Exposure to cis- and trans-regioisomers of S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2-dichlorovinyl)-glutathione result in quantitatively and qualitatively different cellular effects in RPTEC/TERT1 cells.

Bioactivation of trichloroethylene (TCE) via glutathione conjugation is associated with several adverse effects in the kidney and other extrahepatic tissues. Of the three regioisomeric conjugates formed, S-(1,2-trans-dichlorovinyl)-glutathione (1,2-trans-DCVG), S-(1,2-cis-dichlorovinyl)-glutathione and S-(2,2-dichlorovinyl)-glutathione, only 1,2-trans-DCVG and its corresponding cysteine-conjugate, 1,2-trans-DCVC, have been subject to extensive mechanistic studies. In the present study, the metabolism and cellular effects of 1,2-cis-DCVG, the major regioisomer formed by rat liver fractions, and 1,2-cis-DCVC were investigated for the first time using RPTEC/TERT1-cells as in vitro renal model. In contrast to 1,2-trans-DCVG/C, the cis-regioisomers showed minimal effects on cell viability and mitochondrial respiration. Transcriptomics analysis showed that both 1,2-cis-DCVC and 1,2-trans-DCVC caused Nrf2-mediated antioxidant responses, with 3 µM as lowest effective concentration. An ATF4-mediated integrated stress response and p53-mediated responses were observed starting from 30 µM for 1,2-trans-DCVC and 125 µM for 1,2-cis-DCVC. Comparison of the metabolism of the DCVG regioisomers by LC/MS showed comparable rates of processing to their corresponding DCVC. No detectable N-acetylation was observed in RPTEC/TERT1 cells. Instead, N-glutamylation of DCVC to form N-γ-glutamyl-S-(dichlorovinyl)-L-cysteine was identified as a novel route of metabolism. The results suggest that 1,2-cis-DCVC may be of less toxicological concern for humans than 1,2-trans-DCVC, considering its lower intrinsic toxicity and lower rate of formation by human liver fractions.

Transcriptomic-based evaluation of trichloroethylene glutathione and cysteine conjugates demonstrate phenotype-dependent stress responses in a panel of human in vitro models.

Environmental or occupational exposure of humans to trichloroethylene (TCE) has been associated with different extrahepatic toxic effects, including nephrotoxicity and neurotoxicity. Bioactivation of TCE via the glutathione (GSH) conjugation pathway has been proposed as underlying mechanism, although only few mechanistic studies have used cell models of human origin. In this study, six human derived cell models were evaluated as in vitro models representing potential target tissues of TCE-conjugates: RPTEC/TERT1 (kidney), HepaRG (liver), HUVEC/TERT2 (vascular endothelial), LUHMES (neuronal, dopaminergic), human induced pluripotent stem cells (hiPSC) derived peripheral neurons (UKN5) and hiPSC-derived differentiated brain cortical cultures containing all subtypes of neurons and astrocytes (BCC42). A high throughput transcriptomic screening, utilizing mRNA templated oligo-sequencing (TempO-Seq), was used to study transcriptomic effects after exposure to TCE-conjugates. Cells were exposed to a wide range of concentrations of S-(1,2-trans-dichlorovinyl)glutathione (1,2-DCVG), S-(1,2-trans-dichlorovinyl)-L-cysteine (1,2-DCVC), S-(2,2-dichlorovinyl)glutathione (2,2-DCVG), and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC). 1,2-DCVC caused stress responses belonging to the Nrf2 pathway and Unfolded protein response in all the tested models but to different extents. The renal model was the most sensitive model to both 1,2-DCVC and 1,2-DCVG, with an early Nrf2-response at 3 µM and hundreds of differentially expressed genes at higher concentrations. Exposure to 2,2-DCVG and 2,2-DCVC also resulted in the upregulation of Nrf2 pathway genes in RPTEC/TERT1 although at higher concentrations. Of the three neuronal models, both the LUHMES and BCC42 showed significant Nrf2-responses and at higher concentration UPR-responses, supporting recent hypotheses that 1,2-DCVC may be involved in neurotoxic effects of TCE. The cell models with the highest expression of γ-glutamyltransferase (GGT) enzymes, showed cellular responses to both 1,2-DCVG and 1,2-DCVC. Little to no effects were found in the neuronal models from 1,2-DCVG exposure due to their low GGT-expression. This study expands our knowledge on tissue specificity of TCE S-conjugates and emphasizes the value of human cell models together with transcriptomics for such mechanistic studies.

