Enantioselective Metabolism Research Articles

Enantioselective Metabolism of Mefentrifluconazole by Human Liver Microsomes.

A better understanding of the metabolic differences between chiral pesticide enantiomers in organisms is crucial for accurately assessing their risk. The enantioselective metabolism of mefentrifluconazole was investigated by the human liver microsome reaction system. The metabolic rate of S-mefentrifluconazole was found to be 4 times that of R-mefentrifluconazole. The chemical inhibitor method was used to further explore the cause of metabolic difference, and it was found that the inhibitors of CYP2C19 and CYP2C8 significantly reduced the metabolism of S-mefentrifluconazole (70.3-92.0%) and R-mefentrifluconazole (53.0-78.6%), respectively. CYP2C19 is a key metabolic enzyme of S-mefentrifluconazole. Molecular docking indicates that the internal energy of binding of R-mefentrifluconazole to CYP2C19 is too high, resulting in a positive docking fraction (0.1730 kJ/moL). Therefore, R-mefentrifluconazole cannot bind to CYP2C19 under natural conditions. CYP2C8 is the key metabolic enzyme of R-mefentrifluconazole. The lower docking energies (-37.80 kJ/moL for R-mefentrifluconazole and -35.64 kJ/moL for S-mefentrifluconazole) make CYP2C8 more capable of metabolizing R-mefentrifluconazole. This study provides essential data for exploring the toxicological assessment of mefentrifluconazole.

Computational simulations uncover enantioselective metabolism of chiral triazole fungicides by human CYP450 enzymes: A case study of tebuconazole

Tebuconazole (TEB), a prominent chiral triazole fungicide, has been extensively utilized for plant pathogen control globally. Despite experimental evidence of TEB metabolism in mammals, the enantioselectivity in the biotransformation of R- and S-TEB enantiomers by specific CYP450s remains elusive. In this work, integrated in silico simulations were employed to unveil the binding interactions and enantioselective metabolic fate of TEB enantiomers within human CYP1A2, 2B6, 2E1, and 3A4. Molecular dynamics (MD) simulations clearly delineated the binding specificity of R- and S-TEB to the four CYP450s, crucially determining their differences in metabolic activity and enantioselectivity. The primary driving force for robust ligand binding was identified as van der Waals interactions with CYP450s, particularly involving the hydrophobic residues. Mechanistic insights derived from quantum mechanics/molecular mechanics (QM/MM) calculations established C2-methyl hydroxylation as the predominant route of R-/S-TEB metabolism, while C6-hydroxylation and triazol epoxidation were deemed kinetically infeasible pathways. Specifically, the resulting hydroxy-R-TEB metabolite primarily originates from R-TEB biotransformation by 1A2, 2E1 and 3A4, whereas hydroxy-S-TEB is preferentially produced by 2B6. These findings significantly contribute to our comprehension of the binding specificity and enantioselective metabolic fate of chiral TEB by CYP450s, potentially informing further research on human health risk assessment associated with TEB exposure.

Assessing environmental and human health risks: Insight from the enantioselective metabolism and degradation of fenpropidin

Fenpropidin (FPD), a widely employed chiral fungicide, is frequently detected in diverse environments. In an in vitro rat liver microsomes cultivation (RLMs), the metabolism exhibited the order of R-FPD > S-FPD, with respective half-lives of 10.42 ± 0.11 and 12.06 ± 0.15 min, aligning with kinetic analysis results. CYP3A2 has been demonstrated to be the most significant oxidative enzyme through CYP450 enzyme inhibition experiments. Molecular dynamics simulations unveiled the enantioselective metabolic mechanism, demonstrating that R-FPD forms hydrogen bonds with the CYP3A2 protein, resulting in a higher binding affinity (−6.58 kcal mol−1) than S-FPD. Seven new metabolites were identified by Liquid chromatography time-of-flight high-resolution mass spectrometry, which were mainly generated through oxidation, reduction, hydroxylation, and N-dealkylation reactions. The toxicity of the major metabolites predicted by the TEST procedure was found to be stronger than the predicted toxicity of FPD. Moreover, the enantioselective fate of FPD was studied by examining its degradation in three soils with varying physical and chemical properties under aerobic, anaerobic, and sterile conditions. Enantioselective degradation of FPD occurred in soils without enantiomeric transformation, displaying a preference for R-FPD degradation. R-FPD is a low-risk stereoisomer both in the environment and in mammals. The research presented a systematic and comprehensive method for analyzing the metabolic and degradation system of FPD enantiomers. This approach aids in understanding the behavior of FPD in the environment and provides valuable insights into their potential risks to human health.

