Tadashi Ogiso

Promoting effects of various chemicals in rat urinary bladder carcinigenesis initiated by N-nitroso-n-butyl-(4-hydroxybutyl)amine

We studied the capacity of various chemicals to promote urinary bladder cancer in male F344 rats after initiation by N-nitroso-n-butyl-(4-hydroxybutyl)amine (BBN). The rats were given initially 0.01% BBN in the drinking-water for 4 wk and then the test compound in the diet for 34 wk. Effects were judged by measuring the formation of preneoplastic lesions papillary or nodular hyperplasia (PN hyperplasia) of the urinary bladder. Administration of 5%, but not 0.5% (w/w) sodium saccharin in the diet significantly increased the incidence and extent of PN hyperplasia. This finding could be related to the induction of cancers in the rat urinary bladder by high levels of saccharin. Sodium ascorbate (5%). DL-tryptophan (5%) and allopurinol (0.02%) also significantly increased the extent of PN hyperplasia in the affected animals, but other test chemicals, such as acetazolamide (0.35%) and quercetin (5%) did not. The results with sodium saccharin and DL-tryptophan were consistent with previous findings and suggest that sodium ascorbate and allopurinol have promoting activities in urinary bladder carcinogenesis in rats. No correlation was found between the extent of crystalluria and promotion of preneoplastic lesions.

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), β‐cryptoxanthin‐ and hes‐peridine‐rich pulp (CHRP) and 1,1′‐acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium‐term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

Dose-dependent effects of butylated hydroxyanisole, butylated hydroxytoluene and ethoxyquin in induction of foci of rat liver cells containing the placental form of glutathione S-transferase

The dose-dependence effects of 3 antioxidants, butylated hydroxyanisole (BHA: 2.0%, 1.0% and 0.5%), butylated hydroxytoluene (BHT: 1.0%, 0.5% and 0.25%) and ethoxyquin (EQ: 0.5%, 0.25% and 0.125%) combined with partial hepatectomy on the development of preneoplastic lesions in the liver of diethylnitrosamine (DEN, 200 mg/kg body wt)-treated rats were investigated. Feeding of the antioxidants commenced 2 weeks after the single dose of DEN used to initiate the lesions. gamma-Glutamyl transpeptidase (gamma-GT) and the placental form of glutathione S-transferase (GST-P) were used for quantitation of altered focal populations. Results with both markers demonstrated a dose-dependent decrease of foci in BHA-treated rats relative to those in control rats. Morphometric analysis of gamma-GT-positive lesions also revealed decrease in both the number and area of foci in BHT- and EQ-treated groups. The discrepancy between results of quantitation of gamma-GT- and GST-P-positive foci was attributable to the induction of a background, periportal zone staining for gamma-GT, which made differentiation of smaller foci difficult. Comparison of results with the 2 markers suggested that GST-P is the more accurate marker for quantitative studies on enzyme altered foci in rat liver.

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.

Establishment of an in vivo Highly Metastatic Rat Hepatocellular Carcinoma Model

We previously found by chance that N‐nitrosomorpholine (NMOR) given after a multi‐carcinogenic treatment induces liver carcinomas with 56% lung metastasis, and it was confirmed that hepatocellular carcinoma (HCC) with 100% lung metastasis was produced by 24‐week treatment with NMOR and additional treatment with diethylnitrosamine (DEN). In the present study, we modified the duration of NMOR to establish an animal model with a simple experimental protocol and an appropriate experimental duration which would facilitate further study of the mechanisms of metastasis and antimetastatic agents. The results revealed DEN exposure followed by a 16‐week treatment with NMOR to be a most efficient method for the induction of HCC metastasizing to the lung. Loss of cadherin, demonstrated immunohistochemically, occurred in an early stage of carcinogenesis, and this was reflected in malignant conversion of primary lesions. This model, with its essential similarities to malignant tumor behavior in man, should find application not only for elucidation of the mechanisms underlying metastasis, but also in the development of anti‐metastatic agents.

Dose-Dependent Promotion by Phenylethyl Isothiocyanate, a Known Chemopreventer, of Two-Stage Rat Urinary Bladder and Liver Carcinogenesis

The effects of phenylethyl isothiocyanate (PEITC) on urinary bladder and liver carcinogenesis were analyzed in a rat model. Diets containing 0.1%, 0.05%, or 0.01% PEITC were administered for 32 wk to male Fischer 344 rats with and without pretreatment with an injection of diethylnitrosamine (200 mg/kg body wt ip) and 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in the drinking water for 4 wk for initiation. In the initiated groups, PEITC administration significantly increased the incidences of papillary or nodular hyperplasia, dysplasia, and transitional cell carcinomas at higher doses of 0.01%, 0.01%, and 0.05%, respectively, compared with the control group, given initiation alone, in a dose-dependent manner. Without initiation, administration of 0.1% and 0.05% PEITC induced simple and papillary or nodular hyperplasia and dysplasia in the urinary bladder. In the liver, induction of glutathione S-transferase placental form-positive foci was dose dependently enhanced by PEITC administration, but the incidences of liver tumors were not different among the groups. From the present experiment, we can conclude that >0.01% PEITC enhances rat urinary bladder carcinogenesis, while weakly promoting hepatocarcinogenesis. In addition, it is suggested that >0.05% PEITC has tumorigenic potential.

