Toshiya Okamura

Possible participation of oxidative stress in causation of cell proliferation and in vivo mutagenicity in kidneys of gpt delta rats treated with potassium bromate

Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO(3))-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of alpha-tocopherol (alpha-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO(3) were examined. Five male and female gpt delta rats in each group were given KBrO(3) at a concentration of 500ppm in the drinking water for 9 weeks, with 1% of alpha-TP or SAA administered in the diet from 1 week prior to the KBrO(3) treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO(3) were significantly inhibited by SAA, but not alpha-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or alpha-TP. Immunohistochemical and Western blot analyses for alpha(2u)-globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO(3) in both sexes is limited.

Background Data for General Toxicology Parameters in RccHanTM:WIST Rats at 8, 10, 19 and 32 Weeks of Age

Recently, RccHanTM:WIST (Wistar Hannover) rats were introduced to toxicity studies in Japan. The present study was performed to obtain control data for general toxicological parameters as an aid for interpretation of results in toxicity studies using this strain of rats. Four test groups comprising of 25 male and 25 female RccHanTM:WIST rats were housed for 2, 4, 13 or 26 weeks from 6 weeks of age and observed and examined for clinical observation, body weight, food consumption, urinalysis, hematology, blood chemistry, organ weight, necropsy and/or histopathology. Ophthalmological examination was not conducted in this study, and the data in this report were obtained from an ongoing 104-week background study in RccHanTM:WIST rats. These data were compared with the historical control data of CD(SD) (Sprague-Dawley) and/or F344 (Fischer) rats. The body weights of RccHanTM:WIST rats were lower than those of CD(SD) rats and higher than those of F344 rats. The ophthalmological examination revealed a greater incidence of focal corneal opacity. Histopathology revealed focal mineralization of the cornea and Berlin blue-positive pigmentation in the epididymal interstitium as well as hepatocytes. Other than the above, some minor differences were found in urinalysis, hematology, blood chemistry and organ weights as compared with CD(SD) rats.

Oxidative DNA damage and reporter gene mutation in the livers of gpt delta rats given non-genotoxic hepatocarcinogens with cytochrome P450-inducible potency

Previous reports have proposed that reactive oxygen species resulting from induction of cytochrome P450 (CYP) isozymes might be involved in the modes of action of hepatocarcinogens with CYP‐inducible potency. In the present study, we investigated 8‐hydroxydeoxyguanosine (8‐OHdG) levels, in vivo mutagenicity and glutathione S‐transferase placental form (GST‐P)‐positive foci in the livers of gpt delta rats treated with piperonyl butoxide (PBO) or phenobarbital (PhB) for 4 and 13 weeks. Significant elevations in Cyp 1A1 and Cyp 1A2 mRNA levels after PBO treatment, and in Cyp 2B1 mRNA levels after PBO or PhB treatment, appeared together with remarkable hepatomegaly through the experimental period. Time‐dependent and statistically significant increases in 8‐OHdG levels were observed in the PBO treatment group along with significant increases in proliferating cell nuclear antigen (PCNA)‐positive hepatocytes at 4 weeks, while no increase in 8‐OHdG levels was found in PhB‐treated rats. No changes in mutant frequencies of gpt and red/gam (Spi‐) genes in liver DNA from PBO‐ or PhB‐treated rats were observed at 4 or 13 weeks. A 13‐week exposure to either PBO or PhB did not affect the number and area of GST‐P‐positive hepatocytes. CYP 1A1 and 1A2 induction may be responsible for elevated levels of 8‐OHdG in PBO‐treated rats. However, neither GC:TA transversions nor deletion mutations, typically regarded as 8‐OHdG‐related mutations, were observed in any of the treated rats. We conclude that reactive oxygen species, possibly produced through CYP catalytic pathways, likely induced genomic DNA damage but did not give rise to permanent gene mutation. (Cancer Sci 2010; 101: 2525–2530)

Combined Ascorbic Acid and Sodium Nitrite Treatment Induces Oxidative DNA Damage-Associated Mutagenicity In Vitro, but Lacks Initiation Activity in Rat Forestomach Epithelium

Combination treatment with sodium nitrite (NaNO(2)) and ascorbic acid (AsA) is well known to promote forestomach carcinogenesis in rats and weakly enhance esophageal carcinogenesis under acid reflux conditions. Nitric oxide generation and oxidative DNA damage are considered to be related to the enhancement of carcinogenesis. The purpose of the present study was to investigate whether oxidative DNA damage-associated genotoxicity and tumor initiating potential are involved in the carcinogenesis. In the bacterial reverse mutation assay using Escherichia coli deficient in the mutM gene encoding 8-hydroxydeoxyguanosine (8-OHdG) DNA glycosylase, the combination with NaNO(2) and AsA increased the mutation frequency dramatically, slight increase being evident in the parental strain. In vivo, a significant increase in 8-OHdG levels in the rat forestomach epithelium occurred at 24 h after combined treatment. Six-week-old F344 male rats were given drinking water containing 0.2% NaNO(2) and a diet supplemented with 1% AsA in combination, or the chemicals individually or basal diet alone for 12 weeks. After an interval of 2 weeks, they received 1% butylated hydroxyanisole in the diet for promotion until the end of weeks 52 and 78. Although one squamous cell carcinoma was observed in the combined group, there was no significant variation in tumor development among the groups. The study indicated that the combination of NaNO(2) with AsA induces genotoxicity due to oxidative DNA damage in vitro, and elevates 8-OHdG levels in the forestomach epithelium, but lacks initiating activity in the rat two-stage carcinogenesis model.

