Benzyl isothiocyanate and its metabolites inhibit cell proliferation through protein modification in mouse preosteoclast RAW264.7 cells.

Benzyl isothiocyanate (BITC), derived from cruciferous vegetables, is an organosulfur compound exerting antiproliferative effects in several human cancer cells. In this study, we assessed BITC as a potential osteoclastogenesis inhibitor and investigated its underlying mechanism. BITC at 5 µM significantly decreased the viability of the osteoclast-like differentiating RAW264.7 cells, coinciding with the downregulation of the primary biomarkers for osteoclast differentiation, such as the tartrate-resistant acid phosphatase activity and nuclear factor of activated T-cells gene expression. Not only BITC but also its metabolites, inhibited cell proliferation in the normal RAW264.7 cells, suggesting that BITC shows an anti-osteoclastogenesis effect in vivo after its ingestion and metabolism, possibly through an antiproliferative action. Both BITC and its metabolites also enhanced the DNA fragmentation and the caspase-3 activity, whereas their higher concentrations tended to suppress these effects. BITC was intracellularly accumulated when the cells were treated with its metabolites via their degradation into the free form. A quantitative experiment using the proteolysis/high performance liquid chromatography technique showed that the amount of BITC-lysine thiourea in the cells was also increased in a time-dependent manner, suggesting that lysine modification of the cellular proteins actually took place in the cells treated by BITC. Among the cellular proteins, the cleaved caspase-3 was identified as a potential target for lysine modification by BITC. Taken together, BITC dissociated from its metabolites as well as its free form might modulate osteoclastogenesis, possibly through inhibition of cell proliferation by protein modification.

Genetic characterization of Kushum horses in Kazakhstan based on haplotypes of mtDNA and Y chromosome, and genes associated with important traits of the horses.

The Kushum is a relatively new breed of horses in Kazakhstan that was established in the middle of the 20th century through a cross between mares of Kazakhstan local horses and stallions of Thoroughbred, Trotter, and Russian Don breeds to supply military horses. To reveal the genetic characteristics of this breed, we investigated haplotypes of mitochondrial DNA (mtDNA) and single-nucleotide polymorphisms of the Y chromosome, as well as genotypes of five functional genes associated with coat color, body composition, and locomotion traits. We detected 10 mtDNA haplotypes that fell into 8 of the 17 major haplogroups of horse mtDNA, indicating a unique haplotype composition with high genetic diversity. We also found two Y-chromosomal haplotypes in Kushum horses, which likely originated from Trotter and/or Don breeds. The findings regarding the mtDNA and Y-chromosomal haplotypes are concordant with the documented maternal and paternal origins of the Kushum horses. The allele frequencies of ASIP, MC1R, and MATP associated with coat color were consistent with the coat color variations of Kushum horses. The allele frequencies of MSTN associated with endurance performance and those of DMRT3 associated with gait suggested that the observed allele frequencies of these genes were the result of selective breeding for these traits. As a result of this study, we were able to obtain useful information for a better understanding of the origin and breeding history of the Kushum horse breed using molecular markers.

Refining the evolutionary tree of the horse Y chromosome.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski's horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Comparison of the Hepatic Effects of Phenobarbital in Chimeric Mice Containing Either Rat or Human Hepatocytes With Humanized Constitutive Androstane Receptor and Pregnane X Receptor Mice.

Using a chimeric mouse humanized liver model, we provided evidence that human hepatocytes are refractory to the mitogenic effects of rodent constitutive androstane receptor (CAR) activators. To evaluate the functional reliability of this model, the present study examined mitogenic responses to phenobarbital (PB) in chimeric mice transplanted with rat hepatocytes, because rats are responsive to CAR activators. Treatment with 1000 ppm PB for 7 days significantly increased replicative DNA synthesis (RDS) in rat hepatocytes of the chimeric mice, demonstrating that the transplanted hepatocyte model is functionally reliable for cell proliferation analysis. Treatment of humanized CAR and pregnane X receptor (PXR) mice (hCAR/hPXR mice) with 1000 ppm PB for 7 days significantly increased hepatocyte RDS together with increases in several mitogenic genes. Global gene expression analysis was performed with liver samples from this and from previous studies focusing on PB-induced Wnt/ß-catenin signaling and showed that altered genes in hCAR/hPXR mice clustered most closely with liver tumor samples from a diethylnitrosamine/PB initiation/promotion study than with wild-type mice. However, different gene clusters were observed for chimeric mice with human hepatocytes for Wnt/ß-catenin signaling when compared with those of hCAR/hPXR mice, wild-type mice, and liver tumor samples. The results of this study demonstrate clear differences in the effects of PB on hepatocyte RDS and global gene expression between human hepatocytes of chimeric mice and hCAR/hPXR mice, suggesting that the chimeric mouse model is relevant to humans for studies on the hepatic effects of rodent CAR activators whereas the hCAR/hPXR mouse is not.

