Appearance of sex-determining region Y-box 9 (SOX9)- and glutathione S-transferase placental form (GST-P)-positive hepatocytes as possible carcinogenic events in the early stage of furan-induced hepatocarcinogenesis.

Furan, the basic skeleton of various flavoring agents, induces cholangiocellular tumors with higher incidences in the caudate lobe and hepatocellular tumors without the lobe specificity in rats, but the mechanism is unclear. We investigated the lobe distribution of possible carcinogenic events. Furan caused proliferation/infiltration of oval and inflammatory cells prominently in the caudate lobe as early as 4 weeks and cholangiofibrosis in this lobe at 8 weeks. In vivo mutagenicity assays using DNA extracted from the caudate or left lateral lobe of male gpt delta rats, the reporter gene-transgenic rats, treated with 8 mg/kg furan for 4 or 8 weeks showed negative outcomes. The distribution of glutathione S-transferase placental form (GST-P)-positive or sex-determining region Y-box 9 (SOX9)-positive hepatocytes was examined. Significant increases in the number of GST-P-positive hepatocytes were observed in all lobes of furan-treated rats at 8 weeks. By contrast, SOX9-positive hepatocytes, liver injury-inducible progenitor cells, were also found in all lobes of treated rats, the incidences of which were by far the highest in the caudate lobe. In addition, some of these hepatocytes also co-expressed delta like 1 homolog (DLK1), a hepatoblast marker, particularly in areas with a predominant presence of inflammatory cells. Overall, furan induced liver injury, leading to the appearance of SOX9-positive hepatocytes, some of which were subjected to dedifferentiation in the inflammatory microenvironment of a cholangiocarcinoma-prone lobe. Thus, the appearance of SOX9-positive hepatocytes together with GST-P-positive hepatocytes could be initial events in furan-induced hepatocarcinogenesis via non-genotoxic mechanisms.

Site-specific genotoxicity of rubiadin: localization and histopathological changes in the kidneys of rats.

Rubiadin (Rub) is a genotoxic component of madder color (MC) that is extracted from the root of Rubia tinctorum L. MC induces renal tumors and preneoplastic lesions that are found in the proximal tubule of the outer stripe of the outer medulla (OSOM), suggesting that the renal carcinogenicity of MC is site specific. To clarify the involvement of Rub in renal carcinogenesis of MC, we examined the distribution of Rub in the kidney of male gpt delta rats that were treated with Rub for 28 days. We used desorption electrospray ionization quadrupole time-of-flight mass spectrometry imaging (DESI-Q-TOF-MSI), along with the histopathological analysis, immunohistochemical staining, and reporter gene mutation assays of the kidney. DESI-Q-TOF-MSI revealed that Rub and its metabolites, lucidin and Rub-sulfation, were specifically distributed in the OSOM. Histopathologically, karyomegaly characterized by enlarged nuclear and microvesicular vacuolar degeneration occurred in proximal tubule epithelial cells in the OSOM. The ɤ-H2AX- and p21-positive cells were also found in the OSOM rather than the cortex. Although dose-dependent increases in gpt and Spi- mutant frequencies were observed in both the medulla and cortex, the mutant frequencies in the medulla were significantly higher. The mutation spectra of gpt mutants showed that A:T-T:A transversion was predominant in Rub-induced gene mutations, consistent with those of MC. Overall, the data showed that the distribution of Rub and its metabolites resulted in site-specific histopathological changes, DNA damage, and gene mutations, suggesting that the distribution of genotoxic components and metabolites is responsible for the site-specific renal carcinogenesis of MC.

The role of DNA polymerase ζ in benzo[a]pyrene-induced mutagenesis in the mouse lung.

