Yukio Kodama

Long-term toxicity/carcinogenicity of musk xylol in B6C3F1 mice

The long-term toxicity/carcinogenicity of musk xylol, a synthetic nitro musk, was examined in B6C3F1 mice of both sexes. Musk xylol was administered at dietary levels of 0 (control), 0.075 or 0.15% for 80 wk. The overall tumour incidences in all treated groups of both sexes were significantly higher than those in the corresponding controls. Combined malignant and benign liver cell tumours were clearly increased in both sexes, and in males a positive significant trend was also noted for the occurrence of hepatocellular carcinomas. In males the incidence of Harderian gland tumours was also significantly greater in treated groups than in controls. Some other neoplasms, such as lung tumours in both sexes and Harderian gland tumours and lymphomas in females, occurred in greater numbers in the treated groups, although the differences were not statistically significant in comparison with the controls. In addition, the incidences and total numbers of malignant tumours were significantly increased in treated groups of both sexes, although the increases were not dose dependent. The results demonstrated that musk xylol is carcinogenic in B6C3F1 mice when given at dose levels of 0.075 or 0.15% in the diet for 80 wk.

Mechanism of action of benzene toxicity: Cell cycle suppression in hemopoietic progenitor cells (CFU-GM)

The aim of this study was to clarify previously reported controversial data and hypotheses concerning the effect of benzene on the cell cycle of hemopoietic stem cells. In this study, the bromodeoxyuridine UV (BUUV) suicide assay was performed in normal C57BL/6 and p53 knockout (KO) C57BL/6 mice during and after exposure to 300 ppm of benzene for 2 weeks. Our kinetic studies revealed that the cell cycle of hemopoietic myeloid progenitor cells (colony-forming unit granulocyte-macrophage [CFU-GM]), rather than being stimulated, was suppressed by exposure to benzene. The fraction of CFU-GM in S phase was significantly depressed, from 37.1% in controls to 16.3% in normal mice. BrdUrd incorporation in both groups revealed significantly different slopes for untreated and benzene-exposed normal C57BL/6 mice. p53 appeared to induce suppression of both the number and the cycling fraction of hemopoietic progenitor cells, as demonstrated by the lack of benzene-induced suppression of these parameters in p53 KO mice. The likelihood that suppression of bone marrow cellularity and cell cycling is mediated by p53 was supported by the upregulation of p21, a cyclin-dependent kinase inhibitor. Our present study revealed the mechanism of action of benzene hematotoxicity. Benzene suppresses the cell cycle by p53-mediated overexpression of p21, a cyclin-dependent kinase inhibitor, resulting not simply in suppression of hemopoiesis but rather in a dynamic change of hemopoiesis during and after benzene exposure. Thus, the controversies raised by previously reported data are resolved by our present findings of hemopoietic stem cell kinetics.

Susceptibility of transgenic mice carrying human prototype c-Ha-ras gene in a short-term carcinogenicity study of vinyl carbamate and ras gene analyses of the induced tumors