Distribution of the mutant allele of the DMRT3 gene associated with ambling gaits in Japanese native horse populations.

There are currently eight native horse populations in Japan, namely, Hokkaido, Kiso, Noma, Taishu, Misaki, Tokara, Miyako, and Yonaguni horses. Since locomotion traits, including gaitedness, are important for riding and packing horses, the genetic properties associated with these traits could be informative for understanding the characteristics and history of these horses. In this study, we investigated the distribution of the mutant allele of DMRT3 gene (DMRT3:p.Ser301Ter) associated with ambling gaits in the Japanese native horse. We also examined haplotypes of SNPs in the 83-kb region including DMRT3 gene by genotyping four SNPs in this region. The results revealed the presence of DMRT3:p.Ser301Ter in the Hokkaido and Yonaguni populations at allele frequencies of 0.18 and 0.02, respectively, and the observed haplotype associated with DMRT3:p.Ser301Ter was estimated as the most common haplotype in the horses in the world. Since DMRT3:p.Ser301Ter has been hypothesized to spread across Eurasian continent from Medieval England after 850 to 900 CE, our findings of the presence of DMRT3:p.Ser301Ter with the common haplotype in the Japanese native horses will provide a new insight into the history of the Japanese native horse, such as considerable level of gene flow from Eurasian continent after 850 to 900 CE.

Genetic characterization of Vietnamese Yellow cattle using mitochondrial DNA and Y-chromosomal haplotypes and genes associated with economical traits.

Vietnamese Yellow cattle are native cattle well adapted to local tropical environment. The aim of this study was to investigate genetic characteristics of the Yellow cattle using molecular markers. We investigated the nucleotide sequences of mitochondrial DNA and SRY gene on Y chromosome, and genotyped SREBP-1, SCD1, EDG1, NCAPG, DGAT1, MC1R, and HSP70 genes in the Yellow cattle population. The sequence analysis of the mitochondrial DNA showed that most of the cattle possesses zebu (Bos indicus) type I1 haplotype, suggesting relatively low genetic diversity in maternal lineage. The sequence analysis of the SRY gene indicates that while most of the males possess zebu type haplotype, taurine (Bos taurus) type haplotype was also observed, suggesting gene-flow from taurine cattle. The results of the genotyping of the functional genes showed that the NCAPG, SCD, MC1R, and HsSP70 genes are polymorphic in the population, whereas the SREBP-1, EDG1, and DGAT1 genes are monomorphic. Particularly, the presence of the desirable and undesirable alleles of the NCAPG and HSP70 genes, respectively, will be important for the selection of animals by potential performances in meat productivity and fertility. The present findings will be informative for future conservation and breeding of the Vietnamese Yellow cattle.

Candidate genes responsible for early key events of phenobarbital-promoted mouse hepatocellular tumorigenesis based on differentiation of regulating genes between wild type mice and humanized chimeric mice.

Phenobarbital (PB) is a nongenotoxic hepatocellular carcinogen in rodents. PB induces hepatocellular tumors by activating the constitutive androstane receptor (CAR). Some previous research has suggested the possible involvement of epigenetic regulation in PB-promoted hepatocellular tumorigenesis, but the details of its molecular mechanism are not fully understood. In the present study, comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed in mouse hepatocellular adenomas induced by a single 90 mg kg-1 intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumor development-related genes. Pathway enrichment analysis revealed that differentially methylated or hydroxymethylated genes belong mainly to pathways involved in development, immune response and cancer cells in contrast to differentially expressed genes belonging primarily to the cell cycle. Furthermore, overlap was evaluated between the genes with altered expression levels with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma induced by DEN/PB and the genes with altered expression levels in the liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days. With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis. Interestingly, these genes did not overlap with genes altered by the PB treatment of humanized chimeric mice, thus suggesting a species difference between the effects of PB in mouse and human hepatocytes.

Editor's Highlight: Mode of Action Analysis for Rat Hepatocellular Tumors Produced by the Synthetic Pyrethroid Momfluorothrin: Evidence for Activation of the Constitutive Androstane Receptor and Mitogenicity in Rat Hepatocytes.