DNA polymerase zeta (Polζ) is a heterotetramer composed of the catalytic subunit Rev3l, Rev7 and two subunits of Polδ (PolD2/Pol31 and PolD3/Pol32), and this polymerase exerts translesion DNA synthesis (TLS) in yeast. Because Rev3l knockout results in embryonic lethality in mice, the functions of Polζ need further investigation in vivo. Then, we noted the two facts that substitution of leucine 979 of yeast Rev3l with methionine reduces Polζ replication fidelity and that reporter gene transgenic rodents are able to provide the detailed mutation status. Here, we established gpt delta mouse knocked in the constructed gene encoding methionine instead of leucine at residue 2610 of Rev3l (Rev3l L2610M gpt delta mice), to clarify the role of Polζ in TLS of chemical-induced bulky DNA adducts in vivo. Eight-week-old gpt delta mice and Rev3l L2610M gpt delta mice were treated with benzo[a]pyrene (BaP) at 0, 40, 80, or 160 mg/kg via single intraperitoneal injection. At necropsy 31 days after treatment, lungs were collected for reporter gene mutation assays. Although the gpt mutant frequency was significantly increased by BaP in both mouse genotypes, it was three times higher in Rev3l L2610M gpt delta than gpt delta mice after treatment with 160 mg/kg BaP. The frequencies of G:C base substitutions and characteristic complex mutations were significantly increased in Rev3l L2610M gpt delta mice compared with gpt delta mice. The BaP dose-response relationship suggested that Polζ plays a central role in TLS when protective mechanisms against BaP mutagenesis, such as error-free TLS, are saturated. Overall, Polζ may incorporate incorrect nucleotides at the sites opposite to BaP-modified guanines and extend short DNA sequences from the resultant terminal mismatches only when DNA is heavily damaged.

Chromosome aberrations induced by the non-mutagenic carcinogen acetamide involve in rat hepatocarcinogenesis through micronucleus formation in hepatocytes.

Chromosome aberrations (CAs), i.e. changes in chromosome number or structure, are known to cause chromosome rearrangements and subsequently tumorigenesis. However, the involvement of CAs in chemical-induced carcinogenesis is unclear. In the current study, we aimed to clarify the possible involvement of CAs in chemical carcinogenesis using a rat model with the non-mutagenic hepatocarcinogen acetamide. In an in vivo micronucleus (MN) test, acetamide was revealed to induce CAs specifically in rat liver at carcinogenic doses. Acetamide also induced centromere-positive large MN (LMN) in hepatocytes. Immunohistochemical and electron microscopic analyses of the LMN, which can be histopathologically detected as basophilic cytoplasmic inclusion, revealed abnormal expression of nuclear envelope proteins, increased heterochromatinization, and massive DNA damage. These molecular pathological features in LMN progressed with acetamide exposure in a time-dependent manner, implying that LMN formation can lead to chromosome rearrangements. Overall, these data suggested that CAs induced by acetamide play a pivotal role in acetamide-induced hepatocarcinogenesis in rats and that CAs can cause chemical carcinogenesis in animals via MN formation.

In vivo mutagenicity and tumor-promoting activity of 1,3-dichloro-2-propanol in the liver and kidneys of gpt delta rats.

1,3-Dichloro-2-propanol (1,3-DCP), a food contaminant, exerts carcinogenic effects in multiple organs, including the liver and kidneys, in rats. However, the underlying mechanisms of 1,3-DCP-induced carcinogenesis remain unclear. Here, the in vivo mutagenicity and tumor-promoting activity of 1,3-DCP in the liver and kidneys were evaluated using medium-term gpt delta rat models previously established in our laboratory (GPG and GNP models). Six-week-old male F344 gpt delta rats were treated with 0 or 50 mg/kg body weight/day 1,3-DCP by gavage for 4 weeks. After 2 weeks of cessation, partial hepatectomy or unilateral nephrectomy was performed to collect samples for in vivo mutation assays, followed by single administration of diethylnitrosamine (DEN) for tumor initiation. One week after DEN injection, 1,3-DCP treatment was resumed, and tumor-promoting activity was evaluated in the residual liver or kidneys by histopathological analysis of preneoplastic lesions. gpt mutant frequencies increased in excised liver and kidney tissues following 1,3-DCP treatment. 1,3-DCP did not affect the development of glutathione S-transferase placental form-positive foci in residual liver tissues, but enhanced atypical tubule hyperplasia in residual kidney tissues. Detailed histopathological analyses revealed glomerular injury and increased cell proliferation of renal tubular cells in residual kidney tissues of rats treated with 1,3-DCP. These results suggested possible involvement of genotoxic mechanisms in 1,3-DCP-induced carcinogenesis in the liver and kidneys. In addition, we found that 1,3-DCP exhibited limited tumor-promoting activity in the liver, but enhanced clonal expansion in renal carcinogenesis via proliferation of renal tubular cells following glomerular injury.

