Yoko Hirabayashi

Aryl hydrocarbon receptor biology and xenobiotic responses in hematopoietic progenitor cells

Studying the biological functions of the aryl hydrocarbon receptor (AhR) other than its function in xenobiotic drug metabolism may answer the questions as to why AhR orthologues have long been conserved phylogenically widely in the animal kingdom, and why homologues have diverged from nonvertebrate species such as nematodes and drosophila to all the vertebrate species. In this review, we focused on the mechanism of longevity possibly derived from evolution of AhRs and compared the functional difference of hematopoietic progenitors between wild-type (AhR(+/+)) mice and AhR-deficiencies (AhR(+/-), AhR(-/-)). Particular advantages found in wild-type mice compared with AhR-deficiencies were as follows: first, higher antioxidative function in the hematopoietic microenvironment with low oxidative tension seemed to have developed with the evolution of AhR; second, primitive hematopoietic progenitor-cell-specific deceleration and dormancy of cell-cycle regulation may have developed also with AhR evolution, which keeps hematopoietic progenitor cell compartment dormant without extinction by continuous differentiation; third, the consequent evolution of genomic stabilization with a longer lifespan in wild-type mice developed with the evolution of AhR. Experimentally, mice showed a significant extension of lifespan in a gene-dosage-dependent manner with a delayed onset of leukemogenicity. Another possible additional advantage observed in wild-type mice, the mechanism of which is not yet clarified, is an improved efficiency of fertilization in wild-type mice as compared with AhR-deficiencies, which seems to have developed with the evolution of AhR. Four advantages altogether, including the anti-aging feature mentioned above may have induced the AhR molecule to diverge various of species in the animal kingdom.

Benzene-induced bone-marrow toxicity: a hematopoietic stem-cell-specific, aryl hydrocarbon receptor-mediated adverse effect.

Benzene-induced hematopoietic toxicity is an aryl hydrocarbon receptor (AhR)-related adverse effect that is not exhibited in AhR-knockout (KO) mice. In the hematopoietic system, the steady-state expression of AhRs is limited in the hematopoietic progenitor cells; thus, a hierarchical hematopoietic impairment starts from hematopoietic progenitor cells after benzene exposure. When one looks at wild-type recipient mice that have been lethally irradiated and repopulated with AhR-KO bone marrow cells, owing to reconstruction by the marrow from AhR-KO mice, no impairment is observed in the assay of granulo-macrophage colony-forming units (CFU-GMs) in the bone marrow after benzene exposure of the reconstituted mice. In contrast, in mature white blood cells concern, benzene-induced hematopoietic cytotoxicity is observed in the same reconstituted mice; however, this benzene-induced hematopoietic cytotoxicity in mature white blood cells is not induced in the case of AhR-KO mice repopulated with wild-type bone marrow cells after a lethal dose of irradiation. The mechanism of benzene-induced hematopoietic toxicity in the mature blood cells in AhR-KO mice is assumed to be based on metabolites such as phenol and hydroquinone derived from hepatic AhR. Thus, the former toxicity in mature white blood cells is assumed to be based on the metabolites of the wild-type hepatic AhR, whereas the latter lack of toxicity in mature blood cells in AhR-KO mice is due to the lack of benzene-induced metabolism in the liver. Global gene expression analysis of bone marrow cells after benzene exposure reveals that MEF2c, the functions of which are known to maintain lymphocyte differentiation and promote proliferation of hematopoietic progenitor cells, is commonly downmodulated not only in C57BL/6 but also in C3H/He mice. In response to these impairments of the hematopoietic progenitor cells and the niches, stochastic and reciprocal upregulations of integrin beta 2 and the Runx family are observed, which are known to stabilize hematopoietic niches during the steady-state. Direct observation of the hematopoietic progenitor cells, particularly the Lin(-)c-kit(+)Sca-1(+) (LKS) fraction, after benzene exposure revealed an increased amount of intracytoplasmic reactive oxygen species (ROS) detected by ROS-reacting dye as compared with other blood cell fractions.

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.