High dietary levels of momfluorothrin, a nongenotoxic synthetic pyrethroid, induced hepatocellular tumors in male and female Wistar rats in a 2-year bioassay. The mode of action (MOA) for rat hepatocellular tumors was postulated to occur via activation of the constitutive androstane receptor (CAR), as momfluorothrin is a close structural analogue of the pyrethroid metofluthrin, which is known to produce rat liver tumors through a CAR-mediated MOA. To elucidate the MOA for rat hepatocellular tumor formation by momfluorothrin, this study was conducted to examine effects on key and associative events of the CAR-mediated MOA for phenobarbital based on the International Programme on Chemical Safety framework. A 2-week in vivo study in Wistar rats revealed that momfluorothrin induced CYP2B activities, increased liver weights, produced hepatocyte hypertrophy and increased hepatocyte replicative DNA synthesis. These effects correlated with the dose-response relationship for liver tumor formation and also showed reversibility upon cessation of treatment. Moreover, momfluorothrin did not increase CYP2B1/2 mRNA expression and hepatocyte replicative DNA synthesis in CAR knockout rats. Using cultured Wistar rat hepatocytes and the RNA interference technique, knockdown of CAR resulted in a suppression of induction of CYP2B1/2 mRNA levels by momfluorothrin. Alternative MOAs for liver tumor formation were excluded. A global gene expression profile analysis of the liver of male Wistar rats treated with momfluorothrin for 2 weeks also showed similarity to the prototypic CAR activator phenobarbital. Overall, these data strongly support that the postulated MOA for momfluorothrin-induced rat hepatocellular tumors as being mediated by CAR activation.

Evaluation of the human relevance of the constitutive androstane receptor-mediated mode of action for rat hepatocellular tumor formation by the synthetic pyrethroid momfluorothrin.

High dietary levels of the non-genotoxic synthetic pyrethroid momfluorothrin increased the incidence of hepatocellular tumors in male and female Wistar rats. Mechanistic studies have demonstrated that the mode of action (MOA) for momfluorothrin-induced hepatocellular tumors is constitutive androstane receptor (CAR)-mediated. In the present study, to evaluate the potential human carcinogenic risk of momfluorothrin, the effects of momfluorothrin (1-1,000 µM) and a major metabolite Z-CMCA (5-1,000 µM) on hepatocyte replicative DNA synthesis and CYP2B mRNA expression were examined in cultured rat and human hepatocyte preparations. The effect of sodium phenobarbital (NaPB), a prototypic rodent hepatocarcinogen with a CAR-mediated MOA, was also investigated. Human hepatocyte growth factor (hHGF) produced a concentration-dependent increase in replicative DNA synthesis in rat and human hepatocytes. However, while NaPB and momfluorothrin increased replicative DNA synthesis in rat hepatocytes, NaPB, momfluorothrin and Z-CMCA did not increase replicative DNA synthesis in human hepatocytes. NaPB, momfluorothrin and Z-CMCA increased CYP2B1/2 mRNA levels in rat hepatocytes. NaPB and momfluorothrin also increased CYP2B6 mRNA levels in human hepatocytes. Overall, while momfluorothrin and NaPB activated CAR in cultured human hepatocytes, neither chemical increased replicative DNA synthesis. Furthermore, to confirm whether the findings observed in vitro were also observed in vivo, a humanized chimeric mouse study was conducted. Replicative DNA synthesis was not increased in human hepatocytes of chimeric mice treated with momfluorothrin or its close structural analogue metofluthrin. As human hepatocytes are refractory to the mitogenic effects of momfluorothrin, in contrast to rat hepatocytes, the data support the hypothesis that the MOA for momfluorothrin-induced rat liver tumor formation is not relevant for humans.

Genotype distribution and allele frequencies of the genes associated with body composition and locomotion traits in Myanmar native horses.

Myanmar native horses are small horses used mainly for drafting carts or carriages in rural areas and packing loads in mountainy areas. In the present study, we investigated genotype distributions and allele frequencies of the LCORL/NCAPG, MSTN and DMRT3 genes, which are associated with body composition and locomotion traits of horses, in seven local populations of Myanmar native horses. The genotyping result of LCORL/NCAPG showed that allele frequencies of C allele associated with higher withers height ranged from 0.08 to 0.27, and 0.13 in average. For MSTN, allele frequencies of C allele associated with higher proportion of Type 2B muscular fiber ranged from 0.05 to 0.23, and 0.09 in average. For DMRT3, allele frequencies of A allele associated with ambling gait ranged from 0 to 0.04, and 0.01 in average. The presences of the minor alleles of these genes at low frequencies suggest a possibility that these horse populations have not been under strong selection pressure for particular locomotion traits and body composition. Our findings of the presence of these minor alleles in Southeast Asian native horses are also informative for considering the origins of these minor alleles associated with body composition and locomotion traits in horse populations.

Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver.

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45ß, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.

Antitumor NK activation induced by the Toll-like receptor 3-TICAM-1 (TRIF) pathway in myeloid dendritic cells.

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR(-/-), MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.