A 90-day subchronic toxicity study of Myrrh in F344 rats.

Myrrh is a flavoring agent and food additive. Here, we performed a subchronic toxicity study of Myrrh in male and female F344 rats by feeding at 5,000, 15,000 and 50,000 ppm for 90 days. No deaths or clinical signs were observed. Suppression of body weight gain was observed from the early phase of administration in both males and females in the 50,000 ppm group. Because there were no obvious changes in food intake in any of the Myrrh groups compared with the control group, suppression of body weight gain was considered an adverse effect of Myrrh. Hematology and serum biochemistry parameters with significant changes observed in the Myrrh groups were considered to have no toxicological significance. We observed a significant increase in relative kidney weight in male rats treated with 50,000 ppm Myrrh; this effect was considered to be related to the appearance of hyaline droplets in the epithelium of the proximal tubules histopathologically observed in this group. Immunohistochemical staining with anti-α2u-globulin antibodies suggested that these hyaline droplets were caused by factors other than α2u-globulin deposition. Thus, the no-observed-adverse-effect level of Myrrh was determined to be 15,000 ppm (males: 0.85 g/kg/day, females: 0.95 g/kg/day).

Background data of 2-year-old male and female F344 gpt delta rats.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

Furan Induced Characteristic Glutathione S-Transferase Placental Form-Positive Foci in Terms of Cell Kinetics and Gene Expression.

Glutathione S-transferase placental form-positive (GST-P+) foci are markers of preneoplastic lesions in rat hepatocarcinogenesis. Our previous studies using reporter gene transgenic rats showed that furan, a hepatocarcinogen in rodents, rapidly induces the formation of GST-P+ foci after short exposure without reporter gene mutation. We hypothesized that GST-P+ foci induced by furan may have biological characteristics different from those induced by diethylnitrosamine (DEN), a genotoxic hepatocarcinogen. Accordingly, we compared the cell kinetics of GST-P+ foci after cessation of DEN treatment and performed comprehensive gene expression in DEN- or furan-induced GST-P+ foci. The number and area of DEN-induced GST-P+ foci were increased after cessation of treatment, whereas furan decreased these parameters. Size distribution analysis showed that large furan-induced GST-P+ foci disappeared after cessation of treatment. Hierarchical cluster analysis showed that all samples from GST-P+ foci induced by furan were separated from those induced by DEN. SOX9 expression was upregulated in furan-induced GST-P+ foci and was detected by immunohistochemistry in large furan-induced GST-P+ foci. Our results indicated that large furan-induced GST-P+ foci were quite different from DEN-induced GST-P+ foci at the molecular and cellular levels. And one of the properties of disappearing large GST-P+ foci were characterized by inclusion of hepatocytes expressing SOX9.

Molecular Pathological Differences in Global Gene Expression between Two Sustained Proliferative Lesions, Nodular Regenerative Hepatocellular Hyperplasia and Hepatocellular Adenoma, in Mice.

Long-term exposure to piperonyl butoxide (PBO) induces multiple nodular masses along with hepatocellular tumors in the liver of mice. The histopathological features of the nodules led to our diagnosis of nodular regenerative hepatocellular hyperplasia (NRH). However, because of the lack of data on the biological characteristics of NRH, whether this lesion is truly nonneoplastic remains unknown. In this study, the molecular characteristics of NRH were compared with those of hepatocellular adenoma (HCA) by global gene expression analysis. Six-week-old male ICR mice were fed a diet containing 6,000 ppm PBO for 43 weeks to induce NRH and HCA development. Complementary DNA microarray analysis was performed using messenger RNA extracted from NRH and HCA frozen sections collected by laser microdissection. Hierarchical cluster analysis showed that all NRH samples clustered together but were separate from the HCA cluster. Pathway analysis revealed activation of the cell cycle and Delta-Notch signaling in both lesions, but the latter was more upregulated in HCA. Downregulation of cytochrome p450 enzymes was observed in NRH, but not in HCA. These results imply that NRH differs from HCA in terms of not only morphological but also molecular characteristics.

