Yamaguchi

Modifying Effects of Various Chemicals on Preneoplastic and Neoplastic Lesion Development in a Wide‐spectrum Organ Carcinogenesis Model Using F344 Rats

Modifying potentials of various chemicals on tumor development were investigated in a wide‐spectrum organ carcinogenesis model using male F344/DuCrj rats. The animals were treated with N‐nitroso‐diethylamine (100 mg/kg body weight, ip, single injection at the commencement of the study), N‐methyl‐N‐nitrosourea (20 mg/kg body weight, ip, 4 times during weeks 1 and 2) and N‐bis(2‐hydroxypropyl)nitrosamine (0.1% in drinking water, during weeks 3 and 4) for multi‐organ initiation and then were given one of 14 test chemicals including 6 hepatocarcinogens, 7 non‐hepatocarcinogens and 1 non‐carcinogen, or basal diet for 16 weeks. All rats were killed at the end of week 20, and the major organs were carefully examined for preneoplastic and neoplastic lesions. Immunohistochemical demonstration of glutathione S‐transferase‐positivc foci was also used for quantitative assessment of liver preneoplastic lesion development. Modifying effects were shown for 11 out of 14 test agents in the liver, forestomach, glandular stomach, lung, urinary bladder or thyroid, 7 of them targeting more than two organs. This was the first demonstration to our knowledge that cloflbrate possesses enhancing potential for urinary bladder carcinogenesis and an inhibiting effect on thyroid carcinogenesis. Caprolactam showed no effect in any organ, in agreement with its established inactivity. The results indicated that the system could be reliably applied as a medium‐term multiple organ bioassay for assessment of the modification potential of test agents in unknown target sites.

Effects of naturally occurring antioxidants on combined 1,2-dimethylhydrazine- and 1-methyl-1-nitrosourea-initiated carcinogenesis in F344 male rats

The effects of treatment with naturally occurring antioxidants, selenium, beta-carotene, ferulic acid, esculin and eugenol during the promotional phase of tumor development were investigated in male F344 rats pre-treated with 1,2-dimethylhydrazine (DMH) and 1-methyl-1-nitrosourea (MNU). Animals were given 3 subcutaneous injections of DMH at a dose of 40 mg/kg body wt. within 1 week and then were injected with MNU i.p. at a dose of 20 mg/kg body wt. 2 times per week, for 2 weeks. Thereafter, the rats were maintained on diet containing either 0.2% beta-carotene, 2 ppm selenium, 1% ferulic acid, 1% esculin or 0.8% eugenol. At week 52, surviving rats were killed and complete histological examinations were performed. Administration of eugenol enhanced the development of both hyperplasia and papillomas in the forestomach. Although treatment with beta-carotene tended to decrease the incidence and number of large intestinal carcinomas, beta-carotene, selenium, esculin and eugenol all decreased the incidence of kidney nephroblastomas, the differences were not statistically significant. The results thus showed that eugenol exerts promoting activity for forestomach carcinogenesis while the other antioxidants might have weak organ-specific inhibitory effects under these experimental conditions.

Synergistic Effects of Low‐dose Hepatocarcinogens in Induction of Glutathione S‐Transferase P‐positive Foci in the Rat Liver

The effects of combined administration of hepatocarcinogens at low doses on the development of glutathione S‐transferase P‐form (GST‐P)‐positive foci of rat liver were examined utilizing a bioassay model which consists of a single injection of diethylnitrosamine (DEN, 200 mg/kg, ip), two‐thirds partial hepatectomy at week 3 and a 6‐week administration of test compounds. The chemicals used, 2‐acetylaminofluorene (2‐AAF), 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐Me‐DAB), phenobarbital (PB), thioacetamide (TAA), N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN), benzo[a]pyrene (B[a]P), carbazole, and α‐hexachlorocyclohexane (α‐HCH) were incorporated in the diet, except for EHEN which was dissolved in the drinking water, at levels of 1/6 of the doses usually used. The combinations were: I) 2‐AAF, 3′‐Me‐DAB, PB, TAA, EHEN and B[a]P, II) 2‐AAF, 3′‐Me‐DAB and PB, III) TAA, EHEN and B[a]P, IV) 2‐AAF, 3′‐Me‐DAB, carbazole, TAA, EHEN and a‐HCH, V) 2‐AAF, 3′‐Me‐DAB and carbazole, and VI) TAA, EHEN and α‐HCH. All combinations, except for II, caused an increase in the area of the foci as evaluated by the ratios of areas in the combined administration groups to the sum totals of 3 or 6 individual data: I) 1.75, II) 0.81, III) 2.01, IV) 3.62, V) 1.34 and VI) 2.91. The non‐synergistic effect in combination II might be related to PB induction of hepatic microsomal enzymes leading to enhanced enzymatical detoxification of 2‐AAF and 3′‐Me‐DAB. The present results indicate that exposure to several chemicals of similar organotropism, even at doses lower than the apparent carcinogenic levels, might be critical to the carcinogenic process.