To determine if hemizygous transgenic mice carrying the human c‐Ha‐ras gene (CB6F1‐Tg Hras2 mice (Hras2 mice)) are susceptible to the carcinogenic potential of known murine carcinogens, male and female Hras2 mice and their non‐transgenic CB6F1 littermates (non‐Tg mice) were each given a single intraperitoneal injection of 60 mg of vinyl carbamate (VC)/kg body weight or saline (vehicle control) and monitored for 16 wk without further treatment. At necropsy, grossly visible tumors were fixed for histopathologic diagnosis and, when of sufficient size, portions were frozen for subsequent molecular analysis. Nine of 31 male and nine of 29 female Hras2 mice treated with VC died within 16 wk as a result of lung tumor burden. At the termination of the study, lung tumors (alveolar‐bronchiolar epithelial neoplasms and hemangiosarcomas) and focal alveolar‐bronchiolar hyperplasias were present in both sexes of Hras2 and non‐Tg mice treated with VC; there were significantly more proliferative lung lesions in Hras2 than non‐Tg mice. Splenic hemangiosarcomas and squamous cell tumors of the forestomach were induced in male and female VC‐treated Hras2 mice but not in VC‐treated non‐Tg mice. Polymerase chain reaction–single‐strand conformation polymorphism analysis and DNA sequencing of the induced lung tumors revealed point mutations at codon 61 of the transgene in two of 29 lung tumors (one of 16 in males and one of 13 in females) from VC‐treated Hras2 mice; no mutations in murine Ki‐ras were found in these tumors. Point mutations at codons 12 and 61 of the murine Ki‐ras gene were observed, however, in one of 10 and six of 10 lung tumors respectively, from VC‐treated non‐Tg mice. These findings indicate that Hras2 mice are highly sensitive to pulmonary neoplasms and splenic and lung hemangiosarcomas after treatment with VC. The molecular analyses suggest that point mutations of the transgene and the murine Ki‐ras gene do not play a major role in VC induction of pulmonary neoplasms in these transgenic mice. Mol. Carcinog. 20:298–307, 1997.

Increased susceptibility to hepatocarcinogenicity of Nrf2-deficient mice exposed to 2-amino-3-methylimidazo[4,5-f]quinoline

To elucidate the roles of the transcription factor NF‐E2‐related factor (Nrf2) in hepatocarcinogenesis induced by 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), a mutagenic and carcinogenic heterocyclic amine, Nrf2‐deficient mice were treated with 300 p.p.m. IQ in their diet for 1, 4 or 52 weeks. In the long‐term experiment, the multiplicity and incidence of liver tumors in male and female IQ‐treated Nrf2 deficient (–/–) mice were significantly higher than those in their counterpart wild‐type (+/+) mice exposed to IQ. In the short‐term experiment, although IQ exposure to Nrf2(+/+) mice of both sexes did not modify UDP‐glucuronosyltransferase values, glutathione S‐transferase values were significantly increased due to IQ treatment, in contrast to no alteration in male and female Nrf2(–/–) mice. Levels of oxidative stress markers such as 8‐hydroxydeoxyguanosine and thiobarbituric acid reactive substances in the livers of all treated mice were not changed by IQ treatment. IQ‐specific DNA adduct levels were elevated only in female Nrf2(–/–) mice, although the increase was not significant. IQ treatment caused an increase in proliferating cell nuclear antigen labeling indices only in male Nrf2(–/–) mice. The present data clearly show that Nrf2(–/–) mice of both sexes are susceptible to IQ hepatocarcinogenicity, which might result from IQ accumulation due to failure of metabolizing enzyme induction. In addition, inconsistent results concerning IQ‐specific adducts and proliferating cell nuclear antigen labeling indices in male and female Nrf2(–/–) mice suggest the existence of different contributions of Nrf2 to IQ hepatocarcinogenesis between mice of the two sexes. (Cancer Sci 2007; 98: 19–24)

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.

Lack of toxicity/carcinogenicity of monosodium succinate in F344 rats

The toxicity/carcinogenicity of monosodium succinate, a food additive, was examined in F344 rats. The oral LD50 was greater than 8 g/kg body weight. In a 13-wk subchronic oral toxicity study, the only toxicological finding was suppression of body-weight gain in groups given greater than or equal to 2.5% monosodium succinate in the drinking-water. Histological examination revealed no toxic lesions specifically caused by the compound in any organs of any of the treated rats. The maximum tolerated dose was determined to be 2-2.5% on the basis of body-weight depression. In a long-term (2-yr) toxicity/carcinogenicity study, monosodium succinate was given ad lib. in drinking-water (distilled water) at levels of 0, 1 or 2% to groups of 50 male and 50 female rats. No toxic lesion specifically caused by long-term administration of monosodium succinate was detected. No dose-related increase was found in the incidences of tumours in any organ or tissue except for C-cell tumours of the thyroid gland of females. The incidence of these tumours in females given the 2% dose was higher than that in controls but not significantly so, and a positive trend for this tumour was noted in females. C-Cell tumour is one of the most commonly observed spontaneous tumours in ageing female rats of this strain and occurs at a variable incidence. There was no difference between the female control and treated groups in the incidence of preneoplastic change of the thyroid gland. Furthermore, the incidence of C-cell tumours in the female control group was lower than that in our historical controls. It is concluded that the increase in C-cell tumours in the female high-dose group and the detection of a positive trend for this tumour in females were probably a function of experimental variability and were not related to treatment. The results indicate that monosodium succinate had neither toxic nor carcinogenic activity in F344 rats when it was given continuously at levels of 1 or 2% in the drinking-water for 2 yr.

