Yuichi Kuroiwa

In vivo mutagenicity and initiation following oxidative DNA lesion in the kidneys of rats given potassium bromate

To clarify the role of 8‐OHdG formation as a starting point for carcinogenesis, we examined the dose‐dependence and time‐course of changes of OGG1 mRNA expression, 8‐OHdG levels and in vivo mutations in the kidneys of gpt delta rats given KBrO3 in their drinking water for 13 weeks. There were no remarkable changes in OGG1 mRNA in spite of some increments being statistically significant. Increases of 8‐OHdG occurred after 1 week at 500 p.p.m. and after 13 weeks at 250 p.p.m. Elevation of Spi− mutant frequency, suggestive of deletion mutations, occurred after 9 weeks at 500 p.p.m. In a two‐stage experiment, F344 rats were given KBrO3 for 13 weeks then, after a 2‐week recovery, treated with 1% NTA in the diet for 39 weeks. The incidence and multiplicity of renal preneoplastic lesions in rats given KBrO3 at 500 p.p.m. followed by NTA treatment were significantly higher than in rats treated with NTA alone. Results suggest that a certain period of time might be required for 8‐OHdG to cause permanent mutations. The two‐step experiment shows that cells exposed to the alteration of the intranuclear status by oxidative stress including 8‐OHdG formation might be able to form tumors with appropriate promotion. (Cancer Sci 2006; 97: 829–835)

A Possible Role of Nrf2 in Prevention of Renal Oxidative Damage by Ferric Nitrilotriacetate

To ascertain the possible roles of nuclear erythroid 2 p45-related factor 2 (Nrf2), a key transcription factor of phase 2 drug-metabolizing enzymes, in renal cellular defense against oxidative stress, wild-type and Nrf2-knockout (–/–) mice were treated with ferric nitrilotriacetate (Fe-NTA) at doses of 3 or 6 mg iron/kg body weight. After Fe-NTA treatment, Nrf2 (–/–) mice consistently showed lower levels of glutathione (GSH) in the kidney at the low dose and the liver at the high dose than the wild-type mice. Gamma-glutamylcysteine ligase (GCL) activity in the kidney and liver of Nrf2 (–/–) mice was also consistently lower than in wild-type mice after the Fe-NTA treatment. Histopathological examination revealed that nephrotoxicity of Fe-NTA, reflected in necrosis of renal tubule epithelial cells following nuclear damage, was more severe in the Nrf2 (–/–) mice than in their wild-type counterparts. Overall, the data suggest that Nrf2 (–/–) mice are unable to compensate for depletion of renal GSH because of oxidative stress, being more susceptible to Fe-NTA-induced nephrotoxicity. In conclusion, the present study showed that Nrf2 might play an important role in protecting cells from oxidative stress in the kidney through its regulation of antioxidant enzymes.

Combined treatment with green tea catechins and sodium nitrite selectively promotes rat forestomach carcinogenesis after initiation with N‐methyl‐N′‐ nitro‐N‐nitrosoguanidine

Combined treatment with several phenolic antioxidants and sodium nitrite (NaNO2) has already shown to enhance rat forestomach carcinogenesis. In the present experiment, effects of green tea catechins (GTC) alone or in combination with NaNO2 on gastric carcinogenesis were investigated in a rat two‐stage carcinogenesis model. Groups of eight, 6‐week‐old F344 male rats were given 0.01%N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) in their drinking water and 5% NaCl in the diet for 10 weeks for glandular stomach initiation and a single intragastric administration of 100 mg/kg/bodyweight of MNNG at week 9 for forestomach initiation. From week 11, they received either drinking water containing 0.2% NaNO2 and a diet supplemented with 1% GTC in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, incidences and multiplicities of neoplastic lesions were clearly increased by the combined treatment, in spite of GTC alone suppressing the occurrence of papillomas. In a short‐term experiment with similar protocol without MNNG pretreatment, a significant increase of 8‐hydroxydeoxyguanosine (8‐OHdG) levels in forestomach DNA occurred 24 h after the combined treatment, concomitant with erosion and inflammatory cell infiltration. In an in vitro study, electron spin resonance demonstrated hydroxyl radical formation after incubation of epigallocatechin gallate or epicatechin gallate with the NO generator, NOC‐7. Thus, GTC alone showed a weak chemopreventive effect on forestomach carcinogenesis, but in the presence of NaNO2 it exerted a promotive effect which might involve hydroxyl‐radical‐associated oxidative DNA damage. However, no influence was exerted in the glandular stomach. (Cancer Sci 2007; 98: 949–957)

Transition of historical control data for high incidence tumors in f344 rats.