Cysteine-Based Protein Covalent Binding and Hepatotoxicity Induced by Emodin.

Emodin (EMD) is a major ingredient of Polygonum multiflorum Thunb. (PMT), which has shown adverse liver reactions. Despite multiple pharmacological activities, EMD is reported to show various toxicities. Our early study demonstrated the reactivity of EMD to glutathione. This study aimed to determine the covalent interaction of hepatic protein with EMD and the correlation of the protein modification with hepatotoxicity induced by EMD. EMD-derived protein adduction was detected in an incubation mixture containing mouse liver homogenates and EMD. Such protein adduction was also observed in hepatic protein obtained from mice exposed to EMD. The protein covalent binding occurred in time- and dose-dependent manners. Pre-treatment of l-buthionine-sulfoximine significantly potentiated EMD-induced adduction and hepatotoxicity caused by EMD and lipopolysaccharide co-treatment. As expected, EMD-derived protein modification was observed in mouse primary hepatocytes treated with EMD. The increase in EMD exposure concentration intensified EMD-derived protein adduction and increased EMD-induced cell death. The susceptibility of hepatocytes to EMD cytotoxicity and the intensity of EMD-induced protein adduction were attenuated by the co-treatment of hepatocytes with N-acetyl cysteine. A good association of protein modification with hepatotoxicity induced by EMD was illustrated, which facilitates the understanding of the mechanism of hepatotoxicity induced by EMD.

Localization and Bioreactivity of Cysteine-Rich Secretions in the Marine Gastropod Nucella lapillus.

Marine biodiversity has been yielding promising novel bioproducts from venomous animals. Despite the auspices of conotoxins, which originated the paradigmatic painkiller Prialt, the biotechnological potential of gastropod venoms remains to be explored. Marine bioprospecting is expanding towards temperate species like the dogwhelk Nucella lapillus, which is suspected to secrete immobilizing agents through its salivary glands with a relaxing effect on the musculature of its preferential prey, Mytilus sp. This work focused on detecting, localizing, and testing the bioreactivity of cysteine-rich proteins and peptides, whose presence is a signature of animal venoms and poisons. The highest content of thiols was found in crude protein extracts from the digestive gland, which is associated with digestion, followed by the peribuccal mass, where the salivary glands are located. Conversely, the foot and siphon (which the gastropod uses for feeding) are not the main organs involved in toxin secretion. Ex vivo bioassays with Mytilus gill tissue disclosed the differential bioreactivity of crude protein extracts. Secretions from the digestive gland and peribuccal mass caused the most significant molecular damage, with evidence for the induction of apoptosis. These early findings indicate that salivary glands are a promising target for the extraction and characterization of bioactive cysteine-rich proteinaceous toxins from the species.

Transport and Toxicity of Methylmercury-Cysteine in Cultured BeWo Cells.

Mercury is a heavy metal toxicant that is prevalent throughout the environment. Organic forms of mercury, such as methylmercury (MeHg), can cross the placenta and can lead to lasting detrimental effects in the fetus. The toxicological effects of MeHg on the placenta itself have not been clearly defined. Therefore, the purpose of the current study was to assess the transport of MeHg into placental syncytiotrophoblasts and to characterize the mechanisms by which MeHg exerts its toxic effects. Cultured placental syncytiotrophoblasts (BeWo) were used for these studies. The transport of radioactive MeHg was measured to identify potential mechanisms involved in the uptake of this compound. The toxicological effects of MeHg on BeWo cells were determined by assessing visible pathological change, autophagy, mitochondrial viability, and oxidative stress. The findings of this study suggest that MeHg compounds are transported into BeWo cells primarily by sodium-independent amino acid carriers and organic anion transporters. The MeHg altered mitochondrial function and viability, decreased mitophagy and autophagy, and increased oxidative stress. Exposure to higher concentrations of MeHg inhibited the ability of cells to protect against MeHg-induced injury. The findings show that MeHg is directly toxic to syncytiotrophoblasts and may lead to disruptions in the fetal/maternal transfer of nutrients and wastes.

