Keisuke Ozaki

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochem- ical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed; however, the altered cells were negative for both immunohistochemica l markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisome s were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

Association of Adrenal Pheochromocytoma and Lung Pathology in Inhalation Studies with Particulate Compounds in the Male F344 Rat—The National Toxicology Program Experience

Systemic hypoxemia, occurring in space-occupying lung pathologies such as inflammation and neoplasms, reduces the gas exchange area and stimulates catecholamine secretion from the adrenal medulla where chronic endocrine hyperactivity may lead to hyperplasia and neoplasia. We investigated the possible correlation between nonneoplastic chronic pulmonary lesions and adrenal pheochromocytom a in 9 recent, NTP, 2-year particulate inhalation studies in male F344 rats. Re-evaluation for chronic active inflammation, interstitial fibrosis, alveolar epithelial hyperplasia, squamous metaplasia, proteinosis, and histiocytosis revealed significant associations of pheochromocytom a only with the severity of inflammation and fibrosis. Nickel oxide, cobalt sulfate, indium phosphide, talc, and nickel subsulfide studies showed chemical-related incidences of adrenal pheochromocytom a and significant (p < 0.01) associations with inflammation and fibrosis. Gallium arsenide, vanadium pentoxide, molybdenum trioxide, and nickel sulfate hexahydrate studies revealed an increased incidence and/or severity of nonneoplastic lung lesions, but no increased incidence of pheochromocytoma . Although gallium arsenide and molybdenum trioxide showed no dose-related increase in pheochromocytoma, a significant (p < 0.01) correlation of the latter with the severity of fibrosis and inflammation occurred. In the vanadium pentoxide and nickel sulfate hexahydrate studies, no relationship between nonneoplastic lung lesions and pheochromocytoma was manifested. Our investigation assessed the strength of these various associations and supports the possible roles of 2 chronic pulmonary lesions—fibrosis and inflammation—and hypoxemi a in the induction of pheochromocytom a in the F344 male rat.

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.

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.

Synergism between Sodium Chloride and Sodium Taurocholate and Development of Pepsinogen‐altered Pyloric Glands: Relevance to a Medium‐term Bioassay System for Gastric Carcinogens and Promoters in Rats

In an approach to early detection of gastric carcinogens and promoters in an in vivo test system, promotion by sodium chloride (NaCl) and the synergistic effects of NaCl and sodium taurocholate (Na‐TC) on development of pepsinogen‐altered pyloric glands (PAPG) in rat glandular stomach after initiation with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) were investigated. A total of 205 male WKY/NCrj rats were divided into 8 groups. Group 1 was given a single dose of MNNG of 160 mg/ kg body weight by gastric intubation, and starting 2 weeks later basal diet containing Na‐TC for 18 weeks. In addition, 1 ml doses of saturated NaCl solution were given by gastric intubation at weeks 4, 6, 8 and 10. Similarly, group 2 was treated with MNNG and Na‐TC, while group 3 animals received MNNG and NaCl. Group 4 was given MNNG alone. Groups 5–8 served as equivalent controls without MNNG initiation. The results revealed significantly enhanced induction of immunohisto‐chemically defined PAPG in the Na‐TC + NaCl (P< 0.001), Na‐TC (P<0.01) and NaCl (P<0.01) treated animals initiated with MNNG. Sodium chloride demonstrated a clear synergistic effect with Na‐TC in promoting the development of PAPG, suggesting possible advantage for its use in medium‐term in vivo assays for detection of gastric carcinogens and promoters.

Susceptibilities of p53 Knockout and rasH2 Transgenic Mice to Urethane-Induced Lung Carcinogenesis are Inherited from their Original Strains

In the present study, susceptibility of CB6F1 mice carrying the human prototype c-Ha-ras gene (rasH2 mice) and p53 gene knockout mice (p53 (+/−) mice) to urethane-induced lung carcinogenesis was compared under the same experimental conditions. Both strains were administered 500 ppm urethane in their drinking water for 3 weeks. At week 26, lung adenocarcinomas and adenomas were observed in 53% and 100% of rasH2 mice, respectively, and lung adenomas were observed in 67% of rasH2 littermate (non-Tg) mice. However, lung tumors were not observed in either p53 (+/−) or p53 (+/+) mice. Peliosis hepatis and hepatic hemangiomas were observed in 27% and 67% of p53 (+/−) mice, but only in 6.7% and 6.7% of the rasH2 animals, respectively. Under the same experimental conditions, BALB/c mice, the strain of origin of the rasH2 mice, developed lung adenomas at an incidence of 93%, whereas none of the C57BL/6 original strain for p53 (+/−) mice developed lung tumors. Peliosis hepatis was observed in 40% of the C57BL/6 mice, but not in BALB/c mice; hepatic and splenic hemangiomas were not observed in these animals. These results indicate that organ susceptibility of rasH2 and p53 (+/−) mice is inherited from their strains of origin, the rasH2 and BALB/c lines being much more sensitive to the induction of pulmonary carcinogenesis.

Decreased Dimethylnitrosamine‐induced O6‐ and N7‐Methyldeoxyguanosine Levels Correlate with Development and Progression of Lesions in Rat Hepatocarcinogenesis

Formation and repair of O6‐medG and N7‐medG (O6‐ and N7‐methyldeoxyguanosine) in glutathione S‐transferase‐P form (GST‐P)‐positive liver cell foci, nodules, primary hepatocellular carcinoma (HCC) and transplanted hepatocellular carcinoma (TRP) induced by N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN) were immunohistochemically assessed following a single exposure to dimethyl‐nitrosamine (DMN). Male Fischer 344 rats received a 0.1% solution of EHEN as their drinking water for 4 weeks and were maintained on basal diet until week 40, when a single 50 mg/kg body weight dose of DMN was administered intraperitoneally. Nude rats (NIH rnu/rnu) bearing TRP were similarly treated. Sequential Killing 6, 12, 24, 48 and 72 h thereafter revealed significantly decreased indices of cells binding antibodies to O6‐medG and N7‐medG adducts in GST‐P‐positive foci and nodules, and particularly HCC and TRP, as compared to background parenchyma values. Similarly, differences between foci/nodules and HCC/TRP were also significant, indicating that decrease in adduct formation is associated with further malignant conversion. The rate of DNA adduct repair in foci and nodules subsequent to the peak found at the 12 h time‐point did not appear to be significantly different from that in the surrounding tissue at the dose of DMN studied. The results indicate decreased formation of DMN‐associated DNA damage, in line with the known metabolic profile of carcinogen‐induced focal liver lesions.

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.

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 tbe same medium‐term bioassay system, in which a known hepatopromoting agent, DCA, 17‐α‐ethynylestradiol, or 2‐acetyIaminofluorene, 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.

Decreased connexin 32 expression is associated with cellular proliferation and progression of hepatocarcinogenesis in the rat

The expression of connexin 32 (C×32), a major liver gap junction protein, during liver cell proliferation, caused by partial hepatectomy(PH), and development and progression of chemicallyinduced hepatocarcinogenesis was immunohistochemically studied in the rat. C×32 showed; 1) remarkable decrease before and during the S-phase; 2) clear decrease corresponding to increase in simultaneous expression of altered enzyme phenotypes in preneoplastic lesions; 3) Progressive decrease along with progression of hepatocarcinogenesis. Therefore, the observed decrease may indicate an important role for gap junctional intercellular communication in cell proliferation, which correlated to cellular independence and growth advantage in liver tumor progression.