Historical control data of tumor incidence were collected from the control groups (215 animals of each sex) in four recent carcinogenicity studies that were started between 2005 to 2009 (terminally sacrificed between 2007 and 2011) at BoZo Research Center Inc. (Gotemba, Shizuoka, Japan) using Fischer 344 rats (F344/DuCrlCrlj). These data were compared to the previous historical control data (from 1990 to 2004, previously reported) in the same facility. In the results, the incidence of C-cell adenoma in the thyroid tended to increase in both sexes in recent years (30.8% for males and 24.4% for females in 2005-2009) as compared with the previous data (17.4% and 20.1% for males and 11.5% and 11.8% for females in 1990–1999 and 2000–2004, respectively). In addition, the incidences of pancreatic islet cell adenoma in males and uterine adenocarcinoma tended to increase from around 2000 and remained high in recent years (incidences of islet cell adenoma in males of 10.5%, 17.1% and 20.5% in 1990–1999, 2000–2004 and 2005–2009; incidences of uterine adenocarcinoma of 3.3%, 12.0% and 13.5% in 1990–1999, 2000–2004 and 2005–2009, respectively). There was no apparent difference in the incidence of other tumors.

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)

Oxidative DNA damage and in vivo mutagenicity caused by reactive oxygen species generated in the livers of p53‐proficient or ‐deficient gpt delta mice treated with non‐genotoxic hepatocarcinogens

Oxidative stress is thought to participate in chemical carcinogenesis and may trigger gene mutations. To accurately assess the carcinogenesis risk posed to humans by chemical exposure, it is important to understand the pathways by which reactive oxygen species (ROS) are generated and the effects of the resulting oxidative stress. In the present study, p53‐proficient and ‐deficient gpt delta mice were given pentachlorophenol (PCP), phenobarbital (PhB) or piperonyl butoxide (PBO), which are classified as non‐genotoxic hepatocarcinogens in rodents, at the respective carcinogenic doses for 13 weeks. Exposure to PCP or PBO, but not PhB, invoked significant increases in liver DNA 8‐hydroxydeoxyguanosine (8‐OHdG) levels. Treatment with PCP significantly increased mRNA levels of the gene encoding NAD(P):quinone oxidoreductase 1 (NQO1) in the liver, suggesting that redox cycling of the PCP metabolite tetrachlorohydroquinone gave rise to ROS. Exposure to PhB or PBO significantly elevated CYP 2B10 mRNA levels while NQO1 levels were also significantly increased in PBO‐treated mice. Therefore, in addition to involvement of the CYP catalytic pathway in the ROS‐generated system of PBO, catechol derivatives produced from the opening of the PBO functional group methylenedioxy ring probably resulted in ROS generation. However, PCP, PBO and PhB failed to increase gpt and red/gam gene mutations in the liver independently of p53. Overall, the action of oxidative stress by ROS derived from the metabolism of these carcinogens might be limited to cancer‐promoting activity, which supports the previous classification of these carcinogens as non‐genotoxic. Copyright

Antigenotoxic effects of p53 on spontaneous and ultraviolet light B–induced deletions in the epidermis of gpt delta transgenic mice