Effects of inhibition of hepatic sulfotransferase activity on renal genotoxicity induced by lucidin-3-O-primeveroside.

Sulfotransferase 1A (SULT1A) expression is lower in the liver of humans than that of rodents. Therefore, species differences should be taken into consideration when assessing the risk of rodent hepatocarcinogens metabolically activated by SULT1A in humans. Although some renal carcinogens require SULT1A-mediated activation, it is unclear how SULT1A activity in the liver affects renal carcinogens. To explore the effects of SULT1A activity in the liver on genotoxicity induced by SULT1A-activated renal carcinogens, B6C3F1 mice or gpt delta mice of the same strain background were given lucidin-3-O-primeveroside (LuP), a hepatic and renal carcinogen of rodents, for 4 or 13 weeks, respectively, and pentachlorophenol (PCP) as a liver-specific SULT inhibitor, was given from 1 week before LuP treatment to the end of the experiment. A 4 week exposure of LuP induced lucidin-specific DNA adduct formation. The suppression of Sult1a expression was observed only in the liver but not in the kidneys of PCP-treated mice, but co-administration of PCP suppressed LuP-induced DNA adduct formation in both organs. Thirteen-week exposure of LuP increased mutation frequencies and cotreatment with PCP suppressed these increases in both organs. Given that intact levels of SULT activity in the liver were much higher than in the kidneys of rodents, SULT1A may predominantly activate LuP in the liver, consequently leading to genotoxicity not only in the liver but also in the kidney. Thus, species differences should be considered in human risk assessment of renal carcinogens activated by SULT1A as in the case of the corresponding liver carcinogens.

Methods for the analysis of organophosphorus flame retardants-Comparison of GC-EI-MS, GC-NCI-MS, LC-ESI-MS/MS, and LC-APCI-MS/MS.

Organophosphorus flame retardants (PFRs) are extensively used as alternatives to banned polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). In this study, we analyzed 14 PFRs by means of four mass-spectrometry-based methods: gas chromatography combined with electron-impact mass spectrometry (GC-EI-MS) or negative-chemical-ionization mass spectrometry (GC-NCI-MS) and liquid chromatography combined with tandem mass spectrometry using electrospray ionization (LC-ESI-MS/MS) or atmospheric pressure chemical ionization (LC-APCI-MS/MS). The limits of quantification (LOQs) for LC-ESI-MS/MS and LC-APCI-MS/MS (0.81-970 pg) were 1-2 orders of magnitude lower than the LOQs for GC-EI-MS and GC-NCI-MS (2.3-3900 pg). LC-APCI-MS/MS showed the lowest LOQs (mean = 41 pg; median = 3.4 pg) for all but two of the PFRs targeted in this study. For LC-APCI-MS/MS, the lowest LOQ was observed for tributyl phosphate (TBP) (0.81 pg), and the highest was observed for tris(butoxyethyl) phosphate (TBOEP) (36 pg). The results of this study indicate that LC-APCI-MS/MS is the optimum analytical method for the target PFRs, at least in terms of LOQ.

Role of oxidative stress in the chemical structure-related genotoxicity of nitrofurantoin in Nrf2-deficient gpt delta mice.

Despite its antimicrobial activity, nitrofurantoin (NFT) is a renal carcinogen in rats. Oxidative stress induced by reduction of the nitro group of NFT may contribute to its genotoxicity. This is supported by our recent results indicating that the structure of the nitrofuran plays a key role in NFT-induced genotoxicity, and oxidative DNA damage is involved in renal carcinogenesis. Nuclear factor erythroid 2-related factor 2 (NRF2) regulates cellular responses to oxidative stress. To clarify the role of oxidative stress in the chemical structure-related genotoxic mechanism of NFT, we performed reporter gene mutation assays for NFT and 5-nitro-2-furaldehyde (NFA) using Nrf2-proficient and Nrf2-deficient gpt delta mice. NFT administration for 13 weeks resulted in a significant increase in 8-hydroxydeoxyguanosine (8-OHdG; a marker of oxidative stress) and gpt mutant frequency only in the kidneys of Nrf2-/- mice. The mutation spectrum, characterized by increased substitutions at guanine bases, suggested that oxidative stress is involved in NFT-induced genotoxicity. However, NFA did not increase the mutation frequency in the kidneys, despite the increased 8-OHdG in NFA-treated Nrf2-/- mice. Thus, it is unlikely that oxidative stress is involved in the genotoxic mechanism of NFA. These results imply that nitro reduction plays a key role in the genotoxicity of NFT, but the lack of a role of oxidative stress in the genotoxicity of NFA indicates a potential role of side chain interactions in oxidative stress caused by nitro reduction. These findings provide a basis for the development of safe nitrofurans.

