This study attempted to identify substances that are driven out of HEK293 cells by methylmercury. Metabolomic analysis revealed that the levels of 3-phenylpropionic acid, citrulline, lactic acid, ornithine, proline and beta-alanine in the cell culture medium were increased by the treatment of cells with methylmercury. Address to the mechanism underlying the release of these substances will provide useful information to elucidate the toxicity mechanism of methylmercury.

HEK293 cells were cultured in medium containing methylmercury (MeHg), followed by replacement with MeHg-free medium and further culturing. Thus, MeHg preconditioning medium (MeHg-PM) were obtained. Untreated HEK293 cells and C17.2 cells (mouse neural stem cells) were placed in the obtained MeHg-PM for culturing, which resulted in significantly inhibited cell growth. This cell growth inhibition was not affected by heating or proteinase K treatment, suggesting that neither proteins nor peptides caused the growth inhibition.

Of antifoulants that are substitutes for organotin compounds such as tributyltin and triphenyltin, N-(2,4,6-trichlorophenyl)maleimide (IT-354) is listed as a much less toxic agent, although the available information concerning IT-354 toxicity is the results of acute toxicity tests in freshwater fish. In this study, the effects of IT-354 on rat thymic lymphocytes were examined using flow-cytometric techniques with appropriate fluorescent probes in order to estimate the effects of IT-354 on mammalian cells. Treatment of cells with 1-10 μM IT-354 for 1 hr did not increase the population of dead cells (cell lethality). However, 10 μM IT-354 significantly increased the population of living, annexin V-positive cells. Annexin V-positive, living cells are expected to be undergoing apoptosis. IT-354 at 3-10 μM significantly elevated intracellular Ca²⁺ and Zn²⁺ levels mainly by increasing Ca²⁺ influx and intracellular Zn²⁺ release. Furthermore, IT-354 significantly depolarized membranes and decreased cellular non-protein thiol content. Assessments using selected antifouling agents showed that the cellular actions of IT-354 are most likely similar to those of other commonly used antifouling agents. Therefore, the toxic potency of IT-354 on wild mammals is speculated to be similar to those of the other tested antifoulants.

Some household products have high levels of the antimicrobial 2-n-octyl-4-isothiazolin-3-one (OIT). Although the diverse effects of OIT are of concern, information regarding its cellular actions is limited. In a previous study, we found that OIT increased intracellular Zn²⁺ levels in rat thymocytes. However, because Ca²⁺ is considered the essential cation that causes cell injury and death, we examined whether Ca²⁺ and Zn²⁺ were involved in OIT-induced cytotoxicity and proposed the mechanisms underlying these results. The effects of OIT on the membrane and cellular parameters of rat thymocytes were examined with a flow cytometer and appropriate fluorescent probes. OIT (0.3-3 μM) increased intracellular Zn²⁺ levels but not intracellular Ca²⁺ levels. Therefore, the involvement of Zn²⁺ was studied further. The simultaneous application of 0.3 μM OIT and 3 μM ZnCl₂ significantly increased cells with phosphatidylserine-exposed membranes without changing the dead cells. In contrast, applications of 0.3 μM OIT or 3 μM ZnCl₂ alone had no effects. The combination of OIT (0.1-1 μM) and ZnCl₂ (1-3 μM) significantly decreased the cellular non-protein thiol contents. These changes that were induced by their combination were completely suppressed by adding an intracellular Zn²⁺ chelator. These results suggested that submicromolar concentrations of OIT induced Zn²⁺-dependent cytotoxicity in the presence of micromolar concentrations of external Zn²⁺. Because the threshold of OIT levels that affected cellular parameters in the presence of micromolar concentrations of Zn²⁺ are much lower than the OIT contents in some household products, the adverse effects of OIT are of great concern.

Perfluorinated compounds (PFCs) have been used widely, detected worldwide in the environment, and have accumulated highly in animals. As far as we know, there have been no reports which relate the PFC concentration in wild animals to the physicochemical properties. Therefore, we measured the concentrations of 15 currently available PFCs (perfluorocarboxylic acids with x carbons: Cx, perfluorosulfonic acids with x carbons: CxS) in medaka and the environmental water where medaka live. Samples were obtained from 7 points in Japan (Iwate, Ibaraki, Niigata, Hyogo, Yamaguchi, Ehime, and Nagasaki) from July to September in 2013. Twenty to forty medaka were collected from each point, as well as 2 L of water in a clean PET bottle. PFCs were extracted and concentrated using a solid-phase cartridge, and were measured by LC/MS/MS. The medaka samples were treated individually. C5-C9 and C8S were detected mainly in the water, C11-C13 and C8S were detected mainly in medaka. C8S was always detected in high concentrations in the water and medaka. The bioconcentration factors (BCFs) of PFCs were calculated from PFC concentrations of the water and the medaka. The BCFs of C8-C11 were increased exponentially with the length of carbon chain. The BCF of C8S (approx. 5,500) was far greater than C8 (approx. 330) or C9 (approx. 480). However, the BCFs of C8-C11 and C8S tended to increase in proportion with octanol/water partition coefficient (log K_ow).