Exposure to Trichloroethylene Metabolite S-(1,2-Dichlorovinyl)-L-cysteine Causes Compensatory Changes to Macronutrient Utilization and Energy Metabolism in Placental HTR-8/SVneo Cells.

Trichloroethylene (TCE) is a widespread environmental contaminant following decades of use as an industrial solvent, improper disposal, and remediation challenges. Consequently, TCE exposure continues to constitute a risk to human health. Despite epidemiological evidence associating exposure with adverse birth outcomes, the effects of TCE and its metabolite S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) on the placenta remain undetermined. Flexible and efficient macronutrient and energy metabolism pathway utilization is essential for placental cell physiological adaptability. Because DCVC is known to compromise cellular energy status and disrupt energy metabolism in renal proximal tubular cells, this study investigated the effects of DCVC on cellular energy status and energy metabolism pathways in placental cells. Human extravillous trophoblast cells, HTR-8/SVneo, were exposed to 5-20 µM DCVC for 6 or 12 h. After establishing concentration and exposure duration thresholds for DCVC-induced cytotoxicity, targeted metabolomics was used to evaluate overall energy status and metabolite concentrations from energy metabolism pathways. The data revealed glucose metabolism perturbations including a time-dependent accumulation of glucose-6-phosphate+frutose-6-phosphate (G6P+F6P) as well as independent shunting of glucose intermediates that diminished with time, with modest energy status decline but in the absence of significant changes in ATP concentrations. Furthermore, metabolic profiling suggested that DCVC stimulated compensatory utilization of glycerol, lipid, and amino acid metabolism to provide intermediate substrates entering downstream in the glycolytic pathway or the tricarboxylic acid cycle. Lastly, amino acid deprivation increased susceptibility to DCVC-induced cytotoxicity. Taken together, these results suggest that DCVC caused metabolic perturbations necessitating adaptations in macronutrient and energy metabolism pathway utilization to maintain adequate ATP levels.

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.

Formation of di-cysteine acrolein adduct decreases cytotoxicity of acrolein by ROS alleviation and apoptosis intervention.

Acrolein (ACR) is a toxic contaminant for humans. Our previous research indicated that l-cysteine (Cys) decreased the cytotoxicity of acrolein possibly via adduct formation, but which adduct contributed to the toxicity-lowering effect remains unknown. In this work, we identified a di-cysteine acrolein adduct (ACR-di-Cys) and investigated its toxicity against human bronchial epithelial cell line HBE and colon cancer cell line Caco-2. ACR-di-Cys tremendously decreased acrolein-induced cytotoxicity via alleviating ROS and apoptosis intervention. In the condition of no presence of free cysteine, however, this adduct can convert to mono-ACR-Cys in PBS solution by losing a molecule of cysteine conjugated at CC bond. ACR-mono-Cys showed much higher toxicity than ACR-di-Cys, and even higher than acrolein after 48 h exposure. This study indicated that cysteine can react with acrolein to form adducts with different acrolein-detoxifying capacity, and a sufficient intake of cysteine or cysteine-containing proteins can maximize the detoxifying effect for acrolein via the formation of a highly detoxifying agent, ACR-di-Cys.

Characterisation of transcription factor profiles in polycystic kidney disease (PKD): identification and validation of STAT3 and RUNX1 in the injury/repair response and PKD progression.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, caused in the majority of the cases by a mutation in either the PKD1 or the PKD2 gene. ADPKD is characterised by a progressive increase in the number and size of cysts, together with fibrosis and distortion of the renal architecture, over the years. This is accompanied by alterations in a complex network of signalling pathways. However, the underlying molecular mechanisms are not well characterised. Previously, we defined the PKD Signature, a set of genes typically dysregulated in PKD across different disease models from a meta-analysis of expression profiles. Given the importance of transcription factors (TFs) in modulating disease, we focused in this paper on characterising TFs from the PKD Signature. Our results revealed that out of the 1515 genes in the PKD Signature, 92 were TFs with altered expression in PKD, and 32 of those were also implicated in tissue injury/repair mechanisms. Validating the dysregulation of these TFs by qPCR in independent PKD and injury models largely confirmed these findings. STAT3 and RUNX1 displayed the strongest activation in cystic kidneys, as demonstrated by chromatin immunoprecipitation (ChIP) followed by qPCR. Using immunohistochemistry, we showed a dramatic increase of expression after renal injury in mice and cystic renal tissue of mice and humans. Our results suggest a role for STAT3 and RUNX1 and their downstream targets in the aetiology of ADPKD and indicate that the meta-analysis approach is a viable strategy for new target discovery in PKD. KEY MESSAGES: We identified a list of transcription factors (TFs) commonly dysregulated in ADPKD. Out of the 92 TFs identified in the PKD Signature, 35% are also involved in injury/repair processes. STAT3 and RUNX1 are the most significantly dysregulated TFs after injury and during PKD progression. STAT3 and RUNX1 activity is increased in cystic compared to non-cystic mouse kidneys. Increased expression of STAT3 and RUNX1 is observed in the nuclei of renal epithelial cells, also in human ADPKD samples.

The trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine induces progressive mitochondrial dysfunction in HTR-8/SVneo trophoblasts.

Trichloroethylene is an industrial solvent and common environmental pollutant. Despite efforts to ban trichloroethylene, its availability and usage persist globally, constituting a hazard to human health. Recent studies reported associations between maternal trichloroethylene exposure and increased risk for low birth weight. Despite these associations, the toxicological mechanism underlying trichloroethylene adverse effects on pregnancy remains largely unknown. The trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC) induces mitochondrial-mediated apoptosis in a trophoblast cell line. To gain further understanding of mitochondrial-mediated DCVC placental toxicity, this study investigated the effects of DCVC exposure on mitochondrial function using non-cytolethal concentrations in placental cells. Human trophoblasts, HTR-8/SVneo, were exposed in vitro to a maximum of 20 µM DCVC for up to 12 h. Cell-based oxygen consumption and extracellular acidification assays were used to evaluate key aspects of mitochondrial function. Following 6 h of exposure to 20 µM DCVC, elevated oxygen consumption, mitochondrial proton leak and sustained energy coupling deficiency were observed. Similarly, 12 h of exposure to 20 µM DCVC decreased mitochondrial-dependent basal, ATP-linked and maximum oxygen consumption rates. Using the fluorochrome TMRE, dissipation of mitochondrial membrane potential was detected after a 12-h exposure to 20 µM DCVC, and (±)-α-tocopherol, a known suppressor of lipid peroxidation, attenuated DCVC-stimulated mitochondrial membrane depolarization but failed to rescue oxygen consumption perturbations. Together, these results suggest that DCVC caused progressive mitochondrial dysfunction, resulting in lipid peroxidation-associated mitochondrial membrane depolarization. Our findings contribute to the biological plausibility of DCVC-induced placental impairment and provide new insights into the role of the mitochondria in DCVC-induced toxicity.

Amino Acid-Capped Water-Soluble Near-Infrared Region CuInS2/ZnS Quantum Dots for Selective Cadmium Ion Determination and Multicolor Cell Imaging.

Although attractive for their low toxicity, CuInS2/ZnS core/shell quantum dots (CIS/ZnS QDs) still suffer from poor luminescence efficiency and poor water solubility. Herein, two amino acids (AAs), i.e., cysteine (Cys) and threonine (Thr), are used to tune the properties of CIS/ZnS QDs by capping them in both core and shell. It is found that Thr can regulate the density of Cys on the surface of QDs, thus causing a synergistic effect on the enhancement of photoluminescence (PL) intensity. Capping in the shell mainly leads to the enhancement of PL intensity, and capping in the core results in a red-shift of PL wavelength. Accordingly, a new kind of near-infrared region CIS/ZnS QDs with improved optical properties has been prepared. In addition, the Cys- and Thr-capped CIS/ZnS QDs possess outstanding water solubility and biocompatibility. In this work, the QDs are further employed in Cd2+ determination and multicolor imaging, indicating their potential applications. Relying on the enhancement of PL intensity via cation exchange, the Cys- and Thr-capped CIS/ZnS QDs can sense Cd2+ sensitively. Notably, because ZnS shells of the QDs will not be affected by Zn2+, the analytical method can discriminate Cd2+ from Zn2+ depending on the inherent characteristics of QDs. Moreover, intercellular Cd2+ can also be evaluated by the bright PL from the QDs, and the QDs can achieve multicolor imaging. Overall, this work demonstrates that various properties of QDs may be tuned by capping with AAs, and AA-capped QDs are of great value in advanced biosensing and bioimaging.

