Fumio Furukawa

NaCl induced ornithine decarboxylase and DNA synthesis in rat stomach mucosa

Inductions of 200-fold increase in ornithine decarboxylase activity within 6 hours and 9-fold increase in DNA synthesis within 3 hours in rat stomach mucosa were observed after a single oral dose of saturated NaCl solution. NaCl caused dose-dependent induction of ornithine decarboxylase activity at doses of 0.25 to 1. 5g /kg body weight, and of DNA synthesis at doses of 0. 5g to 1. 5g /kg body weight. Administration of 1,3-diaminopropane one hour before NaCl inhibited the induction of ornithine decarboxylase activity in the stomach mucosa 3 and 6 hours after NaCl administration, but a single dose of 1,3-diaminopropane itself induced ornithine decarboxylase after 16 hours.

Lack of a Dose-response Relationship for Carcinogenicity in the Rat Liver with Low Doses of 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline or N-Nitrosodiethylamine

For a long period, it has been generally considered that carcinogens, particularly genotoxic ones, have no threshold in exerting their potential for cancer induction. However, the non‐threshold theory can be challenged with regard to assessment of cancer risk to humans. Here we show that a food‐derived, genotoxic hepatocarcinogen, 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline, forms DNA adducts at low doses, but does not induce glutathione S‐transferase placental form (GST‐P)‐positive foci (considered to be preneoplastic lesions) or 8‐hydroxy‐2′‐deoxyguanosine in rat liver. Moreover a N‐nitroso compound, N‐nitrosodiethylamine, at low doses was also found not to induce GST‐P‐positive foci in rat liver. These results imply that there is a no‐observed effect level for hepatocarcinogenesis by these genotoxic carcinogens.

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.

Oral toxicity of a tocotrienol preparation in rats

Tocotrienols are added as antioxidants to food. As there have been no reports of toxicological evaluation, a 13-week oral toxicity study was performed in Fischer 344 rats of both sexes at dose levels of 0 (group 1), 0.19 (group 2), 0.75 (group 3) and 3% (group 4) of a preparation in powdered diet. Suppression of body weight gain was observed in group 4 males. On hematological examination, significant decrease in mean corpuscular volume (MCV) was observed in all treated males. Platelets were significantly reduced in group 3 and 4 males. Hemoglobin concentration, MCV, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were significantly decreased in group 3 and 4 females and hematocrit in group 4 females. On serum biochemical examination, increase in the albumin/globulin ratio (A/G) and alkaline phosphatase in all treated males, elevated alanine transaminase in group 4 of both sexes and increases in asparagine transaminase and gamma-glutamyl transaminase in group 4 females were observed. With regard to relative organ weights, liver weights in group 4 of both sexes and adrenal weights in all treated males demonstrated an increase, and ovary and uterus weights in group 4 females were reduced. Histopathologically, slight hepatocellular hypertrophy in group 3 and 4 males, and reduction of cytoplasmic vacuolation in the adrenal cortical region in group 4 males were observed. Because of pathological changes in male liver and hematological changes in females, the no-observed-adverse-effect level (NOAEL) was concluded to be 0.19% in the diet (120 mg/kg body weight/day for male rats and 130 mg/kg body weight/day for female rats). As a decrease in MCV, an increase in the A/G, elevation of alkaline phosphatase and increase in adrenal weight were observed in all treated males, a no-observed-effect level (NOEL) could not be determined in this examination.

Effects of cacao liquor proanthocyanidins on PhIP-induced mutagenesis in vitro, and in vivo mammary and pancreatic tumorigenesis in female Sprague-Dawley rats.

The effects of cacao liquor proanthocyanidins (CLPr) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis in vitro and on in vivo carcinogenesis in female Sprague-Dawley (SD) rats were investigated. In the Ames assay using Salmonella typhimurium TA98, CLPr showed strong antimutagenic effects against PhIP when assayed in the presence of S-9 mixture. For determination of the influence on initiation and subsequent development of lesions, CLPr (0.025% or 0.25%) were fed during the period of PhIP application (100 mg/kg given to rats via gastric tubes eight times over 4 weeks), or thereafter until the termination at 48 weeks. CLPr treatments did not affect body or organ weights. The incidences, multiplicities and volumes of mammary tumors in the 0.25% CLPr (post-initiation) group showed a tendency to decrease as compared to PhIP alone group values, although without statistical significance. The incidences of preneoplastic eosinophilic foci in the exocrine pancreas were significantly (P<0.05) decreased in a dose-dependent manner when CLPr were given during the initiation period. These results indicate that CLPr inhibit in vitro mutagenicity of PhIP, as well as rat pancreatic carcinogenesis in the initiation stage, but not mammary carcinogenesis induced by PhIP.