Effects of Modifying Agents on Conformity of Enzyme Phenotype and Proliferative Potential in Focal Preneoplastic and Neoplastic Liver Cell Lesions in Rats

Development of preneoplastic lesions in the rat liver under the influence of various modifiers was investigated with particular attention to changes in simultaneous expression of altered enzyme phenotype within the lesions (conformity) and proliferation potential. Degree of conformity of marker enzymes such as glutathione S‐transferase placental form (GST‐P), glucose‐6‐phosphate dehydrogenase (G6PD), glucose‐6‐phosphatase, adenosine triphosphatase and γ‐glutamyltranspeptidase was compared with levels of S‐bromo‐2‐deoxyuridine labeling. After initiation with diethylnitrosamine, rats were administered the hepatopromoter sodium phenobarbital (PB, 0.05%), the antioxidant ethoxyquin (EQ, 0.5%), or a peroxisome proliferator, clofibrate (CF, 1.0%) or di(2‐ethylhexyl)‐phthalate (0.3%) and killed at week 16 or 32. The PB promoting regimen was clearly associated with increase in the numbers of high conformity class lesions simultaneously expressing three to five enzymes, and elevated proliferation potential. The inhibitor, EQ, in contrast, brought about a time‐dependent decrease in conformity so that only 1 or 2 alterations were most commonly observed at week 32. Lesion populations in the peroxisome proliferator‐ and especially CF‐treated cases were characterized by obvious dissociation between degree of conformity and proliferative status. Such treatment‐dependent differences were not always correlated with the size of the lesion. The results thus suggested that the conformity and proliferation potential of preneoplastic lesions are dependent on modification treatment. Overall, GST‐P was found to be the most reliable marker, although G6PD was less influenced in the peroxisome proliferator cases.

Age‐dependent Induction of Preneoplastic Liver Cell Foci by 2‐Acetylammo‐fluorene, Phenobarbital and Acetaminophen in F344 Rats Initially Treated with Diethylnitrosamine

Effects of age on the induction of glutathione S‐transferase placental form (GST‐P)‐positive hepatic foci in rats were examined using a medium‐term liver bioassay system (for carcinogens). F344 male rats aged 6, 26 and 46 weeks were initially given a single intraperitoneal injection of diethylnitro‐samine (DEN, 200 mg/kg) and, beginning 2 weeks later, received 0.02% 2‐acetylaminofluorene (2‐AAF), 0.05% phenobarbital (PB) or 1.3% acetaminophen (AAP) in the diet for 6 weeks. All animals were subjected to two‐thirds hepatectomy 3 weeks after the DEN injection and were killed at week 8. Quantitative analysis of GST‐P‐positive foci revealed significantly (P< 0.001) increased induction over control levels in terms of both numbers and areas for 2‐AAF at all ages (6, 26 and 46 weeks), but especially in the 6‐week‐old case. In the PB‐ and AAP‐treated groups, the respective enhancing and inhibitory influences were most pronounced in the animals aged 6 weeks, and were less marked in older rats. Thus, the response of F344 rats to the modifying effects of chemicals was age‐dependent, the conclusion being drawn that young rats are more susceptible and therefore more appropriate for assessment of carcinogenic, promoting and inhibitory effects of chemicals.

Number of Simultaneously Expressed Enzyme Alterations Correlates with Progression of N‐Ethyl‐N‐hydroxyethylnitrosamine‐induced Hepatocarcinogenesis in Rats

Preneoplastic and neoplastic liver cell lesions, induced by EHEN (N‐ethyl‐N‐hydroxyethylnitrosamine) in rats, were investigated to establish the numbers of simultaneously expressed altered enzyme phenotypes within the lesion cells. The lesions were divided into 5 classes on the basis of altered expression in one or more of the following 5 enzymes: glutathione S‐transferase placental form, glucose‐6‐phosphate dehydrogenase, glucose‐6‐phosphatase, adenosine triphosphatase, and γ‐glutamyl transpeptidase. Class 1 lesions contained cells expressing one altered enzyme. Similarly, class 2, 3, 4 and 5 lesions had cells simultaneously expressing 2, 3, 4, and 5 enzyme alterations, respectively. Four histopathological categories of lesions, ACF (altered cell foci) (274 lesions), HN (hyperplastic nodules) (47 lesions), HCC (hepatocellular carcinomas) (99 lesions) and THC (transplanted hepatocellular carcinomas) (5 lesions) were studied. Proliferation potential was assessed in terms of 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation. The distribution profiles of classes 1 to 5 showed a clear reciprocal change from low class (1 to 2 enzymes) predominance in ACF to high class (4 to 5 enzymes) predominance in HN. Increase of BrdU labeling indices was clearly correlated with progression from HN to HCC. Only a small population of class 5 ACF showed a high BrdU labeling index, indicating particular potential for further development. Thus, the stages of EHEN‐induced neoplasia were found to be characterized by gradual increase in the number of altered enzyme phenotypes, with acquisition of proliferative potential being associated with further progression towards malignant conversion.