Tumor development in the skin may be a multistep process where multiple genetic alterations occur successively. The p53 gene is involved in genome stability and thus is referred to as “the guardian of the genome.” To better understand the antigenotoxic effects of p53 in ultraviolet light B (UVB)‐induced mutagenesis, mutations were measured in the epidermis of UVB‐irradiated p53+/+ and p53−/− gpt delta mice. In the mouse model, point mutations and deletions are separately identified by the gpt and Spi− assays, respectively. The mice were exposed to UVB at single doses of 0.5, 1.0, or 2.0 kJ/m2. The mutant frequencies (MFs) were determined 4 weeks after the irradiation. All doses of UVB irradiation enhanced gpt MFs by about 10 times than that of unirradiated mice. There were no significant differences in gpt MFs and the mutation spectra between p53+/+ and p53−/− mice. The predominant mutations induced by UVB irradiation were G:C to A:T transitions at dipyrimidines. In contrast, in unirradiated p53−/− mice, the frequencies of Spi− large deletions of more than 1 kb and complex‐type deletions with rearrangements were significantly higher than those of the Spi− large deletions in p53+/+ counterparts. The specific Spi− mutation frequency of more than 1 kb deletions and complex types increased in a dose‐dependent manner in the p53+/+ mice. However, no increase of such large deletions was observed in irradiated p53−/− mice. These results suggest that the antigenotoxic effects of p53 may be specific to deletions and complex‐type mutations induced by double‐strand breaks in DNA. Environ. Mol. Mutagen., 2011.

Lack of nrf2 results in progression of proliferative lesions to neoplasms induced by long-term exposure to non-genotoxic hepatocarcinogens involving oxidative stress

To explore the role of oxidative stress in chemical carcinogenesis driven by non-genotoxic mechanisms, nrf2-deficient (nrf2(-/-)) and nrf2-wild-type (nrf2(+/+)) mice were exposed to pentachlorophenol (PCP) at concentrations of 600 or 1200 ppm for 60 weeks, or piperonyl butoxide (PBO) at concentrations of 3000 or 6000 ppm in the diet for 52 weeks, respectively. Additional studies were performed to examine 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA and hepatotoxicological parameters in serum following 8 weeks of exposure of each group to PBO at the same doses as in the long-term study. Exposure to 600 ppm PCP caused cholangiofibrosis (CF) only in nrf2(-/-) mice, while 1200 ppm PCP induced CF in both genotypes. Moreover, cholangiocarcinomas were found with significant incidence only in nrf2(-/-) mice treated with 1200 ppm PCP. Short-term exposure to 6000 ppm PBO caused significant elevation of 8-OHdG levels in both genotypes, while exposure to 3000 ppm caused a significant increase in 8-OHdG only in nrf2(-/-) mice. There were no inter-genotype changes in the incidences of regenerative hepatocellular hyperplasia (RHH) following long-term exposure to PBO. However, the incidence and multiplicity of hepatocellular adenomas, especially those observed in RHH, were much higher in nrf2-/- mice treated with 6000 ppm PBO than in nrf2+/+ mice treated with 6000 ppm PBO. Therefore, oxidative stress generated through PCP or PBO metabolism may promote the proliferation and progression of preneoplastic lesions to neoplasms.

Lung as a new target in rats of 2‐amino‐3‐methylimidazo[4,5‐f]quinoline carcinogenesis: Results of a two‐stage model initiated with N‐bis(2‐hydroxypropyl)nitrosamine

The effects of IQ on the promotion stage of DHPN‐induced lung carcinogenesis and contributions of oxidative stress were investigated in rats. Groups of 20 male 6‐week‐old F344 rats were given 0.1% DHPN in their drinking water for 2 weeks for initiation. From the age of 9 weeks, they were treated with 0, 150 and 300 p.p.m. of IQ in the diet for 27 weeks. Control rats were similarly fed 300 p.p.m. IQ or basal diet alone without the preceding initiation. IQ clearly (P < 0.01) enhanced the multiplicity of lung tumors in a dose‐dependent manner (DHPN alone, 3.63 ± 1.80; DHPN +150 p.p.m. IQ, 11.50 ± 5.04; DHPN +300 p.p.m. IQ, 18.83 ± 4.58 [no./rat]). In addition, the incidence of lung tumors in the 300 p.p.m. IQ alone group (25%) was significantly (P < 0.05) higher than that in the non‐treatment group (0%). In a second experiment, male rats were given IQ at doses of 0 and 300 p.p.m. in the diet for one week in order to analyze 8‐OHdG formation, levels of TBARS and BrdU‐LI in the lungs. There were no changes in 8‐OHdG or TBARS levels, but significant elevation of BrdU‐LI occurred in the IQ administration group. The overall data clearly indicate that IQ is a potent lung carcinogen in rats, in which oxidative stress may not be involved in lung carcinogenesis. (Cancer Sci 2006; 97: 368 –373)