Benzene-induced hematopoietic neoplasms including myeloid leukemia in Trp53-deficient C57BL/6 and C3H/He mice

This research focused on three major questions regarding benzene-induced hematopoietic neoplasms (HPNs). First, why are HPNs induced equivocally and at only threshold level with low-dose benzene exposure despite the significant genotoxicity of benzene even at low doses both in experiments and in epidemiology? Second, why is there no linear increase in incidence at high-dose exposure despite a lower acute toxicity (LD(50) > 1000 mg/kg body weight; WHO, 2003, Benzene in drinking-water. Background document for development of WHO Guidelines for Drinking-Water Quality)? Third, why are particular acute myeloid leukemias (AMLs) not commonly observed in mice, although AMLs are frequently observed in human cases of occupational exposure to benzene? In this study, we hypothesized that the threshold-like equivocal induction of HPNs at low-dose benzene exposure is based on DNA repair potential in wild-type mice and that the limited increase in HPNs at a high-dose exposure is due to excessive apoptosis in wild-type mice. To determine whether Trp53 deficiency satisfies the above hypotheses by eliminating or reducing DNA repair and by allowing cells to escape apoptosis, we evaluated the incidence of benzene-induced HPNs in Trp53-deficient C57BL/6 mice with specific regard to AMLs. We also used C3H/He mice, AML prone, with Trp53 deficiency to explore whether a higher incidence of AMLs on benzene exposure might explain the above human-murine differences. As a result, heterozygous Trp53-deficient mice of both strains showed a nonthreshold response of the incidence of HPNs at the lower dose, whereas both strains showed an increasing HPN incidence up to 100% with increasing benzene exposure dose, including AMLs, that developed 38% of heterozygous Trp53-deficient C3H/He mice compared to only 9% of wild-type mice exposed to the high dose. The detection of AMLs in heterozygous Trp53-deficient mice, even in the C57BL/6 strain, implies that benzene may be a potent inducer of AMLs also in mice with some strain differences.

Membrane channel connexin 32 maintains Lin(-)/c-kit(+) hematopoietic progenitor cell compartment: analysis of the cell cycle.

Membrane channel connexin (Cx) forms gap junctions that are implicated in the homeostatic regulation of multicellular systems; thus, hematopoietic cells were assumed not to express Cxs. However, hematopoietic progenitors organize a multicellular system during the primitive stage; thus, the aim of the present study was to determine whether Cx32, a member of the Cx family, may function during the primitive steady-state hematopoiesis in the bone marrow (BM). First, the numbers of mononuclear cells in the peripheral blood and various hematopoietic progenitor compartments in the BM decreased in Cx32-knockout (KO) mice. Second, on the contrary, the number of primitive hematopoietic progenitor cells, specifically the Lin−/c-kit+/Scal+ fraction, the KSL progenitor cell compartment, also increased in Cx32-KO mice. Third, expression of Cx32 was detected in Lin−/c-kit+ hematopoietic progenitor cells of wild-type mice (0.27% in the BM), whereas it was not detected in unfractionated wild-type BM cells. Furthermore, cell-cycle analysis of the fractionated KSL compartment from Cx32-KO BM showed a higher ratio in the G2/M fraction. Taken together, all these results imply that Cx32 is expressed solely in the primitive stem cell compartment, which maintains the stemness of the cells, i.e., being quiescent and noncycling; and once Cx32 is knocked out, these progenitor cells are expected to enter the cell cycle, followed by proliferation and differentiation for maintaining the number of peripheral blood cells.