Enhancement of esophageal carcinogenesis in acid reflux model rats treated with ascorbic acid and sodium nitrite in combination with or without initiation

Combined treatment with sodium nitrite (NaNO2) and ascorbic acid (AsA) has already been shown to promote rat forestomach carcinogenesis, possibly due to nitric oxide generation under acidic conditions. We hypothesized that a similar effect might occur in the esophagus when the luminal pH is decreased by acid reflux. To clarify this possibility, reflux esophagitis model rats (F344 male) were coadministered 0.2% NaNO2 in the drinking water and 1% AsA in the diet. After 32 weeks of the combined treatment, a significant increase in the incidence of epithelial hyperplasias of the lower‐middle and lowest parts of the esophagus were observed compared with the basal‐diet group, along with exacerbation of dysplasia and extension of the lesions. Additionally, one squamous cell papilloma was found only in the combined‐treatment group. Subsequently, we confirmed the enhancing effects of NaNO2 and AsA cotreatment in the rat N‐bis(2‐hydroxypropyl)nitrosamine‐initiated esophageal tumorigenesis model. The incidence of hyperplasia was enhanced in all segments, along with the incidence and multiplicity of squamous cell papillomas in the lowest segment of the esophagus. Thus, the data demonstrate that combined treatment with NaNO2 and AsA exerts promoting effects on rat esophageal carcinogenesis under acid reflux conditions, as in the forestomach. These findings suggest that the risk of excessive intake of a combination of nitrite and antioxidants for esophageal carcinogenesis is appreciable, particularly in patients with reflux esophagitis. (Cancer Sci 2008; 99: 7–13)

Chemopreventive Effects of 4-Methylthio-3-butenyl Isothiocyanate (Raphasatin) but Not Curcumin against Pancreatic Carcinogenesis in Hamsters

The modifying effects of 4-methylthio-3-butenyl isothiocyanate (MTBITC) and curcumin were investigated in N-nitrosobis(2-oxopropyl)amine (BOP)-initiated hamsters. Male 6-week-old Syrian hamsters were subcutaneously injected with 10 mg/kg body weight (b.w.) of BOP four times a week, and fed a diet supplemented with 80 mg/kg diet of MTBITC, equivalent to 4.6 mg/kg b.w./day for the initiation stage or 3.8 mg/kg b.w./day for the postinitiation stage administration, respectively, or 2000 mg/kg diet of curcumin, equivalent to 118.8 mg/kg b.w./day for the initiation stage or 100.8 mg/kg b.w./day for the postinitiation stage administration, respectively. The incidence of combined pancreatic lesions, including atypical hyperplasias and adenocarcinomas, was significantly decreased to 55% (P < 0.05) by the 80 mg/kg diet MTBITC given during the initiation stage as compared to the BOP alone group (85%) but not by the curcumin administration at 16 weeks after the BOP-treatment. In the second study, the multiplicity of combined pancreatic lesions was also significantly decreased to 0.50 ± 0.51 (P < 0.05) by 700 mg/kg diet MTBITC given in the initiation stage (equivalent to 59.0 mg/kg b.w./day) as compared to the BOP alone group (1.10 ± 1.02). Our results indicate that the naturally occurring isothiocyanate MTBITC may exert preventive effects against BOP-initiation of hamster pancreatic carcinogenesis.

Role of Nrf2 and Oxidative stress on Fenofibrate-Induced Hepatocarcinogenesis in Rats

Regional specific relationships between oxidative stress and the development of glutathione S-transferase placental form (GST-P)-positive or GST-P-negative lesions in rats, induced by fenofibrate (FF), a peroxisome proliferator, were examined using a two-stage hepatocarcinogenesis model in F344 rats. Animals were initiated with a single ip injection of 200 mg/kg N-diethylnitrosamine (DEN) and from 2 weeks later were fed a diet containing 3000 or 0 ppm FF for 28 weeks. Animals were subjected to a two-third partial hepatectomy at week 3 and sacrificed at week 28. The development of hepatocellular proliferative lesions, which were mainly attributed to GST-P-negative lesions, was significantly increased in the FF-treated groups. Immunohistochemically, GST-P-positive lesions were devoid of intracytoplasmic nuclear factor-erythroid 2-related factor 2 (Nrf2) expression, whereas GST-P-negative lesions expressed higher levels of cytoplasmic Nrf2. On the other hand, nuclear accumulation of Nrf2 was observed in some cells of GST-P-positive lesions that were negative for Nrf2 in the cytoplasm and in GST-P-negative lesions of the DEN-FF group that were positive for Nrf2 in the cytoplasm. The mRNA expression levels of Gpx2 or Gsta2, Nrf2-inducible enzymes, were increased in GST-P-positive tumors or GST-P-positive lesions, respectively. These results suggest that the activation of Nrf2, due to nuclear translocation, occurs in the GST-P-positive lesions. In addition, the development of continuous oxidative stress was identified by mRNA expression analyses as well as by measurements of GST activity and 8-hydroxydeoxyguanosine. These results suggest that the relative inhibition of nuclear translocation of Nrf2 in GST-P-negative lesions aggravated the condition of oxidative stress in the liver of rats given FF, resulting in enhanced tumor promotion in FF-induced hepatocarcinogenesis.

