Characterization of Mechanisms of Glutathione Conjugation with Halobenzoquinones in Solution and HepG2 Cells

Abstract

Halobenzoquinones (HBQs) are a class of emerging disinfection byproducts. Chronic exposure to chlorinated drinking water is potentially associated with an increased risk of human bladder cancer. HBQ-induced cytotoxicity involves depletion of cellular glutathione (GSH), but the underlying mechanism remains unclear. Here we used ultrahigh performance liquid chromatography-high resolution mass spectrometry and electron paramagnetic resonance spectroscopy to study interactions between HBQs and GSH and found that HBQs can directly react with GSH, forming various glutathionyl conjugates (HBQ-SG) in both aqueous solution and HepG2 cells. We found that the formation of HBQ-SG varies with the initial molar ratio of GSH to HBQ in reaction mixtures. Higher molar ratios of GSH to HBQ facilitate the conjugation of more GSH molecules to an HBQ molecule. We deduced the reaction mechanism between GSH and HBQs, which involves redox cycling-induced formation of halosemiquinone (HSQ) free radicals and glutathione disulfide, Michael addition, as well as nucleophilic substitution. The proposed reaction rates are in the following order: formation of HSQ radicals > substitution of bromine by GSH > Michael addition of GSH on the benzoquinone ring > substitution of chlorine by GSH > substitution of the methyl group by GSH. The conjugates identified in HBQ-treated HepG2 cells were the same as those found in aqueous solution containing a 5:1 ratio of GSH:HBQs.

3,3′-Dichlorobiphenyl Is Metabolized to a Complex Mixture of Oxidative Metabolites, Including Novel Methoxylated Metabolites, by HepG2 Cells

“…This human hepatoma cell line is readily available, easy, and inexpensive to maintain 61 and widely used as a model for drug metabolism and toxicity studies. 62,63 The expression of cytochrome P450 enzymes in HepG2 cells is lower compared to primary hepatocytes, 44 an earlier toxicity study. 32 Based on earlier metabolism studies, this concentration was expected to yield metabolite levels that could be detected with our analytical methods.…”

“…We used an untargeted approach to characterize the PCB 11 metabolites formed in HepG2 cells as a model system. This human hepatoma cell line is readily available, easy, and inexpensive to maintain and widely used as a model for drug metabolism and toxicity studies. , The expression of cytochrome P450 enzymes in HepG2 cells is lower compared to primary hepatocytes, ,− which represents a limitation of this cell line for metabolism studies. We employed a PCB 11 concentration that was used in an earlier toxicity study .…”

Halobenzoquinone-Induced Developmental Toxicity, Oxidative Stress, and Apoptosis in Zebrafish Embryos

“…The production of ROS is dependent on the semiquinone and hydroxyl radical structural features of HBQs. , Because of their electron-deficient structure, HBQs are reactive electrophiles, which can readily react with various bionucleophiles, such as proteins and nucleic acids. The nucleophilic attack of GSH by quinones and HBQs has been demonstrated. , Thus, the reduction in GSH is likely a result of both GSH oxidation and conjugation …”

“…The nucleophilic attack of GSH by quinones and HBQs has been demonstrated. 16,46 Thus, the reduction in GSH is likely a result of both GSH oxidation and conjugation. 46 DNA Damage and Apoptosis.…”

Two-Dimensional Device with Light-Controlled Capability for Treatment of Cancer-Relevant Infection Diseases

“…Currently, the most commonly used clinical methods for cancer treatment include chemotherapy and radiotherapy, which can also eliminate lymphocytes at the time for killing cancer cells. Collapse of the autoimmune system significantly suppresses the immune cell-mediated immunity and consequently accelerates the metastasis of cancer cells. − More importantly, at this period, the foreign microbial could easily attack the host cells and trigger serious whole-body infectious diseases, further increasing the cancer cells diffusion rates and recurrence of disease after surgery treatment. − Therefore, the high cancer mortality is often due to concurrent infections caused by chemotherapy and radiotherapy. Typically, at the early stages of infection, the empirical broad-spectrum antibiotic therapy is the most commonly used therapeutic strategy, while the broad-spectrum antibiotics fail to treat target-specific pathogens effectively and often lead to the emergence of antibiotic-resistant pathogens. − In this regard, to realize targeted pathogen treatment, it is critical and necessary to isolate trace amounts of pathogen (1–100 colony-forming units (CFU) mL –1 ) in the blood and then release the pathogen for downstream drug-resistance analysis at a molecular scale.…”