Serial transplantation of p53-deficient hemopoietic progenitor cells to assess their infinite growth potential.

Thirty-five years ago, Siminovitch et al. (Siminovitch L, Till JE, McCulloch EA. J Cell Com Physiol 64:23–32, 1964), using serially transplanted mouse spleens at 14-day intervals, observed a markedly progressive decline in the proliferative capacity of bone marrow (BM) cells, with the loss of cionogenicity by the fourth transplant generation. Using the same protocol, we assessed the proliferative capacity of p53-deficient mouse BM cells transplanted serially at the same 14-day intervals into lethally irradiated mice, which was a useful tool for understanding the characteristics of hemopoletic stem cells lacking solely the p53 gene function. BM cells from p53-deficient homozygous (p53–/–), p53-heterozygous (p53+/–), and wild-type (p53+/+) C57BL/6 mice were transplanted into lethally irradiated C57BL/6 recipients. Fourteen days later, the repopulated spleens were harvested, and 107 cells were retransplanted into secondary recipients. Serial transplantation was continued at 14-day intervals until hemopoietic repopulation failure. The number of heterozygous and homozygous p53-deficient spleen cells increased logarithmically up to the fourth and fifth passages, respectively, whereas wild-type spleen cells ceased to proliferate by the third passage. The number of macroscopic spleen colonies increased logarithmically until the third passage in recipients of heterozygous and homozygous p53-deficient cells, but ceased to grow by the second passage in recipients of wild-type cells. The numbers of heterozygous and homozygous p53-deficient colony forming units in spleen (CFUs-S) remained stable during the first four transplant generations, whereas that of wild-type CFUs-S decreased progressively from the first transplant generation onward. The clonogenicity of p53-deficient cells was lost when the number of CFUs-S per spleen decreased to below 10. This suggests that one out of 10 CFUs-S might be long-term repopulating cells (LTRCs), and that p53-deficient LTRCs may proliferate more rapidly than wild-type LTRCs. Longer passages that were possible in the p53-deficient groups were considered to be due to the faster cell cycle of the p53-deficient hemopoietic progenitor cells, as determined by bromodeoxyuridine incorporation with purging by UV light exposure, followed by hemopoietic colony assay (BUUV assay).

Role of p53 in the Progression from Ochratoxin A-Induced DNA Damage to Gene Mutations in the Kidneys of Mice

Carcinogenic doses of ochratoxin A (OTA) cause increases of mutant frequencies (MFs) of the red/gam gene (Spi(-)) in the kidneys of p53-deficient gpt delta mice, but not in p53-proficient mice. Here, we investigated the role of p53 in the progression from OTA-induced DNA damage to gene mutations. To this end, p53-proficient and -deficient mice were administered 5 mg/kg OTA for 3 days or 4 weeks by gavage. After 3 days of administration, comet assays were performed and there were no differences in the degrees of OTA-induced DNA damage between p53-proficient and -deficient mice. However, the frequencies of γ-H2AX-positive tubular epithelial cells in p53-deficient mice were significantly higher than those in p53-proficient mice, implying that p53 inhibited the progression from DNA damage to DNA double-strand breaks (DSBs). Evaluation of global gene expression and relevant mRNA/protein expression levels demonstrated that OTA increased the expression of Cdkn1a, which encodes the p21 protein, in p53-proficient mice, but not in p53-deficient mice. Moreover, in p53-deficient mice, mRNA levels of cell cycle progression and DSB repair (homologous recombination repair [HR])-related genes were significantly increased. Thus, G1/S arrest due to activation of the p53/p21 pathway may contribute to the prevention of DSBs in p53-proficient mice. In addition, single base deletions/insertions/substitutions were predominant, possibly due to HR. Overall, these results suggested that OTA induced DSBs at the carcinogenic target site in mice and that p53/p21-mediated cell cycle control prevented an increase in the formation of DSBs, leading to gene mutations.