Absorption, distribution, metabolism, and elimination of epoxiconazole enantiomers in lizards (Eremias argus)

Epoxiconazole (EPX) is a world widely used chiral triazole fungicide in the agriculture field. The excessive application of this triazole may cause damage to lizards. However, limited information is known about the toxicokinetics of EPX on lizards. Our study aimed to investigate the enantioselective absorption, distribution, metabolism, and elimination (ADME) of EPX in lizards following low and high dose exposure (10 and 100 mg kg−1 bodyweitht (bw)). The results demonstrated that (+)-EPX was easier absorbed than (−)-EPX in lizard plasma. Both (+)-EPX and (−)-EPX were detected in the liver, gonad, kidney, skin, brain, and intestine, with (+)-EPX preferentially distributed in these tissues. The elimination of (−)-EPX was faster than that of (+)-EPX in lizard liver and kidney in the high dose groups. Chiral conversion was found between EPX enantiomers in lizard skin. Simultaneously, five metabolites including M2, M4, M10, M18 and M19 were detected in lizard liver and kidney after EPX enantiomers exposure. The relative concentrations of M2, M4, and M10 were higher in the liver and kidney of (−)-EPX groups than those produced from (+)-EPX groups. The metabolic enzymes CYP3A4 and SULT1A1 primarily mediated enantioselective metabolism of EPX. The conclusions drawn from this study significantly enhance our understanding of the enantioselective behaviors of chiral triazole fungicides in reptiles, offering essential guidance for assessing the risks associated with different enantiomers of triazole fungicides.

Enantioselective metabolism of fenpropathrin enantiomers by carboxyl/choline esterase 6 in Tetranychus cinnabarinus.

Tetranychus cinnabarinus is a polyphagous pest mite commonly found in agriculture. As an excellent acaricide, fenpropathrin (FEN) is frequently used to control T. cinnabarinus in agriculture. However, commercial FEN is a racemate with two enantiomers, R-FEN and S-FEN. Considering that investigations on the metabolism of FEN by T. cinnabarinus are based on racemate FEN, it is important to investigate the enantioselective metabolism of FEN in T. cinnabarinus. S-FEN was more toxic to T. cinnabarinus than R-FEN by more than 68.8-fold. Moreover, the synergist bioassay revealed that carboxylesterase and cytochrome P450 were the primary enzymes engaged in the detoxification of FEN in T. cinnabarinus, with carboxylesterase playing a leading role. Seven genes were substantially different after the induction of S-FEN and R-FEN. TcCCE06 was screened and selected as a key gene that related to FEN metabolism in T. cinnabarinus. The metabolic results showed that the recombinant TcCCE06 effectively metabolized 32.1% of the R-FEN and 13.8% of the S-FEN within 4 h of incubation. Moreover, R-FEN was demonstrated to have a higher affinity for the TcCCE06 protein than S-FEN based on molecular docking. Our results indicated that TcCCE06 mediates the enantioselective metabolism of FEN in T. cinnabarinus. Our findings will contribute to a more comprehensive understanding of the mechanisms underlying the differential toxicity of the FEN enantiomers against T. cinnabarinus. Furthermore, they also provide a new perspective for the development of enantiomer-enriched acaricides with higher activity and lower pesticide dosage and pollution risks. © 2023 Society of Chemical Industry.

