A practice of expert review by read-across using QSAR Toolbox.

The International Council for Harmonisation of Technical Requirement for Pharmaceuticals for Human Use (ICH) M7 guideline on 'Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk' provides the application of two types of quantitative structure-activity relationship (QSAR) systems (rule- and statistics-based) as an alternative to the Ames test for evaluating the mutagenicity of impurities in pharmaceuticals. M7 guideline also states that the expert reviews can be applied when the outcomes of the two QSAR analyses show any conflicting or inconclusive prediction. However, the guideline does not provide any information of how to conduct expert reviews. Therefore, a conservative approach was chosen in this study, which is based on the intention to capture any mutagenic chemical substances. The 36 chemical substances, which are the model chemical substances in which positive mutagenicity was not observed according to the two types of QSAR analyses (i.e. the results are either conflicting or both negative), were selected from the list of chemical substances with strong mutagenicity known as the reported chemicals under the Industrial Safety and Health Act in Japan. The QSAR Toolbox was used in this study to rationally determine the positive mutagenicity of the 36 model chemical substances by applying a read-across method, a technique to evaluate the endpoint of the model chemical substances using the endpoint information of chemicals that are structurally similar to the model chemical substances. Resulting from the expert review by the read-across method, the 23 model chemical substances (63.8%) were rationally concluded as positive. In addition, 9 out of 11 model chemical substances that were assessed as negative for mutagenicity by both of the QSAR systems had positive analogues, supporting their mutagenicity. These results suggested that the read-across is a useful method, when conducting a conservative approach intended to capture any mutagenic chemical substances.

Investigating toxicity specific to adjuvanted vaccines.

In an attempt to understand the unique toxicity of adjuvanted vaccines, we studied how toxicity develops over time following vaccine administration. In addition to on- and off-target toxicity typically observed with general pharmaceuticals, we observed toxicity associated with both the generation and the broad action of effectors (antibodies and/or cytotoxic T lymphocytes, CTLs). The impact on effector generation appears to be related to local tolerance specific to the adjuvant. The vaccine immune response by effectors serves to demonstrate species relevance as outlined in the recent WHO guideline on the nonclinical evaluation of adjuvanted vaccines. When regarded as pharmaceuticals that function at sites of local administration, adjuvants have inherent on- and off-target toxicity. On-target toxicity of the adjuvant is typically associated with effector generation, and could vary depending on animal species. Therefore, the use of species with sensitivity to adjuvants described in the WHO guidelines is required to evaluate the toxicity of the vaccine associated with effector generation. Changes in safety pharmacology endpoints would be considered off-target and further studies are conducted only if changes in these endpoints are observed in nonclinical or clinical studies. Thus our decision tree does not recommend the routine conduct of stand-alone safety pharmacology studies.

Novel sex-dependent differentially methylated regions are demethylated in adult male mouse livers.

In mammalian livers, sexual dimorphisms are observed in tissue-specific functions and diseases such as hepatocellular carcinoma. We identified sex-dependent differentially methylated regions (S-DMRs) which had been previously been characterized as growth hormone- STAT5 dependent. In this study, we performed genome-wide screening and identified ten additional hypomethylated S-DMR gene regions in male livers. Of these S-DMRs, Uggt2 and Sarnp were hypomethylated in both male and female livers compared to brain and embryonic stem (ES) cells. Similarly, Adam2, Uggt2, and Scp2 were hypomethylated in female embryonic germ (EG) cells and not in male EG cells, indicating that these S-DMRs are liver-specific male hypo-S-DMRs. Interestingly, the five S-DMRs were free from STAT5 chromatin immunoprecipitation (ChIP) signals, suggesting that S-DMRs are independent of the growth hormone-STAT5-pathway. Instead, the DNA methylation statuses of the S-DMRs of Adam2, Snx29, Uggt2, Sarnp, and Rnpc3 genes were under the control of testosterone. Importantly, the hypomethylated S-DMRs of the Adam2 and Snx29 regions showed chromatin decondensation. Epigenetic factors could be responsible for the sexual dimorphisms in DNA methylation status and chromatin structure, as the expression of Dnmt1, Dnmt3b, and Tet2 genes was lower in male mice compared to female mice and TET2 expression recovered following orchidectomy by testosterone treatment. In conclusion, we identified novel male-specific hypomethylated S-DMRs that contribute to chromatin decondensation in the liver. S-DMRs were tissue-specific and the hypomethylation is testosterone-dependent.

DNA methylation profile dynamics of tissue-dependent and differentially methylated regions during mouse brain development.