Identification of Novel Metabolites of Vildagliptin in Rats: Thiazoline-Containing Thiol Adducts Formed via Cysteine or Glutathione Conjugation.

Vildagliptin (VG), a dipeptidyl peptidase-4 inhibitor, is used for treating type 2 diabetes. On rare occasions, VG causes liver injury as an adverse reaction. One case report suggested the involvement of immune responses in the hepatotoxicity, but the underlying mechanisms are unknown. We recently reported that VG binds covalently in vitro to l-cysteine to produce a thiazoline acid metabolite, M407, implying that the covalent binding may trigger the immune-mediated hepatotoxicity. There was no evidence, however, that such a thiazoline acid metabolite was formed in vivo. In the present study, we administered a single oral dose of VG to male Sprague-Dawley rats, and detected M407 in plasma. The sum of urinary and fecal excretions of M407 reached approximately 2% of the dose 48 hours postdosing. Using bile duct-cannulated rats, we demonstrated that M407 was secreted into bile as a glucuronide, designated as M583. Another newly identified thiazoline metabolite of VG, the cysteinylglycine conjugate M464, was detected in urine, feces, and bile. The formation of M464 was confirmed by in vitro incubation of VG with glutathione even in the absence of metabolic enzymes. A glutathione adduct against the nitrile moiety M611 was also detected in vitro but not in vivo. In summary, we found three new thiazoline-containing thiol adduct metabolites in VG-administered rats. Nonenzymatic covalent binding of VG would likely occur in humans, and it may be relevant to predicting adverse reactions.

Potential mechanisms of cellular injury following exposure to a physiologically relevant species of inorganic mercury.

Mercury is a toxic metal that is found ubiquitously in the environment. Humans are exposed to different forms of mercury via ingestion, inhalation, and/or dermal absorption. Following exposure, mercuric ions may gain access to target cells and subsequently lead to cellular intoxication. The mechanisms by which mercury accumulation leads to cellular injury and death are not understood fully. Therefore, purpose of this study was to identify the specific intracellular mechanisms that are altered by exposure to inorganic mercury (Hg2+). Normal rat kidney (NRK) cells were exposed to a physiologically relevant form of Hg2+, as a conjugate of cysteine (10 µM or 50 µM). Alterations in oxidative stress were estimated by measuring lipid peroxidation and mitochondrial oxidative stress. Alterations in actin and tubulin were measured using specific fluorescent dyes. Calcium levels were measured using Fluo-3 AM Calcium Indicator while autophagy was identified with Premo™ Autophagy Sensor LC3B-GFP. The current findings show that exposure to Hg2+ leads to enhanced oxidative stress, alterations in cytoskeletal structure, increases in intracellular calcium, and enhanced autophagy. We have established a more complete understanding of intoxication and cellular injury induced by a relevant form of Hg2+ in proximal tubule cells.

The Uptake and Release of Polysulfur Cysteine Species by Cells: Physiological and Toxicological Implications.

Hydropersulfides and related polysulfides have recently become topics of significant interest due to their physiological prevalence and proposed biological functions. Currently, examination of the effects of hydropersulfide treatment on cells is difficult due to their lack of inherent stability with respect to disproportionation. Herein, it is reported that the treatment of a variety of cell types with cysteine trisulfide (also known as thiocystine; Cys-SSS-Cys), results in an increase in intracellular hydropersulfide levels (e.g., cysteine hydropersulfide; Cys-SSH, and glutathione hydropersulfide; GSSH). Thus, Cys-SSS-Cys represents a possible pharmacological agent for examining the effects of hydropersulfides on cell function/viability. It has also been found that cells with increased intracellular hydropersulfide levels can export Cys-SSH into the extracellular media. Interestingly, the Cys-SSH is the major hydropersulfide exported by cells, although GSSH is the predominant intracellular species. The possible implications of cellular export are discussed.

Self-Assembled Copper-Amino Acid Nanoparticles for in Situ Glutathione "AND" H2O2 Sequentially Triggered Chemodynamic Therapy.