Development of enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its phase-1 metabolites in human biological fluids

In the present study enantioselective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed for the quantitative determination of 3,4-methylenedioxy-methamphetamine (MDMA) and its major phase-1 metabolites 4-hydroxy-3-methoxyamphetamine (HMA), 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA) in human plasma, sweat, oral fluid (OF) and urine. The simultaneous separation of all these compounds and their respective enantioseparation was accomplished on two polysaccharide-based chiral columns. The Lux AMP column with a proprietary chiral selector enabled baseline separation of the enantiomers of MDMA, HMA and HMMA while MDA enantiomers could not be separated with this column under the experimental conditions used in this study. The Lux i-Amylose-3 column based on amylose tris(5-chloro-3-methylphenylcarbamate) as chiral selector baseline-separated the enantiomers of MDMA, HMMA and MDA while the enantiomers of HMA could not be separated. Thus, the various samples were analyzed by using both columns alternatively in combinations with acetonitrile containing 25% (v/v) 5 mM ammonium bicarbonate buffer at pH 11.0 as mobile phase. Analysis time was less than 4 min with the Lux AMP column and less than 6 min with the Lux i-Amylose-3 column. Both methods were validated and applied to the enantioselective determination of MDMA and its phase-I metabolites in human biological fluids, and enantioselective metabolism of MDMA was confirmed.

Two-dimensional chromatography for enantiomeric analysis of mitotane and its metabolite o,p′-DDA in patients with adrenocortical carcinoma indicates enantioselective metabolism

Mitotane is a chiral drug used to treat adrenocortical carcinoma, being metabolized to the o,p’-dichlorodiphenyl acetic acid (o,p’-DDA), also a chiral compound. Despite of its therapeutic significance, the overall ratios and enantiomers have not been known. In this study, we analyzed the enantiomers of mitotane and o,p’-DDA in the plasma of patients by a newly developed chiral-phase method employed in two-dimensional chromatography. Important differences were observed in the ratio of (S)/(R)-mitotane, which varied substantially from 1:1.2 to 1:10 whereas the (S)/(R)-o,p’-DDA ratio was relatively conserved, at approximately 2:1. These findings provide evidence for the enantioselective metabolism and provide a method for further analyses of mitotane and metabolites, which can explain the variation in the therapeutic response.

Enantioselective metabolism of novel chiral insecticide Paichongding by human cytochrome P450 3A4: A computational insight

As a novel chiral neonicotinoid insecticide, Paichongding (IPP) has been widely applied in agriculture due to its excellent insecticidal activity. However, the enantioselective metabolism of IPP stereoisomers (5R7R-IPP, 5S7S-IPP, 5R7S-IPP, and 5S7R-IPP) mediated by enzymes in non-target organisms, especially the cytochrome P450s (CYPs), remains unknown. To address this knowledge gap, we developed an integrated computational framework to elucidate the binding interactions and enantioselective metabolism of IPP stereoisomers in human CYP3A4. The results reveal that 5R7R-IPP shows much stronger binding affinity to CYP3A4 than 5S7S-IPP, while enantiomers 5R7S-IPP and 5S7R-IPP have no essential difference in their binding potential, owing to their specific interactions with key CYP3A4 residues. Although enantiomers 5R7R-IPP and 5S7S-IPP feature distinct binding modes resulting from the chiral differences, their transformation activities are slightly different, with C5 and C13 being the primary metabolic sites, respectively. In contrast, CYP3A4 preferably metabolizes 5R7S-IPP over 5S7R-IPP. The metabolism of epimers 5R7R-IPP and 5R7S-IPP share C5-hydroxylation routes due to the conserved 5R-conformaitons, but differ with the transformation routes at C11/C13 and C3 sites. The 7R-chirality of 5S7R-IPP significantly reduces the metabolic potency compared to 5S7S-IPP. CYP3A4-catalyzed hydroxylation and desaturation of IPP stereoisomers generate various chiral metabolites, with C5- and C13-hydroxyIPPs further transforming into depropylated products. Furthermore, the toxicity assessment reveals that IPP, along with the majority of its hydroxylated, desaturated, and depropylated metabolites, can potentially induce adverse effects on human health, specifically hepatotoxicity, respiratory toxicity, and carcinogenicity. This study provides valuable insights into the enantioselective fate of chiral IPP metabolism by CYP3A4, and the identified metabolites can serve as potential biomarkers for monitoring IPP exposure and associated health risk in human body.