BACKGROUND: Tissues and their component cells have unique DNA methylation profiles comprising DNA methylation patterns of tissue-dependent and differentially methylated regions (T-DMRs). Previous studies reported that DNA methylation plays crucial roles in cell differentiation and development. Here, we investigated the genome-wide DNA methylation profiles of mouse neural progenitors derived from different developmental stages using HpyCH4IV, a methylation-sensitive restriction enzyme that recognizes ACGT residues, which are uniformly distributed across the genome. RESULTS: Using a microarray-based genome-wide DNA methylation analysis system focusing on 8.5-kb regions around transcription start sites (TSSs), we analyzed the DNA methylation profiles of mouse neurospheres derived from telencephalons at embryonic days 11.5 (E11.5NSph) and 14.5 (E14.5NSph) and the adult brain (AdBr). We identified T-DMRs with different DNA methylation statuses between E11.5NSph and E14.5NSph at genes involved in neural development and/or associated with neurological disorders in humans, such as Dclk1, Nrcam, Nfia, and Ntng1. These T-DMRs were located not only within 2 kb but also distal (several kbs) from the TSSs, and those hypomethylated in E11.5NSph tended to be in CpG island (CGI-) associated genes. Most T-DMRs that were hypomethylated in neurospheres were also hypomethylated in the AdBr. Interestingly, among the T-DMRs hypomethylated in the progenitors, there were T-DMRs that were hypermethylated in the AdBr. Although certain genes, including Ntng1, had hypermethylated T-DMRs 5' upstream, we identified hypomethylated T-DMRs in the AdBr, 3' downstream from their TSSs. This observation could explain why Ntng1 was highly expressed in the AdBr despite upstream hypermethylation. CONCLUSION: Mouse adult brain DNA methylation and gene expression profiles could be attributed to developmental dynamics of T-DMRs in neural-related genes.

DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells.

BACKGROUND: Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model. RESULTS: We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire(-/-) mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire(-/-) mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire(+/+) mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire(+/+) and Aire(-/-) mTECs but distinct from that of athymic cells and tissues. CONCLUSIONS: These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

DNA methylation status of nuclear-encoded mitochondrial genes underlies the tissue-dependent mitochondrial functions.

BACKGROUND: Mitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed. RESULTS: We analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclear mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain. CONCLUSIONS: A substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.

Enrichment of short interspersed transposable elements to embryonic stem cell-specific hypomethylated gene regions.

Embryonic stem cells (ESCs) have a distinctive epigenome, which includes their genome-wide DNA methylation modification status, as represented by the ESC-specific hypomethylation of tissue-dependent and differentially methylated regions (T-DMRs) of Pou5f1 and Nanog. Here, we conducted a genome-wide investigation of sequence characteristics associated with T-DMRs that were differentially methylated between ESCs and somatic cells, by focusing on transposable elements including short interspersed elements (SINEs), long interspersed elements (LINEs) and long terminal repeats (LTRs). We found that hypomethylated T-DMRs were predominantly present in SINE-rich/LINE-poor genomic loci. The enrichment for SINEs spread over 300 kb in cis and there existed SINE-rich genomic domains spreading continuously over 1 Mb, which contained multiple hypomethylated T-DMRs. The characterization of sequence information showed that the enriched SINEs were relatively CpG rich and belonged to specific subfamilies. A subset of the enriched SINEs were hypomethylated T-DMRs in ESCs at Dppa3 gene locus, although SINEs are overall methylated in both ESCs and the liver. In conclusion, we propose that SINE enrichment is the genomic property of regions harboring hypomethylated T-DMRs in ESCs, which is a novel aspect of the ESC-specific epigenomic information.

Genome-wide DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) residing in mouse pluripotent stem cells.

DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), has elucidated tissue-specific gene function in mouse tissues. Here, we identified and profiled thousands of T-DMRs in embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs). T-DMRs of ESCs compared with somatic tissues well illustrated gene function of ESCs, by hypomethylation at genes associated with CpG islands and nuclear events including transcriptional regulation network of ESCs, and by hypermethylation at genes for tissue-specific function. These T-DMRs in EGCs and iPSCs showed DNA methylation similar to ESCs. iPSCs, however, showed hypomethylation at a considerable number of T-DMRs that were hypermethylated in ESCs, suggesting existence of traceable progenitor epigenetic information. Thus, DNA methylation profile of T-DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells.

DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) in mouse promoter regions demonstrating tissue-specific gene expression.

DNA methylation constitutes an important epigenetic regulation mechanism in many eukaryotes, although the extent of DNA methylation in the regulation of gene expression in the mammalian genome is poorly understood. We developed D-REAM, a genome-wide DNA methylation analysis method for tissue-dependent and differentially methylated region (T-DMR) profiling with restriction tag-mediated amplification in mouse tissues and cells. Using a mouse promoter tiling array covering a region from -6 to 2.5 kb ( approximately 30,000 transcription start sites), we found that over 3000 T-DMRs are hypomethylated in liver compared to cerebrum. The DNA methylation profile of liver was distinct from that of kidney and spleen. This hypomethylation profile marked genes that are specifically expressed in liver, including key transcription factors such as Hnf1a and Hnf4a. Genes with T-DMRs, especially those lacking CpG islands and those with HNF-1A binding motifis in their promoters, showed good correlation between their tissue-specific expression and liver hypomethylation status. T-DMRs located downstream from their transcription start sites also showed tissue-specific gene expression. These data indicate that multilayered regulation of tissue-specific gene function could be elucidated by DNA methylation tissue profiling.