Quantitative analysis of initiating and promoting activities of five mycotoxins in liver carcinogenesis in rats.

The initiating and promoting activities of several mycotoxins were investigated in F344 male rats. N-2-Fluorenylacetamide (2-FAA) was used as an initiator or a promoter and citrinin, ochratoxin A, sterigmatocystin, (+)rugulosin or patulin was administered at a fixed dose for the same period in the initiation stage or the promotion stage. Partial hepatectomy and administration of carbon tetrachloride (CCl4) were examined to increase the induction of hyperplastic nodules. Ochratoxin A, sterigmatocystin and (+)rugulosin administered in either the initiating or the promoting stage significantly increased the induction of hyperplastic nodules. Citrinin and patulin administered in the initiating stage significantly increased the induction of hyperplastic nodules, but were not effective when administered during the promoting stage. The theoretical classification of the mycotoxins examined is discussed.

Enhancing potential of 6 different carcinogens on multi-organ tumorigenesis after initial treatment with N-methyl-N-nitrosourea in rats

The advantages of applying a whole‐body concept to the assessment of carcinogenic potential of compounds in a two‐stage model after initiation by N‐methyl‐N‐nitrosourea (MNU) were investigated. Male, 6‐week‐old F344 rats were injected with MNU (20 mg/kg, i. p.) twice a week for 4 weeks and they then received 3,2′‐dimethyl‐4‐aminobiphenyl (DMAB) (50 mg/kg, s.c., once a week), N,N′‐dibutylnitrosaraine (DBN) (0.05%, in drinking water), N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) (0.1%, in drinking water), diethylstilbestrol (DES) (2.5 ppm, in diet), sodium o‐phenylphenate (S. OPP) (2%, in diet) or captafol (0.15%, in diet) for 20 weeks. All six carcinogens enhanced the incidences of preneoplastic and neoplastic lesions in their respective target organs: liver, pancreas, small intestine and urinary bladder with DMAB; liver, esophagus, forestomach and urinary bladder with DBN; thyroid, lung, liver, esophagus, forestomach, small intestine and urinary bladder with DHPN; liver and forestomach with DES; and thyroid, forestomach, kidney and urinary bladder with S. OPP; liver and forestomach with captafol. The results suggested that prior treatment with MNU sensitized the tissues to the organotropic carcinogenic potential of chemicals given thereafter for as short a period as 20 weeks. Thus, this system could be utilized as a whole‐body medium‐term bioassay system for the screening of environmental carcinogens, bridging the gap between in vitro mutagenicity and long‐term carcinogenicity tests.

Absence of promotion potential for calcium l-ascorbate, l-ascorbic dipalmitate, l-ascorbic stearate and erythorbic acid on rat urinary bladder carcinogenesis

The effects of treatment with calcium L-ascorbate, L-ascorbic dipalmitate, L-ascorbic stearate and erythorbic acid on two-stage urinary bladder carcinogenesis in F344 rats after initiation with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Carcinogen was administered at a dose of 0.05% in drinking water for 4 weeks and thereafter the test chemicals were given as a 5% supplement in the diet for the following 32 weeks. No increase in the induction of preneoplastic lesions, papillomas or carcinomas was apparent and it was concluded that none of the test chemicals possess promoting activity for urinary bladder carcinogenesis.

Carcinogenicities of quinoline derivatives in F344 rats.

In Ames test, the quinoline derivatives, 6-nitroquinoline (6-NQ), 8-nitroquinoline (8-NQ), 6-methylquinoline (6-NQ), 8-methylquinoline (8-MQ) and 8-hydroxyquinoline (8-HQ) are mutagenic, whereas 5,7-dibromo-quinoline (5,7-DQ) is not. These compounds were administered to groups of male and female F344 rats in their diet for 104 weeks. 8-NQ induced forestomach tumors in male and female rats: it induced squamous cell papillomas in 28 (93.3%) of 30 males and in 36 (97.3%) of 37 females, and squamous cell carcinomas in 20 (66.7%) of 30 males and 24 (64.9%) of 37 females. Thus there was no sex difference in its carcinogenicity. 6-NQ induced only focal hyperplasia of the forestomach at an incidence of 30.0% in male rats and 52.8% in female rats. These results indicate a discrepancy between the mutagenicities of quinoline derivatives and their carcinogenicities to rats: only 8-NQ was carcinogenic to F344 rats.