Epigenetics (EG) is a highly regulated biochemical mechanism underlying the expression of genes related to development and cellular differentiation that is disrupted by environmental stressors including chemicals and radiation. Studies of these phenomena are known as environmental epigenetics (EEG). Regulation of gene expression by the epigenetic mechanism is deeply involved in the developmental stages of animals and humans. EEG is, therefore, very important in the field of toxicology because it deals with the state of gene expression in all types of somatic and germ cells disrupted by environmental chemicals. We propose here an “Embryo-originated Epigenetic Toxicology Method (EEGT)”. In this method embryonic somatic and germ cells are treated with test substances and various toxicological phenomena in whole bodies are examined. Observations on transgenerational effects are also important in this method. This new method could unite various toxicological phenomena based on EEG.

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Many researchers propose that invisible internal alterations that occur through exposure to environmental factors during fetal or neonatal stages affect the risk of cancer, hypertension, and diabetes after maturation. Barker's hypothesis, which states that reduced fetal growth is strongly associated with metabolic syndromes including cardiovascular disease and diabetes, has now been widely accepted and expanded into the Developmental Origins of Health and Disease (DOHaD). Potential molecular mechanisms underlying this phenomenon include the alteration and persistence of epigenomic programming. Clear biochemical evidence has not yet been obtained in human studies; however, in laboratory animals, the fetal environment including physical and chemical factors altered epigenomic states such as DNA methylation and histone modification, and persistent changes affected specific gene expression regulation, resulting in disease susceptibility. Furthermore, in recent studies, environmental chemical exposure during pregnancy altered sperm DNA methylation patterns of male offspring, and the altered status and resulting phenotypes were inherited in the next generation. Challenging and eccentric studies focusing on epigenetic transgenerational effects are currently being conducted to demonstrate the existence of Lamarckian inheritance.

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Congenital malformations can be induced in the offspring of laboratory animals treated with the mutagens, ethylnitrosourea, methylnitrosourea, mitomycin C, triethylenemelamine or procarbazine before copulation. The spectra of malformations in the offspring classified as male-mediated malformations after exposure of paternal mice to mutagens showed no evidence of mutagen-specificity or germ-cell stage-dependent variations. Recently, we demonstrated the increased incidence of congenital malformations in the offspring of male mice exposed in utero to synthetic estrogens such as diethylstilbestrol (DES), 17β-estradiol (E₂) or ethynyl estradiol (EE), and that the induction of male-mediated malformations by DES, E₂ or EE showed a clear threshold effect. Developmental exposure to DES, E₂ or EE caused partial atrophy and feminization in the genital tract. They also showed transgenerational effects when applied prenatally at a dose which caused histopathological changes in the testes. Germ-cell series in normal testis have mechanisms to select against spontaneously arising mutation; but these selection mechanisms may not function efficiently in chemically-damaged testes. Based on these results and considerations, we propose as a hypothesis that transgenerational teratogenesis by prenatal exposure to synthetic estrogens may occur as a consequence of testicular toxicity. Moreover, since DES has been reported to be non-genotoxic, epigenetic mechanisms such as DNA methylation may be involved in the transgenerational teratogenesis induced by estrogenic drugs. The expression patterns of DNA methyltransferases (Dnmts) mRNA, global DNA methylation levels in testicular cells of embryos exposed to estrogenic drugs or in epididymal sperm of mature male mice exposed prenatally to estrogenic drugs were different from those in the controls. Results shown in this review support the proposal that, when evaluating the toxicities of environmental chemicals including endocrine disruptors, epigenetic effects such as DNA methylation should be taken into account.

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Methylation of the cytosine C-5 position in the promoter region of tumor suppressor genes is an important mechanism of carcinogenesis in addition to gene mutation. However, the actual mechanisms of de novo methylation in relation to environmental agents are not clear. We found that cytosine C-5 methylation occurred in the monomer nucleoside and DNA by various methyl radical generating systems, including environmental agents. The possible role of this radical-induced DNA methylation in carcinogenesis is discussed in connection with the presently accepted concept of cancer epigenetics.

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In DNA methylation analysis for epigenetic studies, the most frequently used procedure involves bisulfite-mediated conversion of cytosine to uracil. Resistance of 5-methylcytosine against this bisulfite reaction is the basis for identifying the methylated sites. This chemical modification consists of a treatment of single-stranded nucleic acids with high-concentration bisufite at pH 5-6. We report here that the treatment can be performed at pH > 6 by supplementary presence of tetramethylammonium ion. Quaternary ammonium cation appears to be essential in this unique enhancement of the reaction in the near-neutral pH range, as betaine was also effective but trimethylamine was not. Using a tetramethylammonium-bisulfite reagent at pH 6.4, we achieved a complete deamination of cytosine residues in a DNA molecule, without any significant changes taking place for 5-methylcytosine in it. These findings have opened up the possibility of improving and expanding the DNA methylation analysis technology.

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