•Two of the 8 BUVSs (UV-328 and UV-P) showed altered toxicity upon metabolism.•UV-328 showed more potent antiandrogenic activity after CYP3A4-mediated metabolism.•UV-328 can be metabolized into mono- and di-hydroxylated (OH) metabolites.•Sites of metabolism of UV-328 occur mainly at the alicyclic hydrocarbon atom.•Hydroxylation of BUVSs may present risk concern to human health.Benzotriazole ultraviolet stabilizers (BUVSs) are prominent chemicals widely used in industrial and consumer products to protect against ultraviolet radiation. They are becoming contaminants of emerging concern since their residues are frequently detected in multiple environmental matrices and their toxicological implications are increasingly reported. We herein investigated the antiandrogenic activities of eight BUVSs prior to and after human CYP3A4-mediated metabolic activation/deactivation by the two-hybrid recombinant human androgen receptor yeast bioassay and the in vitro metabolism assay. More potent antiandrogenic activity was observed for the metabolized UV-328 in comparison with UV-328 at 0.25 μM ((40.73 ± 4.90)% vs. (17.12 ± 3.00)%), showing a significant metabolic activation. In contrast, the metabolized UV-P at 0.25 μM resulted in a decreased antiandrogenic activity rate from (16.08 ± 0.95)% to (6.91 ± 2.64)%, indicating a metabolic deactivation. Three mono-hydroxylated (OH) and three di-OH metabolites of UV-328 were identified by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS), which were not reported previously. We further surmised that the hydroxylation of UV-328 occurs mainly at the alicyclic hydrocarbon atoms based on the in silico prediction of the lowest activation energies of hydrogen abstraction from C-H bond. Our results for the first time relate antiandrogenic activity to human CYP3A4 enzyme-mediated hydroxylated metabolites of BUVSs. The biotransformation through hydroxylation should be fully considered during the health risk assessment of structurally similar analogs of BUVSs and other emerging contaminants.Download high-res image (154KB)Download full-size image

The fungicide difenoconazole has been frequently detected in agricultural products, soils and surface waters, causing increasingly public concern due to toxicological properties. Although systemic triazole fungicides can inhibit the enzymatic activity of many CYP450 isozymes, how difenoconazole affects the human CYP3A4 still remains largely unknown. We thus investigated the effect of difenoconazole on normal mRNA expression and protein expression of human CYP3A4 by real-time quantitative PCR and western blot, respectively. Results show that the exposure to difenoconazole from 0.01 to 0.5 μM for 24 h down-regulates mRNA expression levels of human CYP3A4 in HepG2 cells. We also found that difenoconazole could inhibit the enzymatic activity of human CYP3A4 in a concentration-dependent mode. The IC50 of difenoconazole for inhibition of CYP3A4 activity is 0.41 µM, showing a stronger inhibitor in comparison with ketoconazole. Overall, our findings indicate the potential risk of difenoconazole for the disruption of human CYP3A4.

Triazole fungicides, one category of broad-spectrum fungicides, are widely applied in agriculture and medicine. The extensive use leads to many residues and casts potential detrimental effects on aquatic ecosystems and human health. After exposure of the human body, triazole fungicides may penetrate into the bloodstream and interact with plasma proteins. Whether they could have an impact on the structure and function of proteins is still poorly understood. By using multispectroscopic techniques and molecular modeling, the interaction of several typical triazole fungicides with human serum albumin (HSA), the major plasma protein, was investigated. The steady-state and time-resolved fluorescence spectra manifested that static type, due to complex formation, was the dominant mechanism for fluorescence quenching. Structurally related binding modes speculated by thermodynamic parameters agreed with the prediction of molecular modeling. For triadimefon, hydrogen bonding with Arg-218 and Arg-222 played an important role, whereas for imazalil, myclobutanil, and penconazole, the binding process was mainly contributed by hydrophobic and electrostatic interactions. Via alterations in three-dimensional fluorescence and circular dichroism spectral properties, it was concluded that triazoles could induce slight conformational and some microenvironmental changes of HSA. It is anticipated that these data can provide some information for possible toxicity risk of triazole fungicides to human health and be helpful in reinforcing the supervision of food safety.

•We investigated the effect of TR heterodimerization on T3 dissociation.•TR heterodimerization causes complete loss of pathway I in TR α-RXR LBD.•The second dominant dissociation pathway switched after TR heterodimerization.•Mechanisms of T3 egress were elucidated at atomic level.Numerous ligands bind tightly to thyroid hormone receptors (TRs), and exploring the binding and dissociation of these ligands from TRs will increase our understanding of their mechanisms of action. TRs form transcriptionally active heterodimers with retinoid X receptor (RXR); whether this heterodimerization affects ligand dissociation is poorly understood. To investigate the effects of heterodimerization, classical molecular dynamics (MD) simulations and random acceleration molecular dynamics (RAMD) simulations were performed to probe the dissociation of triiodothyronine (T3) from a TRα-RXR ligand binding domain (LBD) heterodimer and the TRα and TRβ LBDs at the atomic level. Seven (I–VII) dissociation pathways were identified for T3. Heterodimerization inhibited pathway I in the TRα-RXR LBD heterodimer, which may block the proper orientation of the helix 12 (H12), therefore affecting the biological functions of TRs. Upon TR heterodimerization, the second most dominant dissociation pathway switched from pathway IV for TRα LBD to pathway II for TRα-RXR LBD. No significant effects of TR heterodimerization were observed on the dominant dissociation pathway III that was located between H3, the H1–H2 loop and the β-sheet. Our study revealed that TR heterodimerization significantly affects T3 dissociation, which provides important information for the study of other TR ligands.

