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Environmental Biology Laboratory Faculty of Medicine University of Tsukuba
環境生物学研究室

The 23rd Symposium of the  Japanese Arsenic Society

Environmental Biology Laboratory 
Faculty of Medicine University of Tsukuba
1-1-1 Tennodai, Tsukuba Ibaraki,
305-8575, Japan

Japanese

Reserch Focus

Redox signaling fluctuations and toxicity that result from the diverse environmental electrophile exposome, and the reactive sulfur species
that control them

 As mentioned in the statement of purpose for our research above, there are multiple environmental electrophiles surrounding us, each with different structures. They are taken up by our bodies and form covalent adducts with such nucleophilic substituents as protein cysteine residues. In the past, the negative side of the formation of such protein adducts had been emphasized, such as tissue injury. On the other hand, the existence of various endogenous electrophiles—such as nitrated fatty acids, 8-nitro-cGMP, prostaglandin J2, and 4-hydroxy-2-nonenal—as well as the discovery of the activation of an electrophilic responsive element via the Keap1/Nrf2 pathway suggest that the body has an adaptive response system for electrophilic stress. Though there are 214,000 cysteine residues in the human genome, 80 to 90 percent of them are used for SH groups, S-S bonds, or ligands for zinc, while it is suggested that the remaining 10 to 20 percent exists as deprotonated thiolate ions (Fig. 1). In addition, it has also been reported that multiple redox signaling pathways exist at the steady-state level, consisting of sensor proteins containing thiolate ions in their molecules, as well as effector molecules, such as kinases and transcription factors, that are negatively controlled thereby (Fig. 1).


 An outline of the research results heretofore obtained by our laboratory is as follows: The human body boasts an excellent ability, not elucidated so far, to detect and respond to environmental electrophiles. In those actions, the main role had been assumed to be played by high-molecular-weight nucleophiles (sensor proteins involved in redox signals) and low-molecular-weight nucleophiles (reactive sulfur species with a pKa value lower than that of GSH, and which easily react with environmental electrophiles) (Fig. 1). That results in individual exposures to the environmental electrophiles; at low doses, the substance in question specifically binds covalently to thiolate ions of the sensor protein, inhibiting the activity of the sensor protein. That has been found to result in the activation of the effector molecule, allowing cell survival (the PTEN/Akt signal), cell proliferation (the PTP1B/EGFR signal), quality control of the cellular proteins (the HSP90/HSF1 signal), and detoxification and excretion of electrophiles (the Keap1/Nrf2 pathway) of downstream genetic groups involved in the detoxification and excretion of electrophiles (Fig. 2). In addition, it was also shown that environmental electrophiles chemically modify intracellular proteins nonspecifically, causing cytotoxicity when exposed to high doses of the substance (Fig. 2). Another discovery was that the environmental electrophiles were captured and inactivated to form the respective sulfur adducts (Fig. 2): the agents in those cases were cysteine persulfide (CysSSH) produced from cystathionine γ-lyase (CSE) and mitochondrial cysteine-tRNA synthetase (CARS2), etc., along with glutathione persulfide (GSSH) and their respective polysulfides produced during the transfer of their intramolecular sulfane sulfur (zerovalent S atoms consisting of six valence electrons, which are reversibly bound to other S atoms), as well as reactive sulfur species (RSS) such as hydrogen sulfide (H2S) (Fig. 2). It was found that it resulted in fluctuations in the redox signal transduction caused by environmental electrophilic exposure, along with a higher toxicity threshold (Fig. 2). For details, please refer to the Grant-in-Aid for Scientific Research (S), “Comprehensive study on environmental electrophiles-mediated signal transduction pathways regulated by RSS” (FY2013-17)

 