Mechanisms of oxidative stress-induced in vivo mutagenicity by potassium bromate and nitrofurantoin.

Oxidative stress is well known as a key factor of chemical carcinogenesis. However, the actual role of oxidative stress in carcinogenesis, such as oxidative stress-related in vivo mutagenicity, remains unclear. It has been reported that 8-hydroxydeoxyguanosine (8-OHdG), an oxidized DNA lesion, might contribute to chemical carcinogenesis. Potassium bromate (KBrO3) and nitrofurantoin (NFT) are known as renal carcinogens in rats. Our previous studies showed an increase in mutant frequencies accompanied by an increased level of 8-OHdG in the kidneys of rodents following KBrO3 or NFT exposure. Furthermore, KBrO3 and NFT induced different types of gene mutations. Thus, in the present study, we performed reporter gene mutation assays and 8-OHdG measurements following KBrO3 or NFT exposure using Nrf2-proficient and Nrf2-deficient mice to clarify the relationship between KBrO3- or NFT-induced oxidative stress and subsequent genotoxicity. Administration of 1,500 ppm of KBrO3 in drinking water resulted in an increase in deletion mutations accompanied by an increase in 8-OHdG level, and administration of 2,500 ppm of NFT in diet induced an increase in guanine base substitution mutations without elevation of the 8-OHdG level in Nrf2-deficient mice. These results demonstrated that the formation of 8-OHdG, which resulted from the oxidizing potential of KBrO3, was directly involved in the increase in deletion mutations, although factors related to oxidative stress other than 8-OHdG might be crucial for NFT-induced guanine base substitution mutations. The present study provides new insight into oxidative stress-related in vivo mutagenicity.

Phosphorylation of protein phosphatase 2A facilitated an early stage of chemical carcinogenesis.

Protein phosphatase 2A (PP2A) is a serine-threonine phosphatase that regulates cell signaling pathways. Its inactivation is correlated with tumor malignancy, possibly due to the effects on cell differentiation and malignant cell transformation. Therefore, it has been noted that PP2A could be a promising target for cancer therapy. In our previous study of the hepatocarcinogen estragole (ES), cell proliferation may be required to convert ES-specific DNA adducts to mutations. To explore the trigger for cell proliferation, gpt delta rats were administered ES by gavage at doses of 3, 30 and 300mg/kg/day for 4weeks. ES-induced cell proliferation and gene mutations were observed at only the high dose whereas ES-specific DNA adducts were detected in a dose-dependent manner. Western blot analyses revealed activation of the Akt and ERK pathways without activation of upstream regulators, such as c-Raf, PKC and, PI3K. Phosphorylation of the PP2A C subunit at Tyr307 was found along with phosphorylation of Src. The overall data might imply that PP2A inactivation is responsible for cell cycle progression through activation of the Akt and ERK pathways at high doses of ES. Based on γ-H2AX immunohistochemistry and Western blot analysis for Rad51 protein, the resultant mutation spectra showed large deletion mutations that might result from double strand breaks of DNA. Thus, it is likely that inactivation of PP2A resulted in acceleration and exacerbation of gene mutations. We conclude that PP2A might contribute to an early stage of chemical carcinogenesis, suggesting that PP2A could be a molecular target of primary cancer prevention.

Osteopontin Deficiency Suppresses Intestinal Tumor Development in Apc-Deficient Min Mice.

