A formamidopyrimidine derivative from the deoxyguanosine adduct produced by food contaminant acrylamide induces DNA replication block and mutagenesis.

Acrylamide, a common food contaminant, is metabolically activated to glycidamide, which reacts with DNA at the N7 position of dG, forming N7-(2-carbamoyl-2-hydroxyethyl)-dG (GA7dG). Owing to its chemical lability, the mutagenic potency of GA7dG has not yet been clarified. We found that GA7dG undergoes ring-opening hydrolysis to form N6-(2-deoxy-d-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-[N-(2-carbamoyl-2-hydroxyethyl)formamido]pyrimidine (GA-FAPy-dG), even at neutral pH. Therefore, we aimed to examine the effects of GA-FAPy-dG on the efficiency and fidelity of DNA replication using an oligonucleotide carrying GA-FAPy-9-(2-deoxy-2-fluoro-ß-d-arabinofuranosyl)guanine (dfG), a 2'-fluorine substituted analog of GA-FAPy-dG. GA-FAPy-dfG inhibited primer extension by both human replicative DNA polymerase ε and the translesion DNA synthesis polymerases (Polη, Polι, Polκ, and Polζ) and reduced the replication efficiency by less than half in human cells, with single base substitution at the site of GA-FAPy-dfG. Unlike other formamidopyrimidine derivatives, the most abundant mutation was G:C > A:T transition, which was decreased in Polκ- or REV1-KO cells. Molecular modeling suggested that a 2-carbamoyl-2-hydroxyethyl group at the N5 position of GA-FAPy-dfG can form an additional H-bond with thymidine, thereby contributing to the mutation. Collectively, our results provide further insight into the mechanisms underlying the mutagenic effects of acrylamide.

Identification of kynurenine and quinolinic acid as promising serum biomarkers for drug-induced interstitial lung diseases.

BACKGROUND: Drug-induced interstitial lung disease (DILD) is a lung injury caused by various types of drugs and is a serious problem in both clinical practice and drug development. Clinical management of the condition would be improved if there were DILD-specific biomarkers available; this study aimed to meet that need. METHODS: Biomarker candidates were identified by non-targeted metabolomics focusing on hydrophilic molecules, and further validated by targeted approaches using the serum of acute DILD patients, DILD recovery patients, DILD-tolerant patients, patients with other related lung diseases, and healthy controls. RESULTS: Serum levels of kynurenine and quinolinic acid (and kynurenine/tryptophan ratio) were elevated significantly and specifically in acute DILD patients. The diagnostic potentials of these biomarkers were superior to those of conventional lung injury biomarkers, Krebs von den Lungen-6 and surfactant protein-D, in discriminating between acute DILD patients and patients with other lung diseases, including idiopathic interstitial pneumonia and lung diseases associated with connective tissue diseases. In addition to identifying and evaluating the biomarkers, our data showed that kynurenine/tryptophan ratios (an indicator of kynurenine pathway activation) were positively correlated with serum C-reactive protein concentrations in patients with DILD, suggesting the potential association between the generation of these biomarkers and inflammation. Our in vitro experiments demonstrated that macrophage differentiation and inflammatory stimulations typified by interferon gamma could activate the kynurenine pathway, resulting in enhanced kynurenine levels in the extracellular space in macrophage-like cell lines or lung endothelial cells. Extracellular quinolinic acid levels were elevated only in macrophage-like cells but not endothelial cells owing to the lower expression levels of metabolic enzymes converting kynurenine to quinolinic acid. These findings provide clues about the molecular mechanisms behind their specific elevation in the serum of acute DILD patients. CONCLUSIONS: The serum concentrations of kynurenine and quinolinic acid as well as kynurenine/tryptophan ratios are promising and specific biomarkers for detecting and monitoring DILD and its recovery, which could facilitate accurate decisions for appropriate clinical management of patients with DILD.

Role of CD44 expressed in renal tubules during maladaptive repair in renal fibrogenesis in an allopurinol-induced rat model of chronic kidney disease.

