Kazushi Okazaki

A cyclooxygenase-2 inhibitor, nimesulide, inhibits postinitiation phase of N-nitrosobis(2-oxopropyl)amine-induced pancreatic carcinogenesis in hamsters

The modification effects of nimesulide, a cyclooxygenase (COX)‐2 inhibitor, administration during the postinitiation phase of pancreatic carcinogenesis were investigated in hamsters treated with N‐nitrosobis(2‐oxopropyl)amine (BOP). Male Syrian hamsters were given 4 weekly s.c. injections of BOP at a dose of 10 mg/kg and thereafter administered 0, 100 or 400 ppm nimesulide in the diet for 36 weeks. Additional groups of hamsters were fed 400 ppm nimesulide without prior BOP initiation or nontreated. At week 40, all surviving animals were killed and development of neoplastic and preneoplastic lesions was assessed histopathologically. The incidence of pancreatic adenocarcinomas was significantly (p < 0.05) decreased in the BOP/400 ppm nimesulide group compared to the BOP alone group. The multiplicity of total lesions of pancreatic adenocarcinoma plus atypical hyperplasia was also significantly (p < 0.05) lowered. Immunohistochemically, COX‐2 was clearly expressed in pancreatic and lung tumor cells, whereas expression was not remarkably affected by the 400 ppm nimesulide treatment. Proliferating cell nuclear antigen labeling indices of pancreatic ducts were significantly (p < 0.01) reduced by nimesulide. The incidence and multiplicity of neoplastic lesions in other organs did not significantly differ among the BOP‐treated groups, though only the multiplicity of lung tumors showed a tendency to decrease. No neoplastic lesions were detected in animals receiving nimesulide alone. Our results clearly indicate that nimesulide protects against BOP‐induced pancreatic tumors in hamsters.

Dose‐related changes of oxidative stress and cell proliferation in kidneys of male and female F344 rats exposed to potassium bromate

It is still of importance to investigate renal carcinogenesis by potassium bromate (KBrO3), a by‐product of water disinfection by ozonation, for assessment of the risk to man. Five female F344 rats in each group were given KBrO3 at a dose of 300 mg/kg by single i.g. intubation or at a dose of 80 mg/kg by single i.p. injection, and were killed 48 h after the administration for measurements of thiobarbituric acid‐reactive substances (TBARS) and 8‐oxodeoxyguanosine (8‐oxodG) levels in the kidney. Both levels in the treated animals were significantly elevated as compared with the control values. In a second experiment, 5 male and female F344 rats in each group were administered KBrO3 at concentrations of 0, 15, 30, 60, 125, 250 and 500 ppm in the drinking water for 4 weeks. KBrO3 in the drinking water did not elevate TBARS in either sex at any of the doses examined, but 8‐oxodG formation in both sexes at 250 ppm and above was significantly higher than in the controls. Additionally, the bromodeoxyuridine‐labeling index for proximal convoluted tubules was significantly increased at 30 ppm and above in the males, and at 250 ppm and above in the females. α2u‐Globulin accumulation in the kidneys of male rats was increased with statistical significance at 125 ppm and above. These findings suggest that DNA oxidation induced by KBrO3 may occur independently of lipid peroxidation and more than 250 ppm KBrO3 in the drinking water can exert a carcinogenic effect by way of oxidative stress.

Dose-dependent promotion of rat forestomach carcinogenesis by combined treatment with sodium nitrite and ascorbic acid after initiation with N-methyl-N′-nitro-N-nitrosoguanidine: Possible contribution of nitric oxide-associated oxidative DNA damage