Simultaneous induction of non-neoplastic and neoplastic lesions with highly proliferative hepatocytes following dietary exposure of rats to tocotrienol for 2 years.

It was recently shown that 1-year chronic exposure of rats to tocotrienol (TT) induced highly proliferative liver lesions, nodular hepatocellular hyperplasia (NHH), and independently increased the number of glutathione S-transferase placental form (GST-P)-positive hepatocytes. Focusing attention on the pathological intrinsic property of NHH, a 104-week carcinogenicity study was performed in male and female Wistar Hannover rats given TT at concentrations of 0, 0.4 or 2% in the diet. The high-dose level was adjusted to 1% in both sexes from week 51 because the survival rate of the high-dose males dropped to 42% by week 50. At necropsy, multiple cyst-like nodules were observed, as in the chronic study, but were further enlarged in size, which consequently formed a protuberant surface with a partly pedunculated shape in the liver at the high dose in both sexes. Unlike the chronic study, NHH was not always accompanied by spongiosis, and instead angiectasis was prominent in some nodules. However, several findings in the affected hepatocytes such as minimal atypia, no GST-P immunoreactivity and heterogeneous proliferation, implied that NHH did not harbor neoplastic characteristics from increased exposure despite sustained high cell proliferation. On the other hand, in the high-dose females, the incidence of hepatocellular adenomas was significantly higher than in the control. There was no TT treatment-related tumor induction in any other organs besides the liver. Thus, the overall data clearly suggested that NHH is successively enlarged by further long-term exposure to TT, but does not become neoplastic. In contrast, TT induces low levels of hepatocellular adenomas in female rats.

Dietary catechol causes increased oxidative DNA damage in the livers of mice treated with acetaminophen.

We have shown that direct reaction of catechol with nitric oxide (NO) results in generation of reactive oxygen and nitrogen species (RNS) through semiquinone radical formation, leading to oxidative DNA damage in rat forestomach. In the present study, we investigated whether dietary catechol systemically exerts the same effects under NO-rich circumstances, when given before and during induction of inflammatory lesions. Male ICR mice were treated with or without 0.8% catechol in the diet for 2 weeks followed by acetaminophen (APAP) administration at a dose of 300mg/kg by single i.p. injection. Along with several indicators of APAP-induced hepatitis, 8-hydroxydeoxyguanosine (8-OHdG) levels and immunohistochemistry for 3-nitrotyrosine (NO(2)Tyr) in the livers were examined at 1.5, 4 and 24h after APAP injection. 8-OHdG was significantly increased at 24h in the co-treatment group, but not with either catechol or APAP alone. Elevation of serum ALT and AST activities, decrease of reduced glutathione levels and histopathological liver changes were observed to the same extents in both APAP-treated groups. In view of the finding of positive hepatocytes for NO(2)Tyr prior to generation of 8-OHdG, the process of oxidative DNA damage might involve RNS formation. Precise quantitative analysis of NO(2)Tyr by means of liquid chromatography with tandem mass spectrometry (LC-MS/MS) in an additional study with the same experimental protocol confirmed increase of RNS due to the reaction of catechol with NO produced after APAP-induced hepatitis. The overall data imply that antioxidants with a catechol structure can cause oxidative DNA damage under inflammatory conditions.

Safety assessment of dietary administered paprika color in combined chronic toxicity and carcinogenicity studies using F344 rats.

Combined chronic toxicity and carcinogenicity studies of paprika color, used as a food additive in various countries, were performed in male and female F344 rats. Dietary concentrations of 0%, 0.62%, 1.25%, 2.5% and 5% were applied in a 52-week toxicity study and 0%, 2.5% and 5% in a 104-week carcinogenicity study. Treatment with paprika color caused a significant increase in incidence of hepatocellular vacuolation in 5% males, but no toxicological effects were found with reference to survival rates, body weights, hematological or serum biochemical parameters and organ weights at any dose level in either sex in the chronic toxicity study. Also, paprika color did not induce specific tumors nor did it exert significant influence on the development of spontaneous tumors in any of the organs examined in the carcinogenicity study. In conclusion, based on slight histopathological changes observed in 5% male livers, the no-observed-effect level (NOEL) was estimated to be 2.5% in the diet (1,253 mg/kg bw/day) and the no-observed-adverse-effect level (NOAEL) was determined to be 5% in the diet (2,388 mg/kg bw/day) for male rats, and for females, the NOEL was concluded to be 5% in the diet (2,826 mg/kg bw/day). Additionally, paprika color was not carcinogenic to male and female F344 rats under the present experimental conditions.