The kidney is a major target organ for the adverse effects of pharmaceuticals; renal tubular epithelial cells (TECs) are particularly vulnerable to drug-induced toxicity. TECs have regenerative capacity; however, maladaptive repair of TECs after injury leads to renal fibrosis, resulting in chronic kidney disease (CKD). We previously reported the specific expression of CD44 in failed-repair TECs of rat CKD model induced by ischemia reperfusion injury. Here, we investigated the pathophysiological role of CD44 in renal fibrogenesis in allopurinol-treated rat CKD model. Dilated or atrophic TECs expressing CD44 in fibrotic areas were collected by laser microdissection and subjected to microarray analysis. Gene ontology showed that extracellular matrix (ECM)-related genes were upregulated and differentiation-related genes were downregulated in dilated/atrophic TECs. Ingenuity Pathway Analysis identified CD44 as an upstream regulator of fibrosis-related genes, including Fn1, which encodes fibronectin. Immunohistochemistry demonstrated that dilated/atrophic TECs expressing CD44 showed decreases in differentiation markers of TECs and clear expression of mesenchymal markers during basement membrane attachment. In situ hybridization revealed an increase in Fn1 mRNA in the cytoplasm of dilated/atrophic TECs, whereas fibronectin was localized in the stroma around these TECs, supporting the production/secretion of ECM by dilated/atrophic TECs. Overall, these data indicated that dilated/atrophic TECs underwent a partial epithelial-mesenchymal transition (pEMT) and that CD44 promoted renal fibrogenesis via induction of ECM production in failed-repair TECs exhibiting pEMT. CD44 was detected in the urine and serum of APL-treated rats, which may reflect the expression of CD44 in the kidney.

Comparison of the sensitivity of histopathological and immunohistochemical analyses and blood hormone levels for early detection of antithyroid effects in rats treated with thyroid peroxidase inhibitors.

Although measurements of blood triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) levels in rodent toxicity studies are useful for detection of antithyroid substances, assays for these measurements are expensive and can show high variability depending on blood sampling conditions. To develop more efficient methods for detecting thyroid disruptors, we compared histopathological and immunohistochemical findings in the thyroid and pituitary glands with blood hormone levels. Six-week-old male and female Sprague-Dawley rats (five rats per group) were treated with multiple doses of the thyroid peroxidase inhibitors propylthiouracil (PTU) and methimazole by gavage for 28 days. Significant decreases in serum T3 and T4 and increases in TSH were observed in the ≥1 mg/kg PTU and ≥3 mg/kg methimazole groups. An increase in TSH was also detected in male rats in the 0.3 mg/kg PTU group. Histopathological and immunohistochemical analyses revealed that follicular cell hypertrophy and decreased T4 and T3 expressions in the thyroid gland were induced at doses lower than doses at which significant changes in serum hormone levels were observed, suggesting that these findings may be more sensitive than blood hormone levels. Significant increases in thyroid weights, Ki67-positive thyroid follicular cell counts, and TSH-positive areas in the pituitary gland were detected at doses comparable with those at which changes in serum T4 and TSH levels were observed, indicating that these parameters may also be useful for evaluation of antithyroid effects. Combining these parameters may be effective for detecting antithyroid substances without relying on hormone measurements.

Pathogenesis of chemically induced nasal cavity tumors in rodents: contribution to adverse outcome pathway.

The pathogenesis of nasal cavity tumors induced in rodents has been critically reviewed. Chemical substances that induce nasal cavity tumors in rats, mice, and hamsters were searched in the National Toxicology Program (NTP), International Agency for Research on Cancer (IARC), and Japan Bioassay Research Center (JBRC) databases, in addition to PubMed. Detailed data such as animal species, administration routes, and histopathological types were extracted for induced nasal cavity tumors. Data on non-neoplastic lesions were also extracted. The relationship between the tumor type and non-neoplastic lesions at equivalent sites was analyzed to evaluate tumor pathogenesis. Genotoxicity data were also analyzed. Squamous cell carcinoma was the most frequent lesion, regardless of the dosing route, and its precursor lesions were squamous metaplasia and/or respiratory epithelial hyperplasia, similar to squamous cell papilloma. The precursor lesions of adenocarcinoma, the second most frequent tumor type, were mainly olfactory epithelial hyperplasia, whereas those of adenoma were respiratory epithelial lesions. These pathways were consistent among species. Our results suggest that the responsible lesions may be commonly linked with chemically-induced cytotoxicity in each tumor type, irrespective of genotoxicity, and that the pathways may largely overlap between genotoxic and non-genotoxic carcinogens. These findings may support the documentation of adverse outcome pathways (AOPs), such as cytotoxicity, leading to nasal cavity tumors and the integrated approaches to testing and assessment (IATA) for non-genotoxic carcinogens.

CD44 expression in renal tubular epithelial cells in the kidneys of rats with cyclosporine-induced chronic kidney disease.