Nanoformulations that can respond to the specific tumor microenvironment (TME), such as a weakly acidic pH, low oxygen, and high glutathione (GSH), show promise for killing cancer cells with minimal invasiveness and high specificity. In this study, we demonstrate self-assembled copper-amino acid mercaptide nanoparticles (Cu-Cys NPs) for in situ glutathione-activated and H2O2-reinforced chemodynamic therapy for drug-resistant breast cancer. After endocytosis into tumor cells, the Cu-Cys NPs could first react with local GSH, induce GSH depletion, and reduce Cu2+ to Cu+. Subsequently, the generated Cu+ would react with local H2O2 to generate toxic hydroxyl radicals (·OH) via a Fenton-like reaction, which has a fast reaction rate in the weakly acidic TME, that are responsible for tumor-cell apoptosis. Due to the high GSH and H2O2 concentration in tumor cells, which sequentially triggers the redox reactions, Cu-Cys NPs exhibited relatively high cytotoxicity to cancer cells, whereas normal cells were left alive. The in vivo results also proved that Cu-Cys NPs efficiently inhibited drug-resistant breast cancer without causing obvious systemic toxicity. As a novel copper mercaptide nanoformulation responsive to the TME, these Cu-Cys NPs may have great potential in chemodynamic cancer therapy.

Characterization of inter-tissue and inter-strain variability of TCE glutathione conjugation metabolites DCVG, DCVC, and NAcDCVC in the mouse.

Trichloroethylene (TCE) is a ubiquitous environmental toxicant that is a liver and kidney carcinogen. Conjugation of TCE with glutathione (GSH) leads to formation of nepthrotoxic and mutagenic metabolites postulated to be critical for kidney cancerdevelopment; however, relatively little is known regarding their tissue levels as previous analytical methods for their detection lacked sensitivity. Here, an LC-MS/MS-based method for simultaneous detection of S-(1,2-dichlorovinyl)-glutathione (DCVG), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAcDCVC) in multiple mouse tissues was developed. This analytical method is rapid, sensitive (limits of detection (LOD) 3-30 fmol across metabolites and tissues), and robust to quantify all three metabolites in liver, kidneys, and serum. The method was used to characterize inter-tissue and inter-strain variability in formation of conjugative metabolites of TCE. Single oral dose of TCE (24, 240 or 800 mg/kg) was administered to male mice from 20 inbred strains of Collaborative Cross. Inter-strain variability in the levels of DCVG, DCVC, and NAcDCVC (GSD = 1.6-2.9) was observed. Whereas NAcDCVC was distributed equally among analyzed tissues, highest levels of DCVG were detected in liver and DCVC in kidneys. Evidence indicated that inter-strain variability in conjugative metabolite formation of TCE might affect susceptibility to adverse health effects and that this method might aid in filling data gaps in human health assessment of TCE.

Trichloroethylene metabolite S-(1,2-dichlorovinyl)-l-cysteine induces lipid peroxidation-associated apoptosis via the intrinsic and extrinsic apoptosis pathways in a first-trimester placental cell line.

Trichloroethylene (TCE), a prevalent environmental contaminant, is a potent renal and hepatic toxicant through metabolites such as S-(1, 2-dichlorovinyl)-l-cysteine (DCVC). However, effects of TCE on other target organs such as the placenta have been minimally explored. Because elevated apoptosis and lipid peroxidation in placenta have been observed in pregnancy morbidities involving poor placentation, we evaluated the effects of DCVC exposure on apoptosis and lipid peroxidation in a human extravillous trophoblast cell line, HTR-8/SVneo. We exposed the cells in vitro to 10-100µM DCVC for various time points up to 24h. Following exposure, we measured apoptosis using flow cytometry, caspase activity using luminescence assays, gene expression using qRT-PCR, and lipid peroxidation using a malondialdehyde quantification assay. DCVC significantly increased apoptosis in time- and concentration-dependent manners (p<0.05). DCVC also significantly stimulated caspase 3, 7, 8 and 9 activities after 12h (p<0.05), suggesting that DCVC stimulates the activation of both the intrinsic and extrinsic apoptotic signaling pathways simultaneously. Pre-treatment with the tBID inhibitor Bl-6C9 partially reduced DCVC-stimulated caspase 3 and 7 activity, signifying crosstalk between the two pathways. Additionally, DCVC treatment increased lipid peroxidation in a concentration-dependent manner. Co-treatment with the antioxidant peroxyl radical scavenger (±)-α-tocopherol attenuated caspase 3 and 7 activity, suggesting that lipid peroxidation mediates DCVC-induced apoptosis in extravillous trophoblasts. Our findings suggest that DCVC-induced apoptosis and lipid peroxidation in extravillous trophoblasts could contribute to poor placentation if similar effects occur in vivo in response to TCE exposure, indicating that further studies into this mechanism are warranted.

