Atsunori Yafune

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.

Aberrant activation of M phase proteins by cell proliferation-evoking carcinogens after 28-day administration in rats.

We have previously reported that hepatocarcinogens increase liver cells expressing p21(Cip1), a G1 checkpoint protein and M phase proteins after 28-day treatment in rats. This study aimed to identify early prediction markers of carcinogens available in many target organs after 28-day treatment in rats. Immunohistochemical analysis was performed on Ki-67, p21(Cip1) and M phase proteins [nuclear Cdc2, phospho-Histone H3 (p-Histone H3), Aurora B and heterochromatin protein 1α (HP1α)] with carcinogens targeting different organs. Carcinogens targeting thyroid (sulfadimethoxine; SDM), urinary bladder (phenylethyl isothiocyanate), forestomach (butylated hydroxyanisole; BHA), glandular stomach (catechol; CC), and colon (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and chenodeoxycholic acid) were examined using a non-carcinogenic toxicant (caprolactam) and carcinogens targeting other organs as negative controls. All carcinogens increased Ki-67(+), nuclear Cdc2(+), p-Histone H3(+) or Aurora B(+) carcinogenic target cells, except for both colon carcinogens, which did not increase cell proliferation. On the other hand, p21(Cip1+) cells increased with SDM and CC. HP1α responded only to BHA. Results revealed carcinogens evoking cell proliferation concurrently induced cell cycle arrest at M phase or showing chromosomal instability reflecting aberration in cell cycle regulation, irrespective of target organs, after 28-day treatment. Therefore, M phase proteins may be early prediction markers of carcinogens evoking cell proliferation in many target organs.

Disruptive cell cycle regulation involving epigenetic downregulation of Cdkn2a (p16Ink4a) in early-stage liver tumor-promotion facilitating liver cell regeneration in rats

Cell cycle aberration was immunohistochemically examined in relation to preneoplastic liver cell foci expressing glutathione S-transferase placental form (GST-P) at early stages of tumor-promotion in rats with thioacetamide (TAA), a hepatocarcinogen facilitating liver cell regeneration. Immunoexpression of p16(Ink4a) following exposure to other hepatocarcinogens/promoters and its DNA methylation status were also analyzed during early and late tumor-promotion stages. GST-P(+) liver cell foci increased cell proliferation and decreased apoptosis when compared with surrounding liver cells. In concordance with GST-P(+) foci, checkpoint proteins at G(1)/S (p21(Cip1), p27(Kip1) and p16(Ink4a)) and G(2)/M (phospho-checkpoint kinase 1, Cdc25c and phospho-Wee1) were either up- or downregulated. Cellular distribution within GST-P(+) foci was either increased or decreased with proteins related to G(2)-M phase or DNA damage (topoisomerase IIα, phospho-histone H2AX, phospho-histone H3 and Cdc2). In particular, p16(Ink4a) typically downregulated in GST-P(+) foci and regenerative nodules at early tumor-promotion stage with hepatocarcinogens facilitating liver cell regeneration and in neoplastic lesions at late tumor-promotion stage with hepatocarcinogens/promoters irrespective of regenerating potential. Hypermethylation at exon 2 of Cdkn2a was detected at both early- and late-stages. Thus, diverse disruptive expression of G(1)/S and G(2)/M proteins, which allows for clonal selection of GST-P(+) foci, results in the acquisition of multiple aberrant phenotypes to disrupt checkpoint function. Moreover, increased DNA-damage responses within GST-P(+) foci may be the signature of genetic alterations. Intraexonic hypermethylation may be responsible for p16(Ink4a)-downregulation, which facilitates cell cycle progression in early preneoplastic lesions produced by repeated cell regeneration and late-stage neoplastic lesions irrespective of the carcinogenic mechanism.

Involvement of multiple cell cycle aberrations in early preneoplastic liver cell lesions by tumor promotion with thioacetamide in a two-stage rat hepatocarcinogenesis model

Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16(Ink4a), which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16(Ink4a-) foci in concordance with GST-P(+) foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P(+) foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16(Ink4a) may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.

Reversible effect of maternal exposure to chlorpyrifos on the intermediate granule cell progenitors in the hippocampal dentate gyrus of rat offspring.

To examine the effects of developmental exposure to chlorpyrifos (CPF) on neurogenesis in the hippocampal dentate gyrus, pregnant rats were treated with 2.8, 14 or 70 ppm CPF in the diet from gestational day 10 to day 21 after delivery. Dams had decreased cholinesterase (ChE) activities in red blood cells (RBC) at intakes of ≥2.8 ppm and in brain at 70 ppm. Offspring on postnatal day (PND) 21 had decreased ChE activities in the RBC and brain at 70 ppm. There were no behavioral abnormalities in the offspring. Immunohistochemical analysis showed decreases in the numbers of cells positive for proliferating cell nuclear antigen and T box brain 2 in the subgranular zone (SGZ) of the dentate gyrus on PND 21 at 70 ppm, while other progenitor cell populations and the apoptotic cell number were unaffected in this zone. However, on PND 77 all changes had disappeared. The distribution of the progenitor cell population expressing nicotinic acetylcholine receptor α7 and lacking expression of postmitotic neuron-specific nuclear protein was unchanged by CPF-exposure, suggesting no effect of cholinergic stimulation on neurogenesis. These results suggest that developmental exposure to CPF directly but transiently affect the proliferation of type-2 progenitor cell populations in the hippocampal neurogenesis. The lowest-observed-adverse-effect level (LOAEL) of CPF was determined to be 2.8 ppm (0.36 mg/kg body weight/day) for dams by the inhibition of ChE activity in the RBC at this dose. As for offspring, no-observed-adverse-effect level (NOAEL) was determined to be 14 ppm (1.86 mg/kg body weight/day) by the decrease of type-2 progenitor cell proliferation in the SGZ and the inhibition of ChE activity in the RBC and brain at 70 ppm. The NOAEL of dams based on the offsprings effects was approximately 2800 times higher than the estimated consumption of CPF through food in the general population and in pregnant women as examined in Japan.

