Kazuo Hakoi

Enhancement by non-mutagenic pesticides of GST-P positive hepatic foci development initiated with diethylnitrosamine in the rat

The potential hepatocarcinogenicity of seven pesticides was examined using a rapid bioassay based on the induction of glutathione S-transferase placental form positive foci in the rat liver. Rats were initially injected with diethylnitrosamine and two weeks later were fed on diet supplemented with one of the pesticides for 6 weeks and then killed; all rats were subjected to a partial hepatectomy at week 3. Positive results were seen with chlorobenzilate (2000 ppm), vinclozolin (2000 ppm), malathion (4000 ppm), tecnazene (2000 ppm) and isoproturon (2000 ppm). S,S,S-tributylphosphorotrithioate (DEF, 200 ppm) and dicloran (2000 ppm) were negative in both number and area analyses. Although chlorobenzilate is carcinogenic in mice, malathion and vinclozolin have been reported as non-carcinogens in both rats and mice. Since the present system is based on the two-stage carcinogenesis hypothesis, it is possible that the chemicals showing positive results in this system possess at least tumor-promoting activity in the rat liver. This is very significant, as most carcinogens show tumor-promoting activity in their target organs.

Comparison of Reversibility of Rat Forestomach Lesions Induced by Genotoxic and Non-genotoxic Carcinogens

Reversibility of forestomach lesions induced by genotoxic and non‐genotoxic carcinogens was compared histopathologically. Groups of 30 to 33 male F344 rats were given dietary 0.1% 8‐nitroquinoline, dietary 0.4–0.2% 2‐(2‐furyl)‐3‐(5‐nitro‐2‐furyl)acrylamide, an intragastric dose of 20 mg/kg body weight N‐methyl‐N′‐nitro‐N‐nitrosoguanidine once a week, or 20 ppm N‐methylnitrosourethane in the drinking water as a genotoxic carcinogen, or 2% butylated hydroxyanisole, 2% caffeic acid, 2% sesamol or 2% 4‐methoxyphenol in the diet as a non‐genotoxic carcinogen for 24 weeks. Ten or 11 rats in each group were killed at week 24. Half of the remainder were maintained on basal diet alone for an additional 24 weeks and the other half were given the same chemical for 48 weeks, and then killed. Forestomach lesions induced by genotoxic carcinogens did not regress after removal of carcinogens. In contrast, simple or papillary hyperplasia (SPH), but not basal cell hyperplasia (BCH), induced by non‐genotoxic carcinogens clearly regressed after cessation of insult. SFH labeling indices in the non‐genotoxic carcinogen‐treated cases decreased after removal of the carcinogenic stimulus whereas BCH values were low irrespective of treatment. Atypical hyperplasia (AH), observed at high incidences in rats treated with genotoxic carcinogens, was also evident in animals receiving non‐genotoxic agents, even after their withdrawal, albeit at low incidences. AH labeling indices remained high even without continued insult. These results indicate that even with non‐genotoxic carcinogens, heritable alterations at the DNA level could occur during strong cell proliferation and result in AH development. This putative preneoplastic lesion might then progress to produce carcinomas.

Medium-term bioassay for the hepatocarcinogenicity of hexachlorobenzene.

Hexachlorobenzene (HCB) is an important environmental contaminant derived mainly from industrial and agricultural sources. It is carcinogenic in mice, rats and hamsters. It has now been studied in a medium-term bioassay for carcinogenicity based on the induction of preneoplastic lesions in the liver. We report here that the bioassay can rapidly detect carcinogenic doses of HCB and that there is a clear dose-response relationship. At the lowest dose of HCB administered, the incidence of preneoplastic lesions in the liver was no different from that in controls.

A Rapid in vivo Bioassay for the Carcinogenicity of Pesticides

Eight pesticides were tested in a bioassay based on the induction of preneoplastic lesions in the liver. Rats were given diethylnitrosamine intraperitoneally at 200 mg/kg bw and two weeks later were treated with pesticides for six weeks and then killed; all rats had a partial hepatectomy at week 3. Hepatocarcinogenic potential was assessed by comparing the number and area of glutathione s-transferase (placental form) -positive foci In the liver with those of controls given diethylnitrosamine alone. Positive results were seen with Chinomethionat, Phosmet and Propiconazole; the results obtained with Captan and Prochloraz were borderline; Benomyl, Daminozide and Folpet gave negative results. Our findings provide enough experimental evidence to indicate that great care should be exercised in the use of these compounds.