Dose‐dependent promotion effects of combined treatment with sodium nitrite (NaNO2) and ascorbic acid (AsA) on gastric carcinogenesis were examined in rats pretreated with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). Groups of 15 6‐week‐old F344 male rats were given 0.01% MNNG in their drinking water for 10 weeks to initiate carcinogenesis in the glandular stomach and a single intragastric administration of 100 mg/kg/bodyweight of MNNG by stomach tube at week 9 to initiate carcinogenesis in the forestomach. From week 11, they received either drinking water containing 0.05, 0.1 or 0.2% NaNO2 and a diet supplemented with 0.1 or 0.2% AsA in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, the incidence of hyperplasia was increased dose dependently by the treatment with NaNO2 alone. Incidences of neoplastic lesions were dramatically increased by the combined treatment with NaNO2 and AsA in a dose‐dependent manner, but AsA itself had no effect. In the glandular stomach, only toxicity and regenerative changes were increased by the high‐dose combination. In a second short‐term experiment conducted for sequential observation, necrosis and strong inflammation were found in the forestomach epithelium shortly after commencing combined treatment with 1.0% AsA and 0.2% NaNO2, followed by hyperplasia, whereas there were no obvious effects in the glandular stomach. In addition, after a 4 h treatment with 1.0% AsA and 0.2% NaNO2, a slight increase in the 8‐hydroxy‐deoxyguanosine levels in the forestomach epithelium was observed by high‐performance liquid chromatography and an electrochemical detection system, albeit without statistical significance. In vitro, electron spin resonance demonstrated nitric oxide formation during incubation with NaNO2 and AsA under acidic conditions. Thus, NaNO2 was demonstrated to exert promoter action in the forestomach, with AsA acting as a strong copromoter through cytotoxicity and regenerative cell proliferation, possibly mediated by oxidative DNA damage, but the combined treatment with NaNO2 and AsA had little influence on glandular stomach carcinogenesis. (Cancer Sci 2006; 97: 175 –182)

Simultaneous treatment with benzyl isothiocyanate, a strong bladder promoter, inhibits rat urinary bladder carcinogenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Effects of benzyl isothiocyanate (BITC) on urinary bladder carcinogenesis were examined in rats simultaneously treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Groups of 20 6-wk-old Fischer 344 male rats were given 10, 100, or 1,000 ppm BITC in the diet or a basal diet with 50 ppm BBN in the drinking water for 40 wk and then killed for autopsy. Additional groups consisting of 10 or 9 rats were similarly given BITC or the basal diet alone without BBN treatment. With BBN treatment, dysplasia, papilloma, and carcinoma incidences and multiplicities were dramatically decreased by simultaneous treatment with BITC in a clear dose-dependent manner. In contrast, epithelial hyperplasia was induced in rats treated with 100 and 1,000 ppm BITC without BBN. These results clearly indicate that although BITC may have weak carcinogenic potency, it is a potent chemopreventive agent against bladder tumor induction by BBN.

Enhancement of urinary bladder carcinogenesis by combined treatment with benzyl isothiocyanate and N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine in rats after initiation

Previously we reported that benzyl isothiocyanate (BITC) strongly enhanced rat urinary bladder carcinogenesis after initiation with N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN), while potently inhibiting BBN‐induction of lesions when given simultaneously with the carcinogen. In the present experiment, the effects of simultaneous treatment with BITC and low‐dose BBN on the postinitiation period of rat urinary bladder carcinogenesis were examined. After treatment with 500 ppm BBN for 4 weeks for initiation, groups of 20, 6‐week‐old, F344 male rats were given 25 ppm BBN alone, basal diet alone, or 100 or 1000 ppm BITC in the diet together with or without 25 ppm BBN in their drinking water for 36 weeks and then killed for autopsy. Further groups consisting of 10 rats each were similarly given BITC or the basal diet together with or without 25 ppm BBN, without initiation treatment. In the initiated groups receiving subsequent BBN exposure, papillary and nodular hyperplasia, dysplasia and carcinoma incidences were significantly increased, and they were further increased by the combined treatment with 100 and 1000 ppm BITC in a dosedependent manner. In the non‐initiation groups, carcinomas were only observed in a single rat in each of the BBN‐treated control and BBN/BITC 100 ppm treatment groups. The results indicate that simultaneous treatment with BITC and a low dose of BBN does not inhibit, but rather enhances rat urinary bladder carcinogenesis after appropriate initiation, and further suggest that BITC may be a human risk factor, at least in high‐risk populations.