Renal tubular epithelial cell (TEC) injury is the most common cause of drug-induced kidney injury (DIKI). Although TEC regeneration facilitates renal function and structural recovery following DIKI, maladaptive repair of TECs leads to irreversible fibrosis, resulting in chronic kidney disease (CKD). CD44 is specifically expressed in TECs during maladaptive repair in several types of rat CKD models. In this study, we investigated CD44 expression and its role in renal fibrogenesis in a cyclosporine (CyA) rat model of CKD. Seven-week-old male Sprague-Dawley rats fed a low-salt diet were subcutaneously administered CyA (0, 15, or 30 mg/kg) for 28 days. CD44 was expressed in atrophic, dilated, and hypertrophic TECs in the fibrotic lesions of the CyA groups. These TECs were collected by laser microdissection and evaluated by microarray analysis. Gene ontology analysis suggested that these TECs have a mesenchymal phenotype, and pathway analysis identified CD44 as an upstream regulator of fibrosis-related genes, including fibronectin 1 (Fn1). Immunohistochemistry revealed that epithelial and mesenchymal markers of TECs of fibrotic lesions were downregulated and upregulated, respectively, and that these TECs were surrounded by a thickened basement membrane. In situ hybridization revealed an increase in Fn1 mRNA in the cytoplasm of TECs of fibrotic lesions, whereas fibronectin protein was localized in the stroma surrounding these tubules. Enzyme-linked immunosorbent assay revealed increased serum CD44 levels in CyA-treated rats. Collectively, these findings suggest that CD44 contributes to renal fibrosis by inducing fibronectin secretion in TECs exhibiting partial epithelial-mesenchymal transition and highlight the potential of CD44 as a biomarker of renal fibrosis.

EpCAM and APN expression in combination with γ-H2AX as biomarkers for detecting hepatocarcinogens in rats.

The phosphorylated form of histone H2AX (γ-H2AX) serves as a commonly utilized biomarker for DNA damage. Based on our previous findings, which demonstrated the formation of γ-H2AX foci as a reliable biomarker for detecting bladder carcinogens in repeated dose 28-day study in rats, we hypothesized that γ-H2AX could also function as a biomarker for detecting hepatocarcinogens. However, we found that γ-H2AX foci formation was not effectively induced by hepatocarcinogens that did not stimulate hepatocyte proliferation. Therefore, we explored alternative biomarkers to detect chemical hepatocarcinogenicity and discovered increased expressions of epithelial cell adhesion molecule (EpCAM/CD326)- and aminopeptidase N (APN/CD13) in the hepatocytes of rats administered various hepatocarcinogens. Significant increases in EpCAM- and APN-positive hepatocytes were observed for eight and five of the 10 hepatocarcinogens, respectively. Notably, five and two of them, respectively, were negative for γ-H2AX foci. These results highlight the potential of EpCAM and APN as useful biomarkers in combination with γ-H2AX for the detection of chemical hepatocarcinogenicity.

Oral toxicological study of titanium dioxide nanoparticles with a crystallite diameter of 6 nm in rats.

BACKGROUND: Though titanium dioxide (TiO2) is generally considered to have a low impact in the human body, the safety of TiO2 containing nanosized particles (NPs) has attracted attention. We found that the toxicity of silver NPs markedly varied depending on their particle size, as silver NPs with a diameter of 10 nm exhibited fatal toxicity in female BALB/c mice, unlike those with diameters of 60 and 100 nm. Therefore, the toxicological effects of the smallest available TiO2 NPs with a crystallite size of 6 nm were examined in male and female F344/DuCrlCrlj rats by repeated oral administration of 10, 100, and 1000 mg/kg bw/day (5/sex/group) for 28 days and of 100, 300, and 1000 mg/kg bw/day (10/sex/group) for 90 days. RESULTS: In both 28- and 90-day studies, no mortality was observed in any group, and no treatment-related adverse effects were observed in body weight, urinalysis, hematology, serum biochemistry, or organ weight. Histopathological examination revealed TiO2 particles as depositions of yellowish-brown material. The particles observed in the gastrointestinal lumen were also found in the nasal cavity, epithelium, and stromal tissue in the 28-day study. In addition, they were observed in Peyer's patches in the ileum, cervical lymph nodes, mediastinal lymph nodes, bronchus-associated lymphoid tissue, and trachea in the 90-day study. Notably, no adverse biological responses, such as inflammation or tissue injury, were observed around the deposits. Titanium concentration analysis in the liver, kidneys, and spleen revealed that TiO2 NPs were barely absorbed and accumulated in these tissues. Immunohistochemical analysis of colonic crypts showed no extension of the proliferative cell zone or preneoplastic cytoplasmic/nuclear translocation of ß-catenin either in the male or female 1000 mg/kg bw/day group. Regarding genotoxicity, no significant increase in micronucleated or γ-H2AX positive hepatocytes was observed. Additionally, the induction of γ-H2AX was not observed at the deposition sites of yellowish-brown materials. CONCLUSIONS: No effects were observed after repeated oral administration of TiO2 with a crystallite size of 6 nm at up to 1000 mg/kg bw/day regarding general toxicity, accumulation of titanium in the liver, kidneys, and spleen, abnormality of colonic crypts, and induction of DNA strand breaks and chromosomal aberrations.