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.

Sequential Morphological and Biological Changes in the Glandular Stomach Induced by Oral Administration of Catechol to Male F344 Rats

Histogenesis and mechanisms of catechol-induced rat glandular stomach carcinogenesis were investigated in male F344 rats. Groups of 5 or 6 rats were treated with dietary catechol at doses of 1, 0.5, 0.1, and 0.01% for 12 hr or for 1, 2, 3, or 7 days or at a dose of 0.8% for 1, 2, 4, 12, and 24 wk; rats were then euthanatized. The initial morphological changes were edema of the gastric wall, inflammatory-cell infiltration, erosion in the pyloric region close to the duodenum, and considerable increase in apoptosis at 12 hr; later, changes included augmented DNA synthesis and cell proliferation, as evaluated by bromodeoxyuridine labeling index and thickness of mucosa, respectively, on day 1. Downward hyperplasia due to excess regeneration appeared at edges of ulceration at week 2. This lesion disappeared, and then submucosal hyperplasia appeared in the course of adenoma development. Only slight expression of c-myc or c-fos was apparent after 30-min oral administration or 1-, 3-, and 6-hr oral administration of catechol. No increase in lipid peroxide levels was evident in gastric epithelium fed catechol for 1 wk. The amount of catechol distributed in the glandular stomach and forestomach epithelium, which is not a target for carcinogenesis, did not differ 1, 3, 6, and 24 hr after a single intragastric dose of 75 mg/kg body weight. Amounts of catechol bound to tissue protein were also not specifically high in the glandular stomach. These results indicate that regenerative cell proliferation due to toxicity plays an important role in catechol-induced glandular stomach carcinogenesis. Protein binding and free radicals may not be largely responsible for the toxicity.

Modification of N-butyl-N-(4-hydroxybutyl)nitrosamine-initiated urinary bladder carcinogenesis in rats by phytic acid and its salts

The effects of dietary phytic acid and its salts on the promotion stage of two-stage urinary bladder carcinogenesis were examined. Male F344 rats were initiated by exposure to 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 wk, and then treated with basal diet containing a 2% supplement of phytic acid (PA), phytic acid dodecasodium salt (Na-PA), phytic acid dodecapotassium salt (K-PA), phytic acid hexamagnesium salt n-hydrate (Mg-PA) or no added chemical for 32 wk. Na-PA significantly increased the development of preneoplastic and neoplastic lesions of the urinary bladder. K-PA also brought about a tendency for increase in papillomas, whereas Mg-PA and PA were without effect. Both Na-PA and K-PA caused elevation of urinary pH, and Na+ or K+ concentration, respectively. These results confirm the promoting activity of the sodium salt of phytic acid for urinary bladder carcinogenesis and indicate modulation by urinary components, as demonstrated by increases in urinary pH, and Na+ concentration.

Spontaneous Thymoma in a 10-Week-Old Sprague-Dawley Rat

Spontaneous thymoma was found in the left lobe of the thymus of a male 10-week-old Sprague-Dawley (SD) rat. Microscopically, the thymic mass showed a sheet of dark area with multiple pale foci. The dark area mainly consisted of densely compacted small lymphoid cells with sporadic large epithelioid cells and mitotic figures. The epithelioid cells and mitotic figures were more frequent than those of the normal thymic cortex in this animal. The multiple pale foci were similar to the normal thymic medulla and occasionally had Hassall’s corpuscles; thus, they were regarded as medullary differentiation areas. Furthermore, some perivascular spaces recognized as characteristics of thymoma were present in the center of the mass. Immunohistochemically, the epithelioid cells in the dark area were positive for cytokeratin. Ultrastructurally, desmosomes and tonofilaments were observed in the epithelioid cells. Thus, this tumor was diagnosed as a thymoma. This is a rare case of thymoma occurring spontaneously in young adult SD rat.

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.