Benoxacor is enantioselectively metabolized by microsomes and cytosol from the human liver

Benoxacor is a safener added to current-use herbicide formulations to protect the target crop from herbicidal toxicity. It is an emerging environmental contaminant that has been detected in surface waters, raising the possibility of human exposure via drinking water. Because it is not subject to the same regulations as active pesticide ingredients, its metabolism and toxicity in humans have not been studied. Here we investigate the enantioselective metabolism of benoxacor in human subcellular fractions. Pooled human liver microsomes (pHLM) and cytosol (pHLC) were incubated with racemic benoxacor for up to 30-min. Gas chromatographic analyses were used to measure the enantioselective depletion of benoxacor. pHLMs with and without nicotinamide adenine dinucleotide phosphate (NADPH, co-factor for cytochrome P450 enzymes [CYPs]) and pHLC with glutathione (GSH, co-factor for glutathione S-transferases [GSTs]) metabolized benoxacor. These results demonstrate that microsomal CYPs, microsomal carboxylesterase (CESs), and cytosolic GSTs metabolize benoxacor. Females were predicted to have a higher clearance of benoxacor by GSTs than males. Male and female pHLM incubations with NADPH showed enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). pHLM incubations without NADPH and pHLC incubations with GSH showed an enrichment of the second eluting enantiomer of benoxacor (E2-benoxacor). Our results indicate that human hepatic microsomal and cytosolic enzymes enantioselectively metabolize benoxacor, a fact that needs to be considered when investigating human exposures and toxicities of benoxacor.

The dichloroacetamide safener benoxacor is enantioselectively metabolized by monkey liver microsomes and cytosol

The metabolism and toxicity of current-use herbicide safeners remain understudied. We investigated the enantioselective metabolism of the safener benoxacor in Rhesus monkey subcellular fractions. Benoxacor was incubated with liver microsomes and cytosol from female and male monkeys (≤30 min). Benoxacor levels and enantiomeric fractions were determined with gas chromatography. Benoxacor was metabolized by microsomal cytochrome P450 enzymes (CYPs), cytosolic glutathione-S-transferases (GSTs), and microsomal and cytosolic carboxylesterase (CESs). CES-mediated microsomal metabolism followed the order males > females, whereas the CYP-mediated clearance followed the order females > males. CYP-mediated metabolism initially resulted in an enrichment of the second eluting benoxacor enantiomer (E2-benoxacor), whereas the first eluting benoxacor enantiomer (E1-benoxacor) was enriched after 10 or 30 min in female or male microsomal incubations. Benoxacor metabolism by GSTs was enantiospecific, with a total depletion of E1-benoxacor after approximately 20 min. Thus, the enantioselective metabolism of benoxacor by GSTs and CYPs may affect its toxicity.

Isolation of Sphingomonas sp. AJ-1 and its enantioselective S-methylation of the triazole fungicide prothioconazole