Age-related decline of mast cell regeneration in senescence-accelerated mice (SAMP1) after chemical myeloablation due to senescent stromal cell impairment

An age-related decline in immune functions is referred to as immunosenescence. Mast cells play an important role in the immune system. However, it has not yet been determined if aging may affect mast-cell development. In the present study, we examined the age-related change in mast-cell development after myeloablation with 5-fluorouracil (5-FU) in senescence accelerated mice (SAMP1), which exhibit senescence-mimicking stromal cell impairment after 30 weeks of age. We found that aged mice with stromal cell impairment (30-36 weeks old) showed a lower recovery of the number of femoral mast-cell progenitors (colony-forming unit [CFU]-mast) (64% of steady state), whereas young mice (8-12 weeks old) showed a higher recovery (122% of steady state). Stromal cells influence mast-cell development by producing positive regulators such as stem cell factor (SCF) and negative regulators such as transforming growth factor-beta (TGF-β). The ratio of the gene expression of SCF to that of TGF-β (SCF/TGF-β βratio) indicates the balance of positive and negative regulation of mast-cell development. SCF/TGF-β βratio increased in both the young and aged mice after 5-FU treatment. However, the SCF/TGF-β βratio rapidly decreased in aged mice, whereas it remained high in young mice. The number of femoral CFU-mast in the S-phase after 5-FU treatment reflects the activation of positive-dominant regulation for mast-cell development by stromal cells. Aged mice showed lower recovery of the number of femoral CFU-mast in the S-phase (47% of steady state), whereas young mice showed a higher recovery (205% of steady state). These results suggest that mast-cell development declines with aging due to stromal-cell functional impairment, which contributes to immunosenescence.

Inflammatory biomarker, neopterin, predominantly enhances myelopoiesis, which suppresses erythropoiesis via activated stromal cells.

Neopterin is produced by monocytes and is a useful biomarker for inflammation. We found previously that neopterin enhanced myelopoiesis but suppressed B-lymphopoiesis triggered by the positive and negative regulations of cytokines produced by stromal cells in mice. The effects of neopterin on erythropoiesis during the enhancement of myelopoiesis were determined in the present study using C57BL/6J mice. The intravenous injection of neopterin into mice resulted in a prolonged decrease in the number of femoral erythroid progenitor cells (BFU-Es and CFU-Es), whereas the number of femoral myeloid progenitor cells (CFU-GMs) was increased. Interestingly, the oscillatory changes in the number of erythroid progenitor cells were reciprocal to those of myeloid progenitor cells. The expression of Cdc42, a regulator of the balance between erythropoiesis and myelopoiesis, was down-regulated, implying that the suppression of erythropoiesis is due to myelopoietic predominance. Furthermore, the expression of SDF-1 in stromal cells, a negative regulator of erythropoiesis, was up-regulated. These results suggest that neopterin facilitates myelopoiesis in the bone marrow by suppressing erythropoiesis, thereby contributing to the potential up-regulation of inflammatory process.

Hematopoietic neoplastic diseases develop in C3H/He and C57BL/6 mice after benzene exposure: Strain differences in bone marrow tissue responses observed using microarrays