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.

Mucosal damage and γ-H2AX formation in the rat urinary bladder induced by aromatic amines with structures similar to o-toluidine and o-anisidine.

Although aromatic amines are widely used as raw materials for dyes, some, such as o-toluidine and o-anisidine, have shown concerning results regarding carcinogenicity in the urinary bladder. We have recently developed a short-term detection method for bladder carcinogens using immunohistochemistry for γ-H2AX, a DNA damage marker. Here, using this method, we evaluated aromatic amines with structures similar to o-toluidine and o-anisidine for bladder mucosal damage and potential carcinogenicity. In total, 17 aromatic amines were orally administered to male F344 rats for 28 days, and histopathological examination and γ-H2AX immunostaining of the urinary bladder were performed. Histopathological analysis revealed that seven aromatic amines, including 4-chloro-o-toluidine (4-CT), o-aminoazotoluene, 2-aminobenzyl alcohol (ABA), o-acetotoluidine (o-AT), 3,3'-dimethoxybenzidine, 4-aminoazobenzene (AAB), and 4,4'-methylenedianiline (MDA), induced various bladder lesions, such as hemorrhage, necrosis, and urothelial hyperplasia. The morphological characteristics of mucosal damage induced by these substances were divided into two major types: those resembling o-toluidine and those resembling o-anisidine. Six of these aromatic amines, excluding MDA, also caused significant increases in γ-H2AX formation in the bladder urothelium. Interestingly, 4-CT did not cause mucosal damage or γ-H2AX formation at the lower dose applied in previous carcinogenicity studies. These results showed for the first time that o-AT and ABA, metabolites of o-toluidine, as well as AAB caused damage to the bladder mucosa and suggested that they may be bladder carcinogens. In addition, 4-CT, which was thought to be a noncarcinogen, was found to exhibit bladder toxicity upon exposure to high doses, indicating that this compound may contribute to bladder carcinogenesis.

Increased Cell Proliferation as a Key Event in Chemical Carcinogenesis: Application in an Integrated Approach for the Testing and Assessment of Non-Genotoxic Carcinogenesis.

In contrast to genotoxic carcinogens, there are currently no internationally agreed upon regulatory tools for identifying non-genotoxic carcinogens of human relevance. The rodent cancer bioassay is only used in certain regulatory sectors and is criticized for its limited predictive power for human cancer risk. Cancer is due to genetic errors occurring in single cells. The risk of cancer is higher when there is an increase in the number of errors per replication (genotoxic agents) or in the number of replications (cell proliferation-inducing agents). The default regulatory approach for genotoxic agents whereby no threshold is set is reasonably conservative. However, non-genotoxic carcinogens cannot be regulated in the same way since increased cell proliferation has a clear threshold. An integrated approach for the testing and assessment (IATA) of non-genotoxic carcinogens is under development at the OECD, considering learnings from the regulatory assessment of data-rich substances such as agrochemicals. The aim is to achieve an endorsed IATA that predicts human cancer better than the rodent cancer bioassay, using methodologies that equally or better protect human health and are superior from the view of animal welfare/efficiency. This paper describes the technical opportunities available to assess cell proliferation as the central gateway of an IATA for non-genotoxic carcinogenicity.

Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 µM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

Characterization of serotonin as a candidate biomarker of severity and prognosis of COVID-19 using LC/MS analysis.

The coronavirus disease 2019 (COVID-19) pandemic has been associated with high mortality worldwide. Owing to its complicated pathophysiology, diagnostic and prognostic biomarkers for effective patient management remain scarce. We analyzed kynurenine, tryptophan, and serotonin levels in the serum of patients with COVID-19 via liquid chromatography/mass spectrometry analysis. Serum serotonin levels were decreased in patients with more severe COVID-19, along with increased kynurenine and decreased tryptophan concentrations. Patients with moderate disease who subsequently worsened showed significantly lower serotonin concentrations compared with those who did not experience severe disease. Serum serotonin levels may represent a valuable biomarker for COVID-19 severity and prognosis.

Serum stratifin and presepsin as candidate biomarkers for early detection of COVID-19 disease progression.