Prevention by 2-mercaptoethane sulfonate and N-acetylcysteine of renal oxidative damage in rats treated with ferric nitrilotriacetate

Ferric nitrilotriacetate (Fe‐NTA) is a renal toxicant and carcinogen in rats and mice. We found that its administration results in formation of 4‐hydroxy‐2‐nonenal (HNE) in the renal proximal tubule cells of rats, and 8‐hydroxydeoxyguanosine (8‐OHdG) adducts in their DNA, suggesting a role for oxidative stress. Since 2‐mercaptoethane sulfonate (MESNA) and N‐acetylcysteine (NAC), administered orally, have been shown to increase the kidney levels of free thiol groups, their influence on the renal toxicity and carcinogenicity induced by Fe‐NTA was examined in the present study. Male Wistar rats were intraperitoneally injected with Fe‐NTA (12 mg Fe/kg), and MESNA (100 mg/kg) or NAC (200 mg/kg) was given orally 1 h before and 1 h after this treatment. The animals were killed for tissue analyses 3 h after the Fe‐NTA exposure. In accord with our previous reports, HNE‐modified protein was detected in the proximal tubules of Fe‐NTA‐treated rats by means of immunohistochemistry. Likewise, levels of 8‐OHdG in the renal nuclear DNA, lipid peroxides as thiobarbituric acid‐reactive substances in the kidneys, and blood urea nitrogen and creatinine in the serum were significantly increased by the Fe‐NTA treatment. All of these changes were completely inhibited by oral administration of MESNA or NAC. These results suggest that both of these compounds can prevent the oxidative stress induced by Fe‐NTA.

Involvement of macrophage inflammatory protein 1α (MIP1α) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

Inhibitory Effects of Antioxidants on N‐Bis(2‐hydroxypropyl)nitrosamine‐induced Lung Carcinogenesis in Rats

Potential second‐stage modifying effects of 8 antioxidants on lung tumorigenesis initiated by N‐bis(2‐hydroxypropyl)nitrosamine (DHPN) were examined in male F344 rats. After an initial 2‐week treatment with DHPN (0.1% in drinking water), rats were administered one of the antioxidants supplemented in the diet for 30 weeks. Although the incidences of lung adenomas were not affected, those of carcinomas were lowered by 2% butylated hydroxyanisole (BHA, 2 rats/20 rats), 1% butylated hydroxytoluene (BHT, 1/20), 0.8% ethoxyquin (EQ, 3/20) and 1%α‐tocopherol (α‐TP, 2/20) treatments as compared to the control level (9/20), while 5% sodium l‐ascorbate (SA), 0.8% catechol (CC), 0.8% resorcinol (RN), and 0.8% hydroquinone (HQ) did not exert any significant effect on incidence. Quantitative analysis of adenomas and carcinomas (numbers and areas of lesions per unit area of lung section) revealed obvious inhibitory effects of SA, CC, and RN as well as BHA, BHT, EQ, and α‐TP. Among the antioxidants, BHT exerted the strongest inhibitory activity. In contrast, DHPN‐induced thyroid tumorigenesis was significantly enhanced by BHT (14/20) and EQ (20/20) treatments (control=5/20). Thus the antioxidants showed opposite effects on lung and thyroid carcinogenesis in the rat.

Lack of initiation activity in rat liver of low doses of 2-amino-3,8- dimethylimidazo(4,5-f )quinoxaline

It has been generally accepted that genotoxic carcinogens have no threshold in exerting their potential for cancer induction. However, the non-threshold theory can be challenged for cancer risk assessment in humans. Here we examined low dose carcinogenicity of a food-derived, genotoxic hepatocarcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), using an in vivo medium-term bioassay to detect initiating activity for rat hepatocarcinogenesis. With MeIQx initiation at various doses followed by administration of phenobarbital, a well known hepatopromoter, no induction of glutathione S-transferase placental form-positive foci, assessed as preneoplastic lesions, was noted at doses of 0.001-1 ppm. The results imply a no-observed effect level for hepatocarcinogenicity with this genotoxic agent.

Potent Chemopreventive Agents Against Pancreatic Cancer

Development of pancreatic cancers is clinically so silent in general that at the time of diagnosis, the vast majority of cases are incurable with a very poor prognosis. Therefore, effective preventive approaches against this aggressive disease are urgently required. Experimentally, carcinogenesis process is assumed to consist of at least two stages named initiation and promotion. Using a two-stage model of hamster pancreatic carcinogenesis, we have reported stage-specific inhibitory effects by a number of potent cancer chemopreventive agents. Among them, phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, remarkably blocked the initiation phase of pancreatic as well as lung carcinogenesis in hamsters initiated with N-nitrosobis(2-oxopropyl)amine (BOP). However, PEITC failed to affect both pancreatic and lung carcinogenesis when given during the post-initiation (promotion) phase of carcinogenesis. In contrast, our recent study clearly demonstrated that a cyclooxygenase (COX)-2 inhibitor substantially protects against BOP-induced pancreatic tumors in hamsters in line with decrease in cell proliferative activity of pancreatic ducts when given in the post-initiation phase. Interestingly, trypsin inhibitors inhibited both initiation and post-initiation phases of BOP-induced pancreatic carcinogenesis although they are known to induce hyperplastic acinar lesions in the rat pancreas. Taken together with these data, our review is aimed at looking over mechanistic insights into potent chemopreventive agents against pancreatic cancer.