In this study, Trp53-deficient and wild-type mice of both C57BL/6 and C3H/He strains were exposed to benzene (33, 100, and 300 ppm; 6h/day, 5 days/week for 26 weeks) and then observed for lifetime. As results, first, the incidence of nonthymic lymphomas in C57BL/6 mice and acute myeloid leukemias (AMLs) in C3H/He mice showed linear responses at the lower exposure level in Trp53-deficient mice; second, the incidence of thymic lymphomas in C57BL/6 mice and nonthymic lymphomas in C3H/He mice increased without a plateau-like ceiling; thus, the former equivocal induction of hematopoietic neoplasms (HPNs) in the case of low-dose benzene exposure was assumed to be based on the DNA repair potential in wild-type mice, and the latter limited increase in HPNs in the case of high-dose benzene exposure was considered to be due to excessive apoptosis in wild-type mice. Concerning the incidence of AMLs, though a dose of 300 ppm benzene inhalation induced 9% AMLs in wild-type C3H/He mice-AML-prone, it induced AMLs in 38% of Trp53-deficient C3H/He mice. Because AMLs were also observed in Trp53-deficient mice, including in the C57BL/6 mice, benzene exposure may also be a potent inducer of AMLs in mice with some strain differences. In the present study, to elucidate the hematopoietic stem cell-specific, aryl hydrocarbon-receptor-related low-dose adverse effect, global gene expression in the bone marrow was analyzed at 28 days after 2-week-intermittent exposure to 150 mg/kg b.w. benzene, by gavage, i.e., equivalent to the above inhalation protocol with 300 ppm. We observed two conceptually different gene expression profiles; common gene profiles (CGPs) shared among mice in each group, and stochastic gene profiles (SGPs), i.e., unique union genes from one individual mouse to another. The CGPs of the experimental group and the SGPs of each individual mouse were separately characterized by individual assay. Concerning the CGPs, reciprocal strain differences between C3H/He and C57BL/6 mice in expression gene profiles, both plausible for leukemogenesis, were identified; namely, dominant downmodulations of Sltm and Cryl1, related to suppression of apoptosis and genomic instability in C3H/He mice, respectively, and dominant downmodulations of Atrx/rad54 and Kdm2a, related to a decrease in DNA repair and genomic instability, respectively, in C57BL/6 mice. These findings imply that these reciprocal gene expression differences induced by benzene exposure may lead each strain to undergo different hematopoietic neoplastic pathways. In contrast, each individual mouse often shows a unique SGP. SGPs often include transcription factors, which regulate reciprocal signaling pathways including further SGPs. Among them, apoptosis-related genes expressed in C57BL/6 mice and those in C3H/He mice were attributable to different combinations of SGPs. Such stochastic case-by-case gene expression may be in good agreement with the individual and strain differences observed following benzene exposure. Because gene chip microarray techniques can elucidate stochastic changes in gene expression profiles, possible stochastic toxicology and its future role are discussed.

Neopterin, inflammation-associated product, prolongs erythropoiesis suppression in aged SAMP1 mice due to senescent stromal-cell impairment

Anemia induced by inflammation is well known to be more serious in the elderly than in non-elderly adults; however, the reason why this is so remains unclear. Neopterin produced by monocytes during inflammation promotes myelopoiesis but suppresses B-lymphopoiesis and erythropoiesis, by activating stromal cells in mice. Here, age-related changes in the erythropoietic response to neopterin were determined using senescence accelerated mice (SAMP1) with senescence stromal-cell impairment. Intravenous injection of neopterin into young mice (8–12 weeks old) resulted in a decrease in erythroid progenitor cell number in the bone marrow (BM), concomitant with an increase in myeloid progenitor cell number over one week. Intravenous injection of neopterin into aged mice (30–36 weeks old) resulted in a prolonged decrease in erythroid progenitor cell number in the BM over three weeks and a limited increase in myeloid progenitor cell number over one day. Neopterin treatment induced a decrease in serum erythropoietin concentrations in young mice but not in aged mice. The gene expression of tumor necrosis factor α (TNF-α), a negative regulator of erythropoiesis, was up-regulated in the BM of both young and aged mice, and the degree of TNF-α up-regulation was the same in both groups. The gene expression of interleukin (IL)-11, a positive regulator of erythropoiesis, was also up-regulated over one day in both young and aged mice. However, IL-11 gene expression remained up-regulated thereafter in young mice, whereas it was rapidly down-regulated in aged mice. These data suggest that prolonged suppression of erythropoiesis in aged mice may be due to a decrease in the production of positive regulators rather than to an increase in the production of negative regulators. Our combined data suggest that age-related impairment of stromal cells induces serious anemia in the elderly during inflammation.

Radiation‐induced, cell cycle–related gene expression in aging hematopoietic stem cells: enigma of their recovery

This paper reviews quantitative and qualitative studies conducted to identify changes in the characteristics of hematopoietic stem/progenitor cells (HSCs/HPCs) with or without radiation exposure. The numerical recovery of HSCs/HPCs after radiation exposure is lower than for other types of cells, an effect that may depend on hierarchical ordering of generation age during blood cell differentiation, from primitive HSCs to various differentiated HPCs. Studies are in progress to evaluate gene expression in bone marrow cells and cells in the lineage‐negative, c‐Kit+, stem cell antigen+ (LKS) fraction from 21‐month‐old mice, with or without radiation exposure. Preliminary data suggest that cell cycle–related genes, that is, cyclin D1 (Ccnd1), phosphatidylinositol 3 kinase regulatory subunit polypeptide 1 (PiK3r1), and Fyn, are upregulated solely in the LKS fraction from 21‐month‐old mice irradiated at 6 weeks of age, compared with the LKS fraction from age‐matched nonirradiated control mice. Additional studies may provide evidence that the aging phenotype is exaggerated following exposure to ionizing radiation, specifically in the LKS fraction.