Reproductive phenotypes in mice with targeted disruption of the 20alpha-hydroxysteroid dehydrogenase gene.

In the corpus luteum of rats and mice, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) catalyzes the conversion of progesterone to a biologically inactive metabolite, 20alpha-dihydroprogesterone (20alpha-OHP). The reduction of progesterone by 20alpha-HSD is believed to be important for functional luteolysis in these rodent species. In addition to the corpus luteum, expression of 20alpha-HSD has been demonstrated in tissues such as the placenta, endometrial epithelia, and fetal skin, although the roles it plays in the latter tissues remain to be determined. To determine the contribution of 20alpha-HSD to functional luteolysis and to the rodent reproductive system more generally, we generated a strain of mice with targeted disruption of the 20alpha-HSD gene. In the 20alpha-HSD-/- mice we obtained, which lacked the genomic region essential for catalytic reaction, neither 20alpha-HSD activity in the corpus luteum nor an increase in the serum concentrations of 20alpha-OHP during pseudopregnancy or pregnancy was detected. The durations of the estrous cycle, pseudopregnancy, and pregnancy were significantly prolonged in the 20alpha-HSD-/- mice, although the serum progesterone levels decreased to levels low enough for delivery of pups at term of pregnancy. In addition, the number of pups, especially live pups, was markedly decreased in the 20alpha-HSD-/- mice. These findings suggest that the role of 20alpha-HSD in functional luteolysis is relatively minor but that it is involved in the survival of newborn mice.

Characterization and functional analysis of the 5'-flanking region of the mouse 20alpha-hydroxysteroid dehydrogenase gene.

20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20alpha-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5'-flanking region, revealed that the region between -83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wild-type Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20alpha-HSD gene.

Cloning and chromosomal localization of mouse 20alpha-hydroxysteroid dehydrogenase gene.

20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, metabolizes progesterone to its inactive form, 20alpha-dihydroprogesterone (20alpha-OHP). 20alpha-HSD is associated with functional luteolysis and plays a significant role in the reproductive system of rodents. Here we report cloning and determination of the chromosomal location of the mouse 20alpha-HSD gene. The mouse 20alpha-HSD gene cloned using 129 SvJ mouse genomic library spanned approximately 18 kb from exon 1 to exon 9. A single transcription start site was identified by 5'-rapid amplification of cDNA ends (RACE) at the site of 50 nucleotides upstream from the ATG translation initiation codon. The exon-intron organization of mouse and human 20alpha-HSD genes were similar. Using 17.5 kb of genomic clone as a probe, we determined the chromosomal location by fluorescence in situ hybridization (FISH). The chromosome in which the mouse 20alpha-HSD gene was detected was identified as chromosome (Chr) 13 according to simultaneous G- and R-banding. Because type 5 17beta-HSD gene, a member of the AKR superfamily, is also located on Chr 13, the present result supports the syntenic relationship between mouse Chr 13 and human Chr 10, which was previously suggested at the chromosomal loci of a gene cluster of the AKR superfamily.

Effects of pair-feeding and growth hormone treatment on obese transgenic rats.

BACKGROUND: It has been shown that GH-deficient subjects tend to have fat accumulation. We have produced human GH (hGH) transgenic rats that exhibit low circulating hGH levels and hyperphagia. These rats are also characterized by severe obesity, hyperinsulinemia and hyperlipidemia. OBJECTIVE: The present study was conducted in order to elucidate how excess caloric intake and impaired GH secretion account for fat accumulation and metabolic abnormalities in the transgenic rats. DESIGN AND METHODS: The transgenic rats were subjected to either pair-feeding with non-transgenic controls or hGH treatment from 4 to 12 weeks of age, and the effects on fat accumulation and some metabolic parameters were assessed. RESULTS: At the age of 12 weeks, body weight and food intake were greater in transgenic than in control rats by 10% and 27% respectively. The ratio of epididymal white adipose tissue weight to body weight (WAT/BW) was more than three times greater in transgenic than in control rats. Although pair-feeding for 8 weeks decreased body weight, it did not affect the WAT/BW ratio. Treatment with hGH affected neither body weight nor food intake, while it reduced the WAT/BW ratio by 30%. Serum concentrations of triglyceride, free fatty acid, insulin and leptin were all significantly higher in the transgenic than in the control rats. Pair-feeding decreased serum triglyceride, insulin and leptin levels, but not serum free fatty acid levels. On the other hand, hGH treatment decreased only serum leptin concentrations. CONCLUSIONS: These results suggest that severe fat accumulation in the transgenic rats mainly resulted from the decreased lipolytic action of GH, while metabolic abnormalities mainly resulted from excess caloric intake.