Osteopontin (OPN) is a secreted phosphoglycoprotein, and is a transcriptional target of aberrant Wnt signaling. OPN is upregulated in human colon cancers, and is suggested to enhance cancer progression. In this study, the effect of deficiency of OPN on intestinal tumor development in Apc-deficient Min mice was investigated. At 16 weeks of age, the number of small intestinal polyps in Min/OPN(+/-) and Min/OPN(-/-) mice was lower than that of Min/OPN(+/+) mice. Colorectal tumor incidences and multiplicities in Min/OPN(+/-) and Min/OPN(-/-) mice were significantly lower than those in Min/OPN(+/+) mice, being 48% and 0.6 ± 0.8, 50% and 0.8 ± 0.9 vs. 80% and 1.6 ± 1.7, respectively. OPN expression in colorectal tumors was strongly upregulated in Min/OPN(+/+) compared to adjacent non-tumor parts, but was decreased in Min/OPN(+/-) and not detected in Min/OPN(-/-). Targets of OPN, matrix metalloproteinases (MMPs)-3, -9, and -13 were lowered by OPN deficiency. Macrophage marker F4/80 in colorectal tumors was also lowered by OPN deficiency. MMP-9 expression was observed in tumor cells and tumor-infiltrating neutrophils. These results indicate that induction of OPN by aberrant Wnt signaling could enhance colorectal tumor development in part by upregulation of MMP-3, -9, and -13 and infiltration of macrophage and neutrophils. Suppression of OPN expression could contribute to tumor prevention, but complete deficiency of OPN may cause some adverse effects.

Anti-Inflammatory Effects of Capsaicin and Piperine on Helicobacter pylori-Induced Chronic Gastritis in Mongolian Gerbils.

BACKGROUND: Spices have been used for thousands of years, and recent studies suggest that certain spices confer beneficial effects on gastric disorders. The purpose of this study was to evaluate possible chemopreventive effects of spice-derived compounds on Helicobacter pylori (H. pylori)-induced gastritis. METHODS: We examined the inhibitory effects of curcumin, capsaicin, and piperine on H. pylori in vitro by determining the colony-forming units and real-time RT-PCR in H. pylori stimulated AGS gastric cancer cells. For in vivo analysis, 6-week-old SPF male Mongolian gerbils were infected with H. pylori, fed diets containing 5000 ppm curcumin, 100 ppm capsaicin, or 100 ppm piperine, and sacrificed after 13 weeks. RESULTS: All three compounds inhibited in vitro proliferation of H. pylori, with curcumin being the most effective. Infiltration of neutrophils and mononuclear cells was suppressed by piperine both in the antrum and corpus of H. pylori-infected gerbils. Capsaicin also decreased neutrophils in the antrum and corpus and mononuclear cell infiltration and heterotopic proliferative glands in the corpus. mRNA expression of Tnf-α and formation of phospho-IκB-α in the antrum were reduced by both capsaicin and piperine. In addition, piperine suppressed expression of Il-1ß, Ifn-γ, Il-6, and iNos, while H. pylori UreA and other virulence factors were not significantly attenuated by any compounds. CONCLUSION: These results suggest that capsaicin and piperine have anti-inflammatory effects on H. pylori-induced gastritis in gerbils independent of direct antibacterial effects and may thus have potential for use in the chemoprevention of H. pylori-associated gastric carcinogenesis.

Acrylamide induces specific DNA adduct formation and gene mutations in a carcinogenic target site, the mouse lung.

Acrylamide (AA) is a contaminant in heated foods and is carcinogenic in multiple organs of rodents. There have been many reports regarding AA-induced DNA modification and genotoxicity. However, the data are insufficient to understand fully the relationship between the two events. A recent report demonstrated carcinogenicity in the mouse lung. The lung is advantageous for investigation of AA-induced genotoxicity because DNA adduct levels are relatively high in this organ. In the present study, reporter gene mutation assays and quantitative analyses of specific DNA adducts were performed in the lungs of mature gpt delta mice treated with AA at doses of 100, 200 and 400 p.p.m. in drinking water for 4 weeks. N7-GA-Gua was detected in all AA-treated mice in a dose-dependent manner. gpt mutant frequencies (MFs) were significantly increased in the middle- and high-dose groups. In the analysis of mutation spectra, significant increases in GC-TA transversions and single base deletion mutations were observed in the high-dose group. Spi(-) MFs were significantly increased in the high-dose group. Analysis of Spi(-) mutants revealed significant increases in the frequencies of single base deletion mutation in runs of G/C and A/T. Analyses of immature mice under the same experimental conditions showed that there were no differences of susceptibility to AA-induced genotoxicity in the two age classes. The overall data clearly show the causal relationship between AA-induced DNA adducts and the gene mutations at carcinogenic target sites.