Over 98% of sprayed insecticides and 95% of herbicides reach non-target species in air, soil, and water. Numerous studies have reported that pesticide residues can cause acute and chronic toxicity. Pesticide residues can be carcinogenic, mutagenic, and immunotoxic. There are actually too few studies that bridge the disciplines of chemobioanalysis and environmental toxicology. Here, we assessed the cytotoxicity of a bipyridilium herbicide diquat in rat adrenal pheochromocytoma cells (PC12). Our results show that diquat caused the decrease in cell viability with a lethal concentration 50 (LC50) of 1.4 × 10−5 mol/L. This cytotoxicity may result from diquat-induced apoptosis, characterized by nuclear fragmentation and chromatin condensation by Hoechst 33324 staining. To explore the possible mechanisms, the interaction between herbicide diquat and calf thymus DNA (ctDNA) was further investigated using fluorescence quenching. The detection of static quenching showed that diquat was linked with ctDNA by electrostatic interaction with a binding constant of 9.288 × 104 L/mol. This is the first study on the interaction of DNA with herbicide diquat by fluorometric method as well as on the evaluation of cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst staining. Given the widespread use of synthetic pesticides, the data would be valuable for the risk assessment of pesticide residues.

Up to 25% of the current pesticides are chiral, the molecules have chiral centers, but most of them are used as racemates. In most cases, enantiomers of chiral pesticides have different fates in the environment. Knowledge of the function of amino acids of enzymes involved in enantioselective behaviors contributes to the understanding of the enantioselectivity of chiral pesticides. In this work, Aspergillus niger lipase (ANL, EC3.1.1.3) was chemically modified using bromoacetic acid (BrAc), 2,3-butanedione (BD), N-bromosuccinimide (NBS), and methanal. The enantioselectivity of the enzymatic hydrolysis of 2,4-dichlorprop-methyl (DCPPM) was investigated by chiral GC. The results have suggested that histidine, arginine, and tryptophan are essential for lipase activity and might be involved in the catalytic site of ANL. In addition, histidine and lysine play an important role in determining the observed enantioselective hydrolysis of chiral herbicide dichlorprop methyl. The molecular modeling study revealed that the essential hydrogen bonds formed between DCPPM and catalytic residues of ANL might be responsible for the enantioselectivity of DCPPM. The loss of enantioselectivity can also arise from the fact that the modification of the amino acids may cause changes in both the nature of the ANL enzyme conformation and the binding pattern of DCPPM. Our study provides basic information for the exploration of the enantioselective interaction mechanism of enzymes with chiral pesticides.

•The anti-androgenic activity of five triazole fungicides was evaluated.•No significant changes of anti-androgenic activity occur prior and after the metabolism.•Tebuconazole, uniconazole, hexaconazole, penconazole, bitertanol potently inhibit CYP3A4.•The anti-androgenic activity is highly correlated with the inhibition potency toward CYP3A4.•Our study is helpful to the health risk assessment of triazole fungicides.Triazole fungicides are widely used as broad-spectrum fungicides, non-steroidal antiestrogens and for various industrial applications. Their residues have been frequently detected in multiple environmental and human matrices. The increasingly reported toxicity incidents have led triazole fungicides as emerging contaminants of environmental and public health concern. However, whether triazole fungicides behave as endocrine disruptors by directly mimicking environmental androgens/antiandrogens or exerting potential androgenic disruption indirectly through the inhibition of cytochrome P450 (CYP450) enzyme activity is yet an unresolved question. We herein evaluated five commonly used triazole fungicides including bitertanol, hexaconazole, penconazole, tebuconazole and uniconazole for the androgenic and anti-androgenic activity using two-hybrid recombinant human androgen receptor (AR) yeast bioassay and comparatively evaluated their effects on enzymatic activity of CYP3A4 by P450-Glo™ CYP3A4 bioassay. All five fungicides showed moderate anti-androgenic activity toward human AR with the IC50 ranging from 9.34 μM to 79.85 μM. The anti-androgenic activity remained no significant change after the metabolism mediated by human liver microsomes. These fungicides significantly inhibited the activity of CYP3A4 at the environmental relevant concentrations and the potency ranks as tebuconazole > uniconazole > hexaconazole > penconazole > bitertanol with the corresponding IC50 of 0.81 μM, 0.93 μM, 1.27 μM, 2.22 μM, and 2.74 μM, respectively. We found that their anti-androgenic activity and the inhibition potency toward CYP3A4 inhibition was significantly correlated (R2 between 0.83 and 0.97, p < 0.001). Our results indicated that the risk assessment of triazole pesticides and structurally similar chemicals should fully consider potential androgenic disrupting effects and the influences on the activity of CYP450s.Download high-res image (111KB)Download full-size image