Formation of a Hydroxymethylfurfural-Cysteine Adduct and Its Absorption and Cytotoxicity in Caco-2 Cells.

Adducts of 5-hydroxymethylfurfural (HMF)-amino acids are formed during food processing and digestion; the elimination capacity of in vitro intestinal digests of biscuits, instant noodles, and potato crisps for HMF is 652, 727, and 540 µg/g, respectively. However, the safety of these adducts is unknown. In this study, an HMF-cysteine adduct named 1-dicysteinethioacetal-5-hydroxymehtylfurfural (DCH), which was found to be produced in the gastrointestinal tract after HMF intake, was prepared to test its effect toward Caco-2 cells. Compared with HMF, the adduct displayed lower cytotoxicity against Caco-2 cells with an IC50 value of 31.26 mM versus 14.95 mM (HMF). The DCH did not induce cell apoptosis, whereas HMF significantly increased the apoptosis rate after incubation at concentrations of 16, 32, and 48 mM for 72 h. DCH showed an absorption rate considerably lower than that of HMF by Caco-2 cells. Lower absorption of DCH may result in lower toxicity compared with HMF against Caco-2 cells. Intracellular transformation of DCH has been observed.

High doses of S-methylcysteine cause hypoxia-induced cardiomyocyte apoptosis accompanied by engulfment of mitochondaria by nucleus.

Despite its important role as a medicinal plant, some studies reported a toxic effect for garlic (Allium sativum) when given in higher doses. Herein, we investigated the possible cardiotoxic effects of high doses of S-methylcysteine (SMC), a water soluble organosulfur compound present in garlic. Rats were orally administered SMC at a low dose (50mg), high dose (150mg) and very high dose (300mg)/kg body weight, or saline (control) for 10days. High and very high doses of SMC resulted in a significant increase in serum cardiac injury biomarkers [aspartate transaminase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin T (cTnT)], as well as oxidative stress marker nitric oxide (NO) concentration in heart and a significant decrease in cardiac superoxide dismutase (SOD) activity. Moreover, ultrastructure findings in myocardium of rats treated by high and very high doses showed inter-bundle vacuolation, loss of myofibrils, and centripetal movement of mitochondria towards nucleus. The mitochondria were partially surrounded by nuclear membrane at high dose SMC, and completely engulfed by nucleus at very high dose. This centripetal movement of mitochondria accompanied by cardiomyocytes hypoxia-induced apoptosis as evident by increasing TUNEL positive cells as well as upregulation of apoptotic genes (caspase3 and Bax), hypoxia inducible factor 1 alpha (HIF1α), dynein light chain 1 (DYNLL1) and downregulation of the anti-apoptotic marker, Bcl2. We conclude that high and very high doses of SMC cause hypoxia induced cardiomyocyte apoptosis accompanied by engulfment of mitochondria by nucleus.

Methylmercury Exposure Reduces the Auditory Brainstem Response of Zebra Finches (Taeniopygia guttata ).

Mercury contamination from mining and fossil fuel combustion causes damage to humans and animals worldwide. Mercury exposure has been implicated in mammalian hearing impairment, but its effect on avian hearing is unknown. In this study, we examined whether lifetime dietary mercury exposure affected hearing in domestic zebra finches (Taeniopygia guttata) by studying their auditory brainstem responses (ABRs). Zebra finches exposed to mercury exhibited elevated hearing thresholds, decreased amplitudes, and longer latencies in the ABR, the first evidence of mercury-induced hearing impairment in birds. Birds are a more appropriate model for the human auditory spectrum than most mammals because of similarities in frequency discrimination, vocal learning, and communication behavior. When mercury is considered in combination with other anthropogenic stressors such as noise pollution and habitat alteration, the hearing impairments we document here could substantially degrade avian auditory communication in wild birds.