Ochratoxin A induces DNA double-strand breaks and large deletion mutations in the carcinogenic target site of gpt delta rats.

Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.

Combined application of comprehensive analysis for DNA modification and reporter gene mutation assay to evaluate kidneys of gpt delta rats given madder color or its constituents.

DNA adductome analysis using liquid chromatography-tandem mass spectrometry is a promising tool to exhaustively search DNA modifications. Given that the molecular weight of chemical-specific adducts is determined by the total molecular weights of the active form and nucleotide bases, we developed a new method of comprehensive analysis for chemical-specific DNA adducts based on the principle of adductome analysis. The actual analytical mass range was 50 mass units up or down from the average molecular weight of the four DNA bases plus the molecular weight of the expected active form of the chemical. Using lucidin-3-O-primeveroside (LuP), lucidin-modified bases formed by its active form were exhaustively searched using this new method. Various DNA adducts, including Luc-N (2)-dG and Luc-N (6)-dA, were identified in the kidneys of rats given LuP. Together with measurement of 8-hydroxydeoxyguanosine (8-OHdG) levels, the combined application of this new method with a reporter gene mutation assay was performed to clarify renal carcinogenesis induced by madder color (MC) that includes LuP and alizarin (Alz) as constituent agents. A DNA adductome map derived from MC-treated rats was almost identical to that of LuP-treated rats, but not Alz-treated rats. Although 8-OHdG levels were elevated in MC- and Alz-treated rats, significant increases in gpt and Spi(-) mutant frequencies were observed only in MC- and LuP-treated rats. In addition, the spectrum of gpt mutants in MC-treated rats showed almost the same pattern as those in LuP-treated rats. The overall data suggest that LuP may be responsible for MC-induced carcinogenicity and that the proposed methodology is appropriate for exploring and understanding mechanisms of chemical carcinogenesis.

Possible contribution of 8-hydroxydeoxyguanosine to gene mutations in the kidney DNA of gpt delta rats following potassium bromate treatment.

8-Hydroxydeoxyguanosine (8-OHdG) is well known not only as an effective biomarker of oxidative stress but also as a mutagenic DNA modification. Incorporation of dAMP at the opposite site of 8-OHdG induces G>T or A>C transversions. However, in vivo analyses of gene mutations caused by potassium bromate (KBrO3), which can induce 8-OHdG at carcinogenic target sites, showed that G>T was prominent in the small intestines of mice, but not in the kidneys of rats. Because KBrO3 was a much clearer carcinogen in the kidneys of rats, detailed analyses of gene mutations in the kidney DNA of rats treated with KBrO3 could improve our understanding of oxidative stress-mediated carcinogenesis. In the current study, site-specific reporter gene mutation assays were performed in the kidneys of gpt delta rats treated with KBrO3. Groups of 5 gpt delta rats were treated with KBrO3 at concentrations of 0, 125, 250, or 500 ppm in the drinking water for 9 weeks. At necropsy, the kidneys were macroscopically divided into the cortex and medulla. 8-OHdG levels in DNA extracted from the cortex were dramatically elevated at concentrations of 250 ppm and higher compared with those from the medulla. Cortex-specific increases in mutant frequencies in gpt and red/gam genes were found at 500 ppm. Mutation spectrum and sequence analyses of their mutants demonstrated significant elevations in A>T transversions in the gpt gene and single base deletions at guanine or adenine in the gpt or red/gam genes. While A>T transversions and single base deletions of adenine may result from the oxidized modification of adenine, the contribution of 8-OHdG to gene mutations was limited despite possible participation of the 8-OHdG repair process in guanine deletion.