Comparison of murine gene expression profiles between spontaneous and radiation-induced myelogenous leukemias: Stochastic and probabilistic expression variances in the former vs radiation-specific expression commonalities in the latter

OBJECTIVEnTo elucidate the common characteristics of murine radiation-induced myelogenous leukemias, global gene-chip expression profiles were compared with age-matched steady-state bone marrow tissue profiles and spontaneous myelogenous leukemia profiles.nnnMATERIALS AND METHODSnSix each of C3H/He mice-derived radiation-induced and spontaneously developed myelogenous leukemias were analyzed. Bone marrow cells from five each of 2- and 21-month-old mice were used to subtract nonleukemic information in the analysis. mRNAs from individual mice were analyzed separately using 45,101 gene chips followed by computational biological analysis.nnnRESULTSnFirst, principal component analysis (PCA) was performed to discriminate the gene expression profiles of individual mice with radiation-induced myelogenous leukemia from those of bone marrow cells from 2- or 21-month-old mice. Discriminant union genes for individual leukemias were then selected, which finally yielded 242 genes, among which six are radiation-related genes including Hus-1, Edf1a2, andVegf-c; 16 are apoptosis/cell-death-related genes, 13 are cell-cycle/cell-growth-related genes, and 50 are suppressor/promoter genes. PCA of these 242 genes consistently enabled the discrimination of the radiation-induced leukemias from the spontaneous leukemias. Second, the other components of the same PCA provided four different eigenvector clusters in an unsupervised manner representing four histopathological findings, with which the differential diagnosis in molecular taxonomy was significant as determined by analysis of variance of the global gene expression profiles.nnnCONCLUSIONnDiscriminant union genes in radiation-induced myelogenous leukemias against spontaneous myelogenous leukemias and age-matched nonleukemic bone marrow profilings generated by unsupervised computational analysis essentially represent probabilistic biomarkers for radiation-induced myelogenous leukemias, which may contribute to developing a model for risk of secondary carcinogenesis in patients treated by whole-body irradiation.

Benzene activates caspase-4 and -12 at the transcription level, without an association with apoptosis, in mouse bone marrow cells lacking the p53 gene

Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. However, although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying lympho/leukemogenicity is not fully understood. Caspase-4 (alias caspase-11) and -12 are inflammatory caspases implicated in inflammation and endoplasmic reticulum stress-induced apoptosis. The objectives of this study were to investigate the altered expression of caspase-4 and -12 in mouse bone marrow after benzene exposure and to determine whether their alterations are associated with benzene-induced bone marrow toxicity, especially cellular apoptosis. In addition, we evaluated whether the p53 gene is involved in regulating the mechanism, using both wild-type (WT) mice and mice lacking the p53 gene. For this study, 8-week-old C57BL/6 mice [WT and p53 knockout (KO)] were administered a benzene solution (150xa0mg/kg diluted in corn oil) via oral gavage once daily, 5xa0days/week, for 1 or 2xa0weeks. Blood and bone marrow cells were collected and cell counts were measured using a Coulter counter. Total mRNA and protein extracts were prepared from the harvested bone marrow cells. Then qRT-PCR and Western blotting were performed to detect changes in the caspases at the mRNA and protein level, respectively. A DNA fragmentation assay and Annexin-V staining were carried out on the bone marrow cells to detect apoptosis. Results indicated that when compared to the control, leukocyte number and bone marrow cellularity decreased significantly in WT mice. The expression of caspase-4 and -12 mRNA increased significantly after 12xa0days of benzene treatment in the bone marrow cells of benzene-exposed p53KO mice. However, apoptosis detection assays indicated no evidence of apoptosis in p53KO or WT mice. In addition, no changes of other apoptosis-related caspases, such as caspase-3 and -9, were found in WT or p53KO mice at the level of mRNA and proteins. These results indicated that upregulation of caspase-4 and -12 in mice lacking the p53 gene is not associated with cellular apoptosis. In conclusion, caspase-4 and -12 can be activated by benzene treatment without inducing cell apoptosis in mouse bone marrow, which are partly under the regulation of the p53 gene.