Global DNA methylation screening of liver in piperonyl butoxide-treated mice in a two-stage hepatocarcinogenesis model.

Disruptive epigenetic gene control has been shown to be involved in carcinogenesis. To identify key molecules in piperonyl butoxide (PBO)-induced hepatocarcinogenesis, we searched hypermethylated genes using CpG island (CGI) microarrays in non-neoplastic liver cells as a source of proliferative lesions at 25 weeks after tumor promotion with PBO using mice. We further performed methylation-specific polymerase chain reaction (PCR), real-time reverse transcription PCR, and immunohistochemical analysis in PBO-promoted liver tissues. Ebp4.1, Wdr6 and Cmtm6 increased methylation levels in the promoter region by PBO promotion, although Cmtm6 levels were statistically non-significant. These results suggest that PBO promotion may cause altered epigenetic gene regulation in non-neoplastic liver cells surrounding proliferative lesions to allow the facilitation of hepatocarcinogenesis. Both Wdr6 and Cmtm6 showed decreased expression in non-neoplastic liver cells in contrast to positive immunoreactivity in the majority of proliferative lesions produced by PBO promotion. These results suggest that both Wdr6 and Cmtm6 were spared from epigenetic gene modification in proliferative lesions by PBO promotion in contrast to the hypermethylation-mediated downregulation in surrounding liver cells. Considering the effective detection of proliferative lesions, these molecules could be used as detection markers of hepatocellular proliferative lesions and played an important role in hepatocarcinogenesis.

Identification of epigenetically downregulated Tmem70 and Ube2e2 in rat liver after 28-day treatment with hepatocarcinogenic thioacetamide showing gene product downregulation in hepatocellular preneoplastic and neoplastic lesions produced by tumor promotion.

The present study identified genes showing promoter region hypermethylation by CpG island microarrays in the liver of rats treated with hepatocarcinogen thioacetamide (TAA) for 28days. Among 47 hypermethylated genes, Hist1h2aa, Tmem70, Ube2e2, and Slk were confirmed to show hypermethylation by methylation-specific quantitative polymerase chain reaction (PCR) and pyrosequencing analyses as well as downregulation of transcript levels by real-time reverse transcription-PCR analysis in the livers of rats treated with TAA. All gene products of the 4 selected genes showed decreased immunoreactivity forming negative liver cell foci in a subpopulation of glutathione S-transferase placental form (GST-P)+ foci in TAA-promoted rat livers in a two-stage hepatocarcinogenesis model. Among them, TMEM70 and UBE2E2 showed increased incidences of negative foci in GST-P+ foci by promotion of all examined TAA, β-naphthoflavone, piperonyl butoxide, fenbendazole and phenobarbital, while HIST1H2AA and SLK did not respond to all promotive treatments. In the late stage of tumor promotion by TAA, the incidence of GST-P+ proliferative lesions with downregulation of TMEM70 or UBE2E2 was higher in adenomas and carcinomas than liver cell foci. TMEM70 plays a role in mitochondrial oxidative phosphorylation, and UBE2E2 participates in the stabilization of cell cycle regulatory proteins. Therefore, our results indicate that aberrant epigenetic gene downregulation suggestive of a metabolic shift of cellular respiration from oxidative phosphorylation to glycolysis and aberrant cell cycle regulation facilitating cell proliferation from as early as 28days after hepatocarcinogen treatment contribute to tumor development.

Direct progression of capsular invasive carcinomas from subcapsular focal hyperplasias induced by hypothyroidism-mediated tumor promotion in a rat two-stage thyroid carcinogenesis model.

PurposeSome goitrogens promote thyroid carcinogenesis in rats in an initiation-promotion model; this model frequently produces carcinomas that invade fibrously thickened capsules, termed capsular invasive carcinomas (CICs). The present study tested a hypothesis that CICs originate from parenchymal proliferative lesions located beneath the capsule.MethodsCell proliferation activity, cell-cycle kinetics and cellular invasion were immunohistochemically examined in subcapsular proliferative lesions in male F344 rats treated with an anti-thyroid agent, propylthiouracil or sulfadimethoxine, during the tumor-promotion phase after initiation with N-bis(2-hydroxypropyl)nitrosamine.ResultsFocal follicular cell hyperplasias (FFCHs) were the most commonly observed parenchymal proliferative lesions. Subcapsular FFCHs located near CICs showed more Ki-67+ cells in the capsular side than the contrary parenchymal center side. Most of these FFCHs located near CICs showed accumulated immunoreactivity for cyclin A, cyclin D, cyclin E and cyclin-dependent kinase-2, whereas most subcapsular FFCHs located elsewhere did not show such accumulated expression of cell-cycle molecules. Subcapsular FFCHs immunoreactive at the capsular front for tenascin-C, a tumor invasion marker of extracellular matrix protein, showed high proliferation activity.ConclusionsSubcapsular FFCH-forming cells can potentially spread directly into the fibrously thickened capsule to form CICs by accelerating cell-cycle activity.