The prognosis of patients with severe cases of COVID-19 is poor; thus, biomarkers for earlier prediction of COVID-19 progression are vital. We measured levels of five lung injury-related biomarkers, SP-D, KL-6, presepsin, kallistatin and stratifin, in serum samples collected serially during hospitalization from 31 patients with mild/moderate or severe/critical COVID-19 pneumonia, and their predictive performances were compared. Like the previously reported presepsin, a new biomarker candidate, stratifin, was significantly elevated with the onset of severe or critical symptoms in COVID-19 patients and decreased with symptom improvement. Notably, changes in stratifin and presepsin levels were distinctly earlier than those in SP-D, KL-6 and even SpO2/FiO2 values. Furthermore, serum levels of these biomarkers were significantly higher at the pre-severe stage (before the start of oxygen support) of patients who eventually advanced to severe/critical stages than in the patients who remained at the mild/moderate stage. These results were confirmed in an independent cohort, including 71 mild/moderate and 14 severe/critical patients, for whom the performance of stratifin and presepsin in discriminating between mild/moderate and pre-severe conditions of COVID-19 patients was superior to that of the SpO2/FiO2 ratio. Therefore, we concluded that stratifin and presepsin could be used as prognostic biomarkers for severe COVID-19 progression.

Histopathological and immunohistochemical evaluation for detecting changes in blood hormone levels caused by endocrine disruptors in a 28-day repeated-dose study in rats.

Although measurements of blood hormone levels in rodent toxicological studies can provide important information on the mechanisms of toxicity and extrapolation to humans, there are several difficulties such as large individual differences and limited sample volume. To develop a more simplified method that does not depend solely on blood samples, we examined the possible application of immunohistochemistry for detecting endocrine disruptors in short-term studies. Aminotriazole (AMT), propylthiouracil (PTU), phenobarbital, aminoglutethimide (AGT), estradiol, and vitamin D3 were administered orally to 6-week-old male and female SD rats (five/group) for 28 days. Measurements of serum hormone levels revealed decreases in triiodothyronine (T3) and thyroxine (T4) in the AMT and PTU groups, an increase in thyroid stimulating hormone (TSH) in the AMT, PTU, and AGT groups, and an increase in adrenocorticotrophic hormone in the AGT group. Increased thyroid, pituitary, and adrenal gland weights; histopathological lesions, including follicular hypertrophy/hyperplasia, hypertrophy/vacuolation of anterior pituitary cells, and increased adrenocortical vacuolation were observed in association with the hormone level changes. Immunohistochemical analysis revealed a decreased T4 level in the thyroid gland of the AMT and PTU groups and an increased area of TSH positive immunostaining in the pituitary gland of the AMT, PTU, and AGT groups, consistent with the changes in serum T4 and TSH levels, respectively. These results suggest that histopathological analysis and immunohistochemistry for T4 and TSH might be useful and sensitive methods of detecting thyroid dysfunction, and that combining organ weight measurements is a reliable parameter of detecting endocrine disruptors.

Early detection of urinary bladder carcinogens in rats by immunohistochemistry for γ-H2AX: a review from analyses of 100 chemicals.

In safety evaluations of chemicals, there is an urgent need to develop short-term methods to replace long-term carcinogenicity tests. We have reported that immunohistochemistry for γ-H2AX, a well-established biomarker of DNA damage, can detect bladder carcinogens at an early stage using histopathological specimens from 28-day repeated-dose oral toxicity studies in rats. Given the markedly low level of γ-H2AX formation in the bladder urothelium of untreated rats, an increase in γ-H2AX-positive cells following chemical exposure can be relatively easy to identify. Among the 100 compounds examined to date, bladder carcinogens can be detected with high sensitivity (33/39; 84.6%) and specificity (58/61; 95.1%). As expected, γ-H2AX formation levels tended to be high following exposure to genotoxic bladder carcinogens, whereas nongenotoxic bladder carcinogens also increased the number of γ-H2AX-positive cells, probably through secondary DNA damage associated with sustained proliferative stimulation. γ-H2AX formation in the bladder urothelium reflects species differences in susceptibility to bladder carcinogenesis between rats and mice and shows a clear dose-dependency associated with the intensity of tumor development as well as high reproducibility. Some of the bladder carcinogens that showed false-negative results in the evaluation of γ-H2AX alone could be detected by combined evaluation with immunostaining for bladder stem cell markers, including aldehyde dehydrogenase 1A1. This method may be useful for the early detection of bladder carcinogens, as it can be performed by simple addition of conventional immunostaining using formalin-fixed paraffin-embedded tissues from 28-day repeated-dose toxicity studies in rodents, which are commonly used in safety evaluations of chemical substances.

o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis.

Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

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.