Annual Meeting of the Japanese Society of Toxicology

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Objectives: Acrylamide (ACR), an environmental soft electrophile, is used for synthesis of polyacrylamide in industry and for production of gels in scientific research. Recently ACR contamination has also been reported in certain carbohydrate-rich foods cooked at high temperature. Neurotoxicity of ACR has been reported in humans and experimental animals. However, underlying mechanism of ACR neurotoxicity remains elusive. The present study aims to investigate the role of microglial activation and neuroinflammation in the neurotoxicity of ACR. Methods: In vivo: Male 10-week-old Wistar rats were exposed to ACR by gavage at 0, 0.2, 2, or 20 mg/kg BW, once/day, 7 days/week, for 5 weeks. Expression levels of inflammation related genes and microglial markers in the cerebral cortex were examined by quantitative real-time PCR. In vitro: Murine immortalized microglial BV-2 cells were treated with ACR at different dose (0, 5, 50, or 500 μM), for different time (0, 4, 6, 8, 12, 18, 24, or 36 h). MTS assay was performed to evaluate effects of ACR exposure on cell viability. Expression of inflammation related genes was examined by quantitative real-time PCR. Results: In vivo studies showed that ACR exposure at 20 mg/kg BW for 5 weeks significantly increased expression of pro-inflammatory cytokines IL-1β and IL-6 in rat cerebral cortex. ACR exposure at 2 mg/kg BW for 5 weeks significantly increased IL-18 expression level in the cerebral cortex. ACR exposure at 2 mg/kg BW for 5 weeks significantly increased expression of microglial markers, including CD11b and CD40, in rat cerebral cortex. For in vitro study, MTS assay showed that ACR exposure ranging from 10 to 1000 μM did not decrease BV2 microglia cell viability; while ACR exposure at more than 2000 μM significantly decreased BV2 cell viability. ACR exposure increased expression of pro-inflammatory cytokines IL-1β and IL-18 in BV2 microglia, in a time- and dose- dependent manner. Expression of iNOS, which is also an inflammatory marker, was increased after ACR exposure. Moreover, NLRP3 inflammasome-related genes, including NLRP3, Caspase 1 and ASC, which regulate production of cytokines IL-1β and IL 18, were also increased in the cerebral cortex of rats exposed to ACR at 2 mg/kg BW for 5-weeks. Conclusions: The above in vivo and in vitro results suggest that microglia activation and neuroinflammation play roles in neurotoxicity of ACR, probably through NLRP3 inflammasome pathway.

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Most petroleum substances (PS) are produced at a volume of >100 tonnes/year in the EU, hence need to be assessed for prenatal developmental toxicity (PDT). If this is done following the current OECD guidelines, a large number of experimental animals are needed to fulfil the data gap. The application of in vitro assays, such as the zebrafish embryo test (ZET), may reduce animal experimentation and resources needed to study the PDT potencies of PS for example by providing a way to set priorities. PS are complex materials comprising hundreds to millions of different hydrocarbon compounds, including polycyclic aromatic hydrocarbons (PAHs). PDT as observed with some PS has been associated with the presence of 3-7 ring PAHs (Kamelia et al. 2017). To test this hypothesis to a further extent, DMSO-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid products (GTL), containing no PAHs, were tested in the ZET. The results show that DMSO-extracts of PS caused concentration-dependent inhibition on zebrafish embryos development and their potency appeared to be associated with the amount of 3- to 5- ring PAHs they contain. The observed effects include delayed-development, dorsal curvature, shorter body length, pericardial and yolk sac edema. On the contrary, and as expected, both of the GTL extracts did not affect the development of zebrafish embryo. Our findings showed the applicability of the ZET to assess in vitro PDT of PS and that this potency is proportional to their 3-5 ring PAH content. This supports our hypothesis that PAHs are primary inducers of the PDT of some PS.

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To explore total mercury (THg) and methylmercury (MeHg) bioaccumulation in the Bimastus parvus species earthworm (B. parvus), native to the leachate-contaminated forest soils around a Hg polluted traditional landfill, Japan. General soil properties, concentrations of THg and MeHg in forest soils and in B. parvus were determined. The results indicated that the average THg concentrations in B. parvus and forest soils in the leachate-contaminated sites were 10.21 and 14.90 times higher than the control sites, respectively, whereas similar average MeHg concentrations were observed in forest soils (< 0.01 kg^-1) and B. parvus (0.100 - 0.114 mg kg^-1) across all sampled sites. The average bioaccumulation factors of B. parvus in forest soil THg (BAF_THg) were similar between the leachate-contaminated sites and the control sites. Cluster and regression analyses demonstrated that the B. parvus Hg (THg / MeHg) and soil THg were positively correlated with each other and with soil organic matter (SOM) and clays, but negatively correlated with sand and hardly correlated with silts and pH in leachate-contaminated forest soils. From these results, it is proposed that Hg exposure to food chains is possible through B. parvus, because B. parvus shows a high ability to accumulate THg and MeHg in both leachate contaminated and control forest soils. Together these findings indicated that the role of B. parvus in MeHg production is not clear, and it is possible that the MeHg in B. parvus was firstly formed within forest soils and then accumulated in their tissues.

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Cadmium mechanisms of toxicity, e.g. GSH depletion, ROS and apoptosis induction, have been addressed in different tissues and cell types, including the immune system. However, the various immune cell types could be differentially sensitive to cadmium depending on their ability to deal with the mechanisms of toxicity, based on their cellular physiology, potentially modulating different immune functions. Macrophages and mast cells are two types of innate immune cells that initiate protective immune responses. Macrophages are associated with type 1 immunity against intracellular pathogens and infected cells, while mast cells are associated with type 2 immunity to extracellular bacteria and parasites. In order to study the immunomodulatory effects of cadmium on macrophages and mast cells we carried out a mechanistic study in vitro using two models of murine macrophages, RAW264.7 and NR8383 cell lines, and two models of murine mast cells, MC/9 and RBL-2H3 cell lines. Depletion of GSH was observed in the four cell lines tested, but mast cells showed steeper GSH-depletion compared to macrophages. Functional measurements showed that cadmium leads to pro-inflammatory effects in macrophages, increasing TNFα and nitric oxide in LPS and non-activated cells; while immunosuppressive effects on mast cells, showing dose-response inhibition of histamine in IgE-mediated activation and spontaneous secretion. In this way, cadmium may modulate immune responses by enhancing macrophage and suppressing mast cell functioning, favouring type 1 response at expenses of type 2 response.

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