Chemicals and Drugs Forming Reactive Quinone and Quinone Imine Metabolites.

Quinones and quinone imines are highly reactive metabolites (RMs) able to induce dangerous effects in vivo. They are responsible for all kinds of toxicity, for example, cytotoxicity, immunotoxicity, and carcinogenesis. Furthermore, hepatotoxicity of chemicals/drugs in particular can be induced by quinone and quinone imine metabolites. According to their reactivity, quinones and quinone imines react as Michael's acceptors with cell proteins or DNA and, in this way, cause damage to the cells. Quinones and quinone imines also have high redox potential and, due to their semiquinone radicals, are capable of redox cycling and forming reactive oxygen species (ROS). However, the presence of quinones and quinone imines structures in compounds is not always responsible for a toxic effect. The main question, therefore, is what are the main factors responsible for the toxicity of the chemicals and drugs that form RMs. For this reason, the presence of structural alerts and evidence for the formation of reactive quinones and quinone imines metabolites and their mechanisms of toxicity through cellular effects are discussed in this review, together with examples.

Endocrine disrupting activities and immunomodulatory effects in lymphoblastoid cell lines of diclofenac, 4-hydroxydiclofenac and paracetamol.

A critical literature review reveals that knowledge of side effects of pharmaceuticals diclofenac and paracetamol is extremely important because of their widespread use and occurrence in the environment. In order to delineate whether these compounds have endocrine activity and influence on the immune system, we assessed the potential endocrine disrupting and immunomodulatory activities of: diclofenac (DIC), its metabolite 4-hydroxydiclofenac (4-HD) and paracetamol (PAR). Herein, we report on their impact on estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR) and thyroid hormone receptor (TR). The endocrine disrupting effects were assessed in vitro in MDA-kb2 and GH3.TRE-Luc cell lines and by the XenoScreen YES/YAS assay. Moreover, binding affinity to nuclear receptors (GR and AR) was also measured. Immunomodulatory properties of the compounds were evaluated in lymphoblastoid cell lines. All the tested compounds showed endocrine disrupting and immunomodulatory activities. The results revealed that both DIC and its metabolite 4-HD exhibited significant estrogenic, anti-androgenic (in YAS assay), (anti)-androgenic, (anti)-glucocorticoid and anti-thyroid hormonal activities (in luciferase reporter gene assays). DIC showed direct binding to the GR, while its metabolite 4-HD to the GR and AR. Only metabolite 4-HD showed estrogenic, androgenic (in YAS assay) and thyroid-hormonal activities. PAR had anti-androgenic activity and anti-thyroid hormonal activity. PAR displayed GR agonist activity with competition to its receptor and agonistic activity to AR. All of the compounds significantly modulated pro-inflammatory and immunoregulatory cytokine production in lymphoblastoid cell lines and were thus proven immunomodulatory. The study is useful in determining toxicological effects and contributes to the knowledge of possible side effects of diclofenac, its metabolite and paracetamol.

Endocrine Activity of AVB, 2MR, BHA, and Their Mixtures.

Personal care products are used increasingly, resulting in growing concern concerning their potential disruption of normal hormonal functions. Recent results on the bioaccumulation of cosmetic ingredients in wildlife and humans point to the need for an in-depth analysis for endocrine activity, in particular with respect to their influence on the androgen (AR), glucocorticoid (GR), and thyroid hormone receptors (TRs). Furthermore, humans are commonly exposed simultaneously to complex mixtures of endocrine active compounds. We have therefore examined 3 frequently used cosmetic ingredients: 2-methylresorcinol (2MR), butylated hydroxyanisole (BHA) and avobenzone (AVB), for (anti)-androgen-, (anti)-glucocorticoid-, and (anti)-thyroid hormone-like activities. Their binary and ternary mixtures at EC50 or IC50 concentrations have also been examined for anti-androgen-, glucocorticoid-, and thyroid hormone-like activities. In the MDA-kb2 reporter cell line, compounds possessed anti-androgen-, glucocorticoid-, and anti-glucocorticoid-like activities (except AVB). A new cell line, GH3.TRE-Luc, was used to evaluate anti-thyroid and thyroid hormone-like activities. The combinations 2MR + BHA and 2MR + BHA + AVB have glucocorticoid-like activity: only 2MR + AVB has anti-androgen-like activity. On the other hand, binary and ternary mixtures of compounds showed no thyroid hormone-like activity. Thus, in addition to identifying new endocrine disrupting compounds, it is also necessary to determine the effects of their mixtures in order to assess fully their risk to human health.