Enhancement by Cigarette Smoke Exposure of 2-Amino-3,8-dimethylimidazo(4,5- f )quinoxaline-induced Rat Hepatocarcinogenesis in Close Association with Elevation of Hepatic CYP1A2

The modifying effects of cigarette smoke (CS) exposure on a heterocyclic amine (HCA) 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx)‐induced carcinogenesis were investigated in male F344 rats. Groups 1 and 2 were fed MeIQx at a dose of 300 ppm, and simultaneously received CS and sham smoke (SS) for 16 weeks, respectively. Groups 3–5 were given the MeIQx diet for 4 weeks, and simultaneously exposed to CS for 4 weeks (group 3), exposed to CS for 12 weeks after the MeIQx treatment (group 4) or received SS for 16 weeks (group 5). Groups 6 and 7 were fed basal diet and respectively received CS and SS for 16 weeks. In terms of the mean number or area, the development of glutathione S‐transferase placental form‐positive (GST‐P+) liver cell foci was significantly (P<0.01) greater in group 1 than in group 2. The mean number of colonic aberrant crypt foci (ACFs) per animal was increased by continuous CS exposure regardless of MeIQx feeding, the differences between groups 4 and 5 (P<0.05), and between groups 6 and 7 (P<0.05) being significant. Immunoblot analysis confirmed that the hepatic CYP1A2 level in group 6 was remarkably increased as compared to that in group 7. In addition, liver S9 from rats in group 6 consistently increased the mutagenic activities of six HCAs including MeIQx as compared to those in group 7. Thus, our results clearly indicate that CS enhances hepatocarcinogenesis when given in the initiation phase via increasing intensity of metabolic activation for MeIQx and possibly colon carcinogenesis when given in the post‐initiation phase in rats induced by MeIQx.

Lack of significant inhibitory effects of a plant lignan tracheloside on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female Sprague–Dawley rats

Tracheloside, one of the plant lignans which can be extracted from the debris after safflower oil is produced from the seeds of Carthamus tinctorious, is an analogue of another plant lignan, arctiin, the side-chain C-2 of the five-membered ring being changed from a hydrogen to a hydroxyl group. We have already demonstrated that arctiin has chemopreventive effect on mammary carcinogenesis. Therefore, chemopreventive effects of tracheloside on the initiation or post-initiation period of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female rats were examined. For initiation, female Sprague-Dawley (SD) rats at the 6 weeks of age were given intragastric administrations of 100 mg/kg body weight of PhIP once a week for 8 weeks. The animals were treated with 0.2 or 0.02% tracheloside during or after this carcinogen exposure. Control rats were fed basal diet with PhIP initiation or 0.2% tracheloside or basal diet alone without initiation throughout the experimental period. All surviving animals were necropsied at the week 52 of administration. There were no clear treatment-related changes with statistical significance in all parameters for mammary carcinomas measured in this experiment. These results indicate that tracheloside may not exert significant effects on PhIP-induced mammary carcinogenesis at least under the present experiment condition.

Enhancing effects of simultaneous treatment with sodium nitrite on 2-amino-3-methylimidazo[4,5-f]quinoline-induced rat liver, colon and zymbal's gland carcinogenesis after initiation with diethylnitrosamine and 1,2-dimethylhydrazine