Prothioconazole is a widely used chiral triazole fungicide, and its residue pollution has attracted wide attention in recent years. However, little is known about microbial metabolic processes of prothioconazole enantiomers. In this study, a prothioconazole-degrading strain, Sphingomonas sp. AJ-1, was isolated from activated sludge. The optimal temperature and pH for prothioconazole degradation by strain AJ-1 were 30 °C and 6.0, respectively, and the degradation rate of prothioconazole by strain AJ-1 was negatively correlated with the initial concentration. When supplemented with additional carbon source, the degradation rates of 10 mg/L (Rac)-/(S)-/(R)-prothioconazole by strain AJ-1 were 76.0 %, 100.0 % and 64.8 % within 6 d, respectively. The CS bond of prothioconazole was methylated to produce (S)-/(R)-prothioconazole-S-methyl by strain AJ-1, but the degradation rate of prothioconazole by strain AJ-1 with (S)-enantiomer was 2.54-fold of that with (R)-enantiomer. Moreover, the toxicity of (Rac)-prothioconazole-S-methyl was 5.57 times lower than that of (Rac)-prothioconazole to Pseudokirchneriella subcapitata. The results showed that strain AJ-1 had obvious enantioselective metabolism for prothioconazole, and this metabolism was a detoxification process. This study provides new insights into the enantioselective metabolism of the chiral fungicide prothioconazole in microorganisms.

Differences in Enantioselective Hydroxylation of 2,2',3,6-Tetrachlorobiphenyl (CB45) and 2,2',3,4',6-Pentachlorobiphenyl (CB91) by Human and Rat CYP2B Subfamilies.

Although polychlorinated biphenyls (PCBs) were commercially banned half a century ago, contamination of the environment and organisms by PCBs is still observed. PCBs show high persistence and bioaccumulation, resulting in toxicity. Among PCBs, chiral PCBs with more than three chlorine atoms at the ortho-position exhibit developmental and neurodevelopmental toxicity. Because toxicity is dependent on the atropisomer, atropisomer-specific metabolism is vital in determining toxicity. However, structural information on enantioselective metabolism remains elusive. Cytochrome P450 (CYP, P450) monooxygenases, particularly human CYP2B6 and rat CYP2B1, metabolize separated atropisomers of 2,2',3,6-tetrachlorobiphenyl (CB45) and 2,2',3,4',6-pentachlorobiphenyl (CB91) to dechlorinated and hydroxylated metabolites. Docking studies using human CYP2B6 predict 4'-hydroxy (OH)-CB45 from (aR)-CB45 as a major metabolite of CB45. Di-OH- and dechlorinated OH-metabolites from human CYP2B6 and rat CYP2B1 are also detected. Several hydroxylated metabolites are derived from CB91 by both P450s; 5-OH-CB91 is predicted as a major metabolite. CB91 dechlorination is also detected by identifying 3-OH-CB51. A stable conformation of PCBs in the substrate-binding cavity and close distance to P450 heme are responsible for high metabolizing activities. As hydroxylation and dechlorination change PCB toxicity, this approach helps understand the possible toxicity of chiral PCBs in mammals.

Enantioselective Metabolic Mechanism and Metabolism Pathway of Pydiflumetofen in Rat Liver Microsomes: In Vitro and In Silico Study.

Pydiflumetofen (PYD) has been used worldwide. However, the enantioselective fate of PYD within mammals is not clear. Thus, the enantioselective metabolism and its potential mechanisms of PYD were explored via in vitro and in silico. Consistent results were observed between metabolism and enzyme kinetics experiments, with S-PYD metabolizing faster than R-PYD in rat liver microsomes. Moreover, CYP3A1 and carboxylesterase 1 were found to be major enzymes participating in the metabolism of PYD. Based on the computational results, S-PYD bound with CYP3A1 and carboxylesterase 1 more tightly with lower binding free energy than R-PYD, explaining the mechanism of enantioselective metabolism. Nine phase I metabolites of PYD were identified, and metabolic pathways of PYD were speculated. This study is the first to clarify the metabolism of PYD in mammals, and further research to evaluate the toxicological implications of these metabolites will help in assessing the risk of PYD.

Identification of Cytochrome P450 Isozymes Involved in Enantioselective Metabolism of Fipronil in Fish Liver: In Vitro Metabolic Kinetics and Molecular Modeling.