 Incidentally, as mentioned in the statement of purpose for our research above, it is believed that each of us, depending on our living environment, lifestyle and dietary habits, is exposed to different doses and multiple environmental electrophiles (Fig. 3). In 2005, Christopher Wild, who served as director of the International Agency for Research on Cancer (IARC) from 2009 to 2018, proposed the intriguing concept of the exposome to describe the totality of environmental exposures experienced by humans throughout their lifetime. Although exposomes are classified into general external factors, special external factors and internal factors, it is considered that differences in the dose amount and time of exposure may be related to health, presymptomatic illness or disease. Focusing on the fact that highly reactive electrophiles are recognized as “priority components” in exposome research, and that environmental electrophiles with differing structures are included among the chemicals, pollutants, diet and lifestyle factors that constitute the special external factors, our laboratory began research in FY2018 on the environmental electrophile exposome vis-à-vis our daily lives (Fig. 3). Our expectation is that a combined exposure to different electrophiles will cause each of those substances to covalently bond to the thiolate ions of the sensor protein, resulting in the activation of the response molecule at lower doses than individual exposure. In other words, the facts suggest that toxicity is expressed at a lower dose than individual exposure, in response to the lowering of the sensitivity and response threshold during combined exposure. Therefore, our study has led to the need for combined exposure studies and a rethinking of the environmental risks. For details, please refer to Grant-in-Aid for Scientific Research (S) “Environmental electrophile exposome and reactive sulfur species as its regulator molecule” (2018-22).


 Foreign substances, such as pharmaceuticals and chemicals, are oxidized in cells by Phase I xenobiotic metabolizing enzymes such as cytochrome P450 (CYP) (a Phase I reaction). Generally, while the oxidation (primarily hydroxylation) leads to a decrease in the activity of the mother compound, it is also known to inadvertently become an electrophilic metabolite. As mentioned above, it has been reported that electrophilic metabolites are covalently linked to cysteine residues of proteins, causing tissue injury (metabolic activation). However, Phase II xenobiotic metabolizing enzymes engage in its protection, involved as they are in the conjugation reactions of small polar molecules such as GSH and uridine diphosphate (UDP)-glucuronic acid mediated by GSH transferase (GST) or UDP-glucuronosyl transferase (UGT) (a Phase II reaction). The polar metabolites thus produced are excreted extracellularly by transporters, which recognize polar groups such as multidrug resistance proteins (MRP) (a Phase III reaction). The Phase I to III reactions described above are acknowledged as “dogma” in the field of xenobiotic metabolism (Fig. 4).
 Meanwhile, in 2011, our laboratory identified (MeHg)2S as a new metabolite from rat livers and SH-SY5Y cells exposed to methylmercury (MeHg). It was confirmed that this sulfur adduct, in SH-SY5Y cells and mice, exhibits lower toxicity than methylmercury, and it was found that RSS are involved in its formation. Sulfur adduct formation by RSS was found not only withMeHg, but also the sulfur adducts of 1,2-naphthoquinone (1,2-NQ), 1,4-NQ, cadmium, and NAPQI, which is an electrophilic metabolite of acetaminophen. Especially, in the cases of 1,4-NQ and cadmium, hardly any changes were observed in the redox signal and toxicity caused by exposure to the parent compound for each sulfur adduct. This series of research results can be described as a new detoxification system through the capture and inactivation of RSS, which is different from the GSH adducts of electrophiles that had previously been known.
 The next question that needs to be answered is to identify the process followed by the resulting sulfur adducts of the electrophiles when excreted from the body into the environment. As mentioned above, methylmercury starts from the CysSSH produced from the CSE and CARS2 in cells, then, by transferring the sulfane sulfur of that molecule, it reacts with GSSH as well as such RSS as polysulfides and hydrogen sulfide (H2S), generating bismethylmercury sulfide (MeHg)2S (Fig. 4). Meanwhile, however, if it can be demonstrated that the sulfur adduct in question is generated in the reaction with extracellularly-released CysSSH (RSS) or a sulfane sulfur-binding protein before taken into the cells, we think it will lead to the presence of a Phase zero reaction (Fig. 4). On the other hand, given that the (MeHg)2S produced in cells is more lipid-soluble than methylmercury, it is possibly polarized in some way and excreted from the cells. For further details on the in-vivo fate of (MeHg)2S in cells (as well as in the extracellular environment), please refer to the Grant-in-Aid for Scientific Research (S) “Environmental electrophile exposome and reactive sulfur species as its regulator molecule” (2018-22).