Comparison of in vitro hormone activities of novel flame retardants TBB, TBPH and their metabolites TBBA and TBMEPH using reporter gene assays.

The anti-androgenic and anti-thyroid hormonal activities of the two novel brominated flame retardants, TBB and TBPH and of their metabolites TBBA and TBMEPH have been compared using the luciferase reporter gene assays. Only the parent compounds TBB and TBPH exhibited anti-glucocorticoid activity with IC50 values of 1.9 µM and 0.3 µM. Furthermore, mode of action for these two compounds is by direct competing to the glucocorticoid receptor (GR) with IC50 values of 0.03 µM and 0.002 µM. All four tested compounds possess anti-androgenic and anti-thyroid hormonal activities, without agonist activities on the respective receptors. Anti-androgenic activities with IC50 values of 43.5 µM, 0.1 µM, 47.5 µM and 1.3 µM were found for TBB, TBPH, TBBA and TBMEPH. The anti-thyroid hormonal IC50 values of 37.5 µM, 0.1 µM, 22.8 µM and 32.3 µM for TBB, TBPH, TBBA and TBMEPH, together with the above quoted results, indicate that metabolism can modify anti-androgenic, anti-glucocorticoid and anti-thyroid hormonal effects of these novel brominated flame retardants. Furthermore, the parent flame retardants are shown to be able to disrupt the function of the GR as antagonists by direct competition to the receptor.

Influence of metabolism on endocrine activities of bisphenol S.

Bisphenol S (BPS; bis[4-hydroxyphenyl]sulfone) is commonly used as a replacement for bisphenol A in numerous consumer products. The main goal of this study was to examine the influence of different metabolic reactions that BPS undergoes on the endocrine activity. We demonstrate that hydroxylation of the aromatic ring of BPS, catalyzed mainly by the cytochrome P450 enzymes CYP3A4 and CYP2C9, is its major in-vitro phase I biotransformation. Nevertheless, coupled oxidative-conjugative reactions analyses revealed that glucuronidation and formation of BPS glucuronide is the predominant BPS metabolic pathway. BPS reactive metabolites that can be tracked as glutathione conjugates were not detected in the present study. Two in-vitro systems were used to evaluate the endocrine activity of BPS and its two main metabolites, BPS glucuronide and hydroxylated BPS 4-(4-hydroxy-benzenesulfonyl)-benzene-1,2-diol (BPSM1). In addition, we have tested two structural analogs of BPS, bis[4-(2-hydroxyetoxy)phenyl]sulfone (BHEPS) and 4,4-sulfonylbis(2-methylphenol) (dBPS). The test systems were yeast cells, for evaluating estrogenic and androgenic activities, and the GH3.TRE-Luc reporter cell line for measuring thyroid hormone activity. BPS and BPSM1 were weak agonists of the estrogen receptor, EC50 values of 8.4 × 10(-5) M and 6.7 × 10(-4) M, respectively. Additionally, BPSM1 exhibited weak antagonistic activity toward the thyroid hormone receptor, with an IC50 of 4.3 × 10(-5) M. In contrast to BPSM1, BPS glucuronide was inactive in these assays, inhibiting neither the estrogen nor the thyroid hormone receptors. Hence, glucuronidation appears to be the most important pathway for both BPS metabolism and detoxification.

