Annual Meeting of the Japanese Society of Toxicology

Exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces Cyp1a1, one of the major genes that play a key role in dioxin effects. A previous study showed that in utero TCDD exposure at embryonic day 12.5 dramatically increased Cyp1a1 induction during early development and caused DNA hypomethylation by postnatal day 14 at a specific CpG site, -500 from the Cyp1a1 transcription start site. Although this study showed epigenetic changes in the offspring from maternal TCDD exposure, exact effects in the earliest developmental stage are not known. Using mouse embryonic (ES) cells derived from B6N22^UTR mouse, this study explored the in vitro genetic and epigenetic effects from TCDD exposure. First, ES cells were differentiated into neuronal lineages for 11 days. As differentiation progressed, cells became neuronal-like expressing Map2. Although Cyp1a1 transcription was induced by TCDD exposure, the induction decreased as differentiation progressed. In the second experiment, hepatic cell differentiation method was used as a better candidate to examine Cyp1a1 transcriptional activity. ES cells stably transfected with EGFP driven by Cyp1a1 promoter were cultured into hepatocytes and were observed for 30 days. Fluorescent microscopy determined that some of EGFP-positive cells were clearly induced in TCDD treated cells. However, there was no change in DNA methylation. From these results, it can be concluded that in vitro, TCDD alters DNA methylation differently from in vivo models. Nevertheless, finding the epigenetic similarities between in vitro and in vivo toxic effects will open the door to mechanisms underlying epigenetic alterations arising from toxicological exposures during early development.

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The neonicotinoids are a new class of widely used potent insecticides accounting for over 17% of the global insecticide market. They were developed from the modification of nicotinoids and are thus similar to nicotine in structure and function. Acting primarily and selectively at insect nicotinic acetylcholine receptor (nAChR) in the central nervous system (CNS), they are believed to possess low toxicity to vertebrates. However, several in vivo and in vitro mammalian studies (mouse) have reported neonicotinoids to impair reproduction at almost all levels. The adverse effects of nicotine on pregnancy have also been reported severally and are known to be mediated via the nAChR hence, we investigated for the adverse effects of neonicotinoids on human trophoblast cells and the underlying signaling mechanisms using human choriocarcinoma NJG and BeWo cells. Cells were treated with neonicotinoids for 24, 48 and 72 h with re-exposures every 24 h. This resulted in a time and concentration-dependent growth inhibition in both cell lines. Increased generation of reactive oxygen species (ROS) suggested involvement of oxidative stress in the cytotoxicity of neonicotinoids. Western blot data revealed that neonicotinoid exposure also induced a dose and time dependent activation of ERK1/2 which was inhibited by mecamylamine (a non-selective inhibitor of nAChR). Cleaved caspase-3 expression substantiated neonicotinoid induced cytotoxicity and suggests apoptosis as the pathway through which cell death occurs. Overall, these findings suggest that neonicotinoids and nicotine exert similar cytotoxic effect to human trophoblasts and may adversely affect reproduction in humans through disruption of essential signaling pathways.
Funding source: This work was supported in part by Program for Leading Graduate Schools “HIGO (Health life science: Interdisciplinary and Glocal Oriented), MEXT, Japan.

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Epigenetic modifications on histones and DNA exercise precise regulation of gene expression, and thus have a profound influence on cellular function. Through unclear mechanisms, xenobiotics are known to cause perturbations in the epigenome, resulting in altered biological states and aberrant outcomes.
In this study, 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) administered orally at 3μg/kg bodyweight caused Cyp1a1 promoter demethylation within 24 h in the mouse liver, indicating possible active DNA demethylation. This change in DNA methylation occurred with changes in 5-hydroxymethylcytosine, an active DNA demethylation intermediate, and persisted up to 40 days post treatment. As anticipated, there was a concurrent increase in the transcriptionally permissive H3K4me3 mark. These findings were replicated in vitro in isolated mouse hepatocytes. The epigenetic changes induced by TCDD were Ahr dependent, as Ahr KO mice and derived hepatocytes had intact Cyp1a1 promoter methylation levels. Additionally, TCDD did not change mRNA expression levels of known mediators of active DNA demethylation including Tet1, Tet2, Tet3, Tdg, Apex1, Gadd45a and Apobec1.
We show that TCDD, through Ahr, induces epigenetic switching at the Cyp1a1 promoter which results in memorization of the xenobiotic exposure event.

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Purpose: This study is to review the evidence comparing the efficacy and safety between L-carnitine and extracorporeal elimination therapy in the management of acute valproic acid overdose.

Methods: PubMed, Embase, Cochrane library, Web of science, KoreaMed, KMbase, and KISS were searched, using the terms carnitine and valproic acid. All studies, regardless of design, reporting efficacy or safety endpoints were included. Reference citations from identified publications were reviewed. Both English and Korean languages were included. Two authors extracted primary data elements including poisoning severity, presenting features, clinical management, and outcomes.

Results: Thirty two articles including 33 cases were identified. Poisoning severity was classified as 3 mild, 11 moderate, and 19 severe cases. Nine cases were treated with L-carnitine while 24 cases were performing extracorporeal therapy without L-carnitine. All patients except one expired patient who treated with hemodialysis recovered clinically and no adverse effects were noted. A case report comparing two patients who ingested same amount of valproic acid showed increased ICU stay (3 vs 11 days) in case of delayed extracorporeal therapy.

Conclusion: Published evidence comparing L-carnitine with extracorporeal therapy is limited. Based on the available evidence, it is reasonable to consider L-carnitine for patients with acute valproic acid overdose. In case of severe poisoning, extracorporeal therapy would also be considered in the early phase of treatment.

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