Combined effects of sodium nitrite (NaNO2) and 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ) on liver, colon and Zymbals gland carcinogenesis were assessed using a rat two‐stage carcinogenesis model, with a focus on involvement of oxidative stress. Male 6‐week‐old F344 rats were given a single intraperitoneal injection of 200 mg/kg of diethylnitrosamine and 4 subcutaneous injections of 40 mg/kg of 1,2‐dimethylhydrazine for initiation. Then, they were administered 0 or 300 ppm IQ in the diet or 0, 0.1 or 0.2% NaNO2 in their drinking water for 27 weeks. The treatment with NaNO2 + IQ significantly enhanced colon and Zymbals gland carcinogenesis and tended to enhance hepatocarcinogenesis. The incidence of lung tumors in the IQ‐treated groups was significantly increased as compared with the initiation alone group. In a second experiment, male rats were given IQ or NaNO2 under the same conditions as before for 1 week, and at sacrifice, their liver and colon tissue or mucosa were collected for analysis of 8‐hydroxydeoxyguanosine (8‐OHdG), thiobarbituric acid reactive substances (TBARS), acrolein‐modified protein and the bromodeoxyuridine‐labeling index (BrdU‐LI) (in the colon). In the colon, 8‐OHdG, acrolein‐modified protein levels and BrdU‐LI were significantly increased by the combined treatment. These results indicate that the treatment with NaNO2 enhances IQ‐induced colon and Zymbals gland carcinogenesis in rats and that oxidative DNA damage and lipid peroxidation may partly be involved, especially in the colon. In addition, this experiment showed that IQ can act as a potent lung carcinogen in rats.

Synergistic interaction between excess caffeine and deficient iodine on the promotion of thyroid carcinogenesis in rats pretreated with N-bis(2-hydroxypropyl) nitrosamine

The combined effects of caffeine (1,3,7‐trimethylxanthine) with iodine deficiency (ID) were examined in a rat two‐stage thyroid carcinogenesis model using N‐bis(2‐hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 6 groups each consisting of 10 animals, and received a single s.c. injection of 2800 mg/ kg DHPN. From 1 week after the DHPN initiation, the rats were respectively fed a basal diet in which the protein was exchanged for 20% gluten, containing 1500 ppm caffeine+ID, 300 ppm caffeine+ID, 60 ppm caffeine+ID, 1500 ppm caffeine or ID or a basal diet alone for 12 weeks. Relative thyroid weights were significantly (P<0.05) increased due to the development of prolifera‐tive lesions induced by the ID diet as compared to the DHPN‐alone group value, which was enhanced by caffeine, albeit without statistical significance. Relative pituitary weights were significantly (P<0.05) increased with 300 or 1500 ppm caffeine+ID as compared to the DHPN‐alone group value. Serum thyroid stimulating hormone (TSH) levels were slightly increased by ID, an effect which was further enhanced by 300 or 1500 ppm caffeine. Serum thyroxine (T4) levels were slightly increased by caffeine or ID alone, but decreased by caffeine with ID. Histopathologically, thyroid follicular carcinomas were found only in the 1500 ppm caffeine+ID group, although thyroid follicular adenomas were detected in all the ID‐treated groups. The multiplicity of focal thyroid follicular hyperplasias was significantly (P<0.05) increased by 1500 ppm caffeine. These results indicate that caffeine may synergistically promote thyroid carcinogenesis with ID partially through a pituitary‐dependent pathway in rats, implying the possible implication of routine caffeine intake in the promotion of thyroid carcinogenesis. (Cancer Sci 2003; 94: 334–337)

A subchronic toxicity study of shea nut color in Wistar rats

Shea nut color, obtained from nuts of the shea tree (Butyrospermum parkii), is used as a food-coloring agent. Flavonoid pigments are considered to be the responsible constituents. As there have been no reports of toxicological evaluation, a 13-week subchronic toxicity study was performed in Wistar Hannover rats at dose levels of 0 (control), 0.07, 0.31, 1.25 and 5% in powdered basal diet. The average of daily shea nut color intake was 51.3, 226.1, 986.8 and 3775.5 mg/kg/day for males and 56.4, 272.9, 1166.7 and 4387.7 mg/kg/day for females, respectively. During the administration period, daily observation of clinical signs and weekly measurement of body weights and food consumption were performed. After the end of the treatment, hematology, serum biochemistry, organ weight and histopathological examinations were conducted. No significant toxicological changes were observed in any parameters in this study. Hence, the no adverse effect dose of shea nut color was estimated to be greater than 5.0% for both sexes (3775.5 mg/kg/day for males and 4387.7 mg/kg/day for females).