A quantum chemical study of the reactivity of acetaminophen (paracetamol) toxic metabolite N-acetyl-p-benzoquinone imine with deoxyguanosine and glutathione.

Acetaminophen (APAP) forms some reactive metabolites that can react with DNA. APAP is a potentially genotoxic drug and is classified as a Group 3 drug according to International Agency for Research on Cancer (IARC). One of the possible mechanisms of APAP genotoxicity after long term of use is that its reactive quinone imine (QI) metabolite of acetaminophen (NAPQI), can chemically react with DNA after glutathione (GSH) depletion. A quantum chemical study of the reactions between the NAPQI and deoxyguanosine (dG) or GSH was performed. Activation energies (ΔG(ǂ)) for the reactions associated with the 1, 4-Michael addition were calculated on the M062X/6-311++G (d,p) level of theory. We modeled the reaction with dG as a multi-step process. The first step is rate-limiting (ΔG(ǂ) = 26.7 kcal/mol) and consists of formation of a C-N bond between the C3 atom of the QI moiety and the N7 atom of dG. The second step involves proton transfer from the C3 moiety to the nitrogen atom of the QI with ΔG(ǂ) of 13.8 kcal/mol. The depurination reaction that follows has a ΔG(ǂ) of 25.7 kcal/mol. The calculated ΔG(ǂ) for the nucleophilic attack of the deprotonated S atom of GSH on the C3 atom of the NAPQI is 12.9 kcal/mol. Therefore, the QI will react with GSH much faster than with DNA. Our study gives mechanistic insight into the genotoxicity of the APAP metabolite and will be useful for estimating the genotoxic potential of existing drugs with a QI moiety. Our results show that clinical application of APAP is safe, while in the case of severely depleted GSH levels APAP should be administered with caution.

In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future.

Glucocorticoid-like activity of propylparaben, butylparaben, diethylhexyl phthalate and tetramethrin mixtures studied in the MDA-kb2 cell line.

Endocrine-disrupting compounds can interfere with the endocrine organs or hormone system and cause tumors, birth defects and developmental disorders in humans. The estrogen-like activity of compounds has been widely studied but little is known concerning their possible modulation of the glucocorticoid receptor. Steroidal (synthetic and natural) and non-steroidal endocrine-active compounds commonly occur as complex mixtures in human environments. Identification of such molecular species, which are responsible for modulating the glucocorticoid receptor are necessary to fully assess their risk. We have used the MDA-kb2 cell line, which expresses endogenous glucocorticoid receptor and a stably transfected luciferase reporter gene construct, to quantify the glucocorticoid-like activity of four compounds present in products in everyday use - propylparaben (PP), butylparaben (BP), diethylhexyl phthalate (DEHP) and tetramethrin (TM). We tested all possible combinations of these compounds at two concentrations (1 µM and 10 nM) and compared their glucocorticoid-like activity. At the concentration of 1 µM seven mixtures were identified to have glucocorticoid-like activity except: DEHP+TM, BP+TM, DEHP+PP+TM, BP+PP+TM. At the concentration of 10 nM only three mixtures have glucocorticoid modulatory activity: DEHP+PP, BP+PP, DEHP+BP+PP+TM. Identified glucocorticoid-like activities were between 1.25 and 1.51 fold at the concentration of 1 µM and between 1.23 and 1.44 fold at the concentration of 10 nM in comparison with the solvent control. Individually BP, PP, and DEHP had glucocorticoid-like activity of 1.60, 1.57 and 1.50 fold over the solvent control at the concentration of 1 µM. On the other hand PP and DEHP, at the concentration of 10nM, showed no glucocorticoid-like activity, while BP showed 1.44 fold. The assertion that individual glucocorticoid-like compounds do not produce harm because they are present at low, ineffective levels in humans may be irrelevant when we include mixed exposures. This study emphasizes that risk assessment of compounds should take mixture effects into account.