Persistence of Liver Cirrhosis in Association with Proliferation of Nonparenchymal Cells and Altered Location of α-Smooth Muscle Actin-Positive Cells

This study was carried out to achieve pathological understanding for the persistence of cirrhosis induced by thioacetamide (TAA). Forty-five, male, 21-day-old, F344 rats were randomly allocated to group 1 and received drinking water as a control, and groups 2 and 3 given 0.015% or 0.03% TAA, respectively for 12 weeks. Two-third of animals per group were sacrificed, and remainder were maintained for a further 4 weeks without TAA treatment. Liver cirrhosis was induced in all animals in group 3 at week 12, with obvious increase of collagen content, and this persisted after cessation of TAA. Proliferating cell nuclear antigen (PCNA) positive labeling indices of nonparenchymal cells were increased significantly after cessation in groups 2 and 3 (p < 0.01). RT-RCR analysis of α-smooth muscle actin (α-SMA) showed significant increase in group 3 compared to that of control at both time points (p < 0.05). Immunohistochemical staining of it demonstrated positive cells to mainly be located around regenerating hepatic nodules at week 12, however, they were focused into enlarged portal areas consisting of fibrous tissues and pseudo-bile ductular cells after the cessation. Taken together, we conclude persistence of liver cirrhosis could be associated with the proliferation of nonparenchymal cells and altered location of α-SMA positive cells.

Sex Differences in o-Phenylphenol and Sodium o-Phenylphenate Rat Urinary Bladder Carcinogenesis: Urinary Metabolites and Electrolytes under Conditions of Aciduria and Alkalinuria

F344 male and female rats were administered 1.25%o‐phenylphenol (OPP) or 2% sodium o‐ phenylphenate (Na‐OPP) in combination with 3% NaHCO3 or 1% NI4Cl for 8 weeks and changes in the urinary bladder histopathology and the urinary components were examined. Administration of OPP with NaHCO3 resulted in marked urothelial liyperplasia in the urinary bladder of male rats, the response being less pronounced in females. OPP alone exerted no proliferative effect and NaHCO3 induced only slight hyperplasia in males. Na‐OPP alone induced mild hyperplastic lesions only in males, this being completely prevented by concomitant administration of NH4C1. The findings thus demonstrated a clear correlation between hyperplastic response and reported carcinogenic potential of these treatments. Of the urinary factors examined, increases in levels of pH and sodium ion concentration were positively associated with proliferative lesions especially in males, although the findings failed to explain the sex difference. Urinary concentrations of non‐conjugated forms of OPP metabolites were also not directly correlated with the development of hyperplasias. Thus, changes in individual urinary factors presumably affect nrothelial proliferation in combination rather than separately. The presence of OPP metabolites, including 2‐phenyl‐l,4‐benzoquinone, in the urine may be unimportant in the OPP urinary carcinogenesis even under conditions of alkalinuria and high sodium ion concentration.

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.

Development and Application of an in Vivo Medium-Term Bioassay System for the Screening of Hepatocarcinogens and Inhibiting Agents of Hepatocarcinogenesis

The number of compounds which have been introduced into our environment is now far beyond our capacity to perform costly long-term carcinogenicity tests. For the purpose of performing mass-screening of compounds, in vitro short-term tests such as the Salmonella/microsome assay (Ames’ test) have been developed1,2, and a variety of carcinogenic compounds have been shown to be mutagenic with an apparent good correlation3–6. Recently, however, it has been shown that the mutagenicity results have not always correlated well to carcinogenicity4. Therefore, the development of an appropriate in vivo medium-term assay system which could bridge the gap between mutagenicity and carcinogenicity tests has become an urgent necessity. Use of rat liver utilizing the two-step concept for such a screening assay has the particular advantage of relatively easy quantitative detection of preneoplastic enzyme altered foci7–10. Recent studies in this laboratory indicated that immunohistochemically demonstrated GST-P+ foci show good conformity to previously used hyperplastic nodules or γ-glutamyl transpeptidase-positive foci with the advantage of having far less background hepatocyte staining due to non-specific induction11–15.