Fipronil has been frequently detected in waterways worldwide at concentrations that threaten aquatic organisms, yet the metabolic behavior of fipronil enantiomers in aquatic organisms is largely unknown, which is of significance in enantioselective toxicity evaluation. We quantitatively identified the specific cytochrome P450 (CYP) isozymes involved in metabolizing fipronil enantiomers in tilapia by combining in vitro metabolic kinetic assays and molecular docking. Inhibition studies suggested that CYP1A enzyme was the main isoform catalyzing metabolism of fipronil and that CYP3A contributed in a limited way to the metabolism in fish liver S9. Both the dissipation rate constant and the maximum metabolic velocity of R-(-)-fipronil were greater than those of S-(+)-fipronil in tilapia liver S9, suggesting that tilapia selectively metabolized R-(-)-fipronil. The CYP1A1 isozyme exhibited the highest binding capacity to R-(-)-fipronil and S-(+)-fipronil (binding energy -9.39 and -9.17 kcal/mol, respectively), followed by CYP1A2 (-7.30 and -6.94 kcal/mol, respectively) and CYP3A4 (-7.16 and -6.91 kcal/mol, respectively). The results of in vitro metabolic assays and molecular docking were consistent, that is, CYP1A, specifically CYP1A1, exhibited a higher metabolic capacity to fipronil than CYP3A, and fish liver S9 selectively metabolized R-(-)-fipronil. The present study provides insight into the enantioselective metabolic behavior and toxicological implications of the in vitro metabolic kinetics of fipronil in fish. Environ Toxicol Chem 2022;41:230-239. © 2021 SETAC.

Enantiomer metabolism of acephate and its metabolite methamidophos in in vitro tea (Camellia sinensis L.) systems: Comparison between cell suspensions and excised tissues

Enantioselective metabolism of chiral pesticide in plants is very important. In vitro system has become an effective means to study the metabolism of pesticides in plants, but the study on the metabolism of chiral pesticides has not been reported. This work compared the enantiomer metabolic behavior of acephate and its metabolite methamidophos between tea cell suspensions and excised tea stem with leaves. (±)-Acephate could be absorbed and transferred well to top leaves by the cut end of excised stem after 24 h. (±)-Methamidophos was derived from the metabolism of (±)-acephate in tea plants at 3–5% in leaves and 2–3% in stems at 216 h. The content of (+)-methamidophos was 1.5 times higher than that of (−)-methamidophos in excised leaves. Though both (±)-acephate and (±)-methamidophos could be metabolized well by cell suspension, (±)-acephate and (±)-methamidophos was non-enantioselectively metabolized in cell suspension. It was shown that using the excised tea stem with leaves for chiral pesticide metabolism studies was much closer to intact plant than cell suspensions. This result also established an effective and easily available in vitro metabolic model for the study of enantioselective metabolism of chiral contaminants from environment.

Stereochemistry of chiral pesticide uniconazole and enantioselective metabolism in rat liver microsomes

In this work, stereochemistry of uniconazole enantiomers and their metabolism behaviors in rat liver microsomes have been researched. Significance analysis has been applied in data processing. Absolute configurations of uniconazole enantiomers were identified through vibrational circular dichroism spectroscopy. According to their elution order from the chiral column using the CO2-methanol (80:20, v/v) mixture, two eluted fractions were determined to be (R)-uniconazole and (S)-uniconazole, respectively. A high-efficient and sensitive LC-MS/MS chiral analysis method was established for investigating the metabolism of uniconazole enantiomers in rat liver microsomes. The metabolic half-life of (R)-uniconazole (38.7 min) in rat liver microsomes was half that of (S)-enantiomer (74.5 min), and maximum velocity of metabolism, Michaelis constant of metabolism as well as the intrinsic metabolic clearance of (R)-uniconazole were significantly higher than (S)-enantiomer (p < 0.05), which indicated that (R)-uniconazole was preferentially metabolized in rat liver microsomes. By the virtue of molecular docking, (R)-uniconazole exhibited a higher binding affinity to cytochrome CYP2D2 than (S)-enantiomer, which corroborated well with the metabolism results. This work will shed light on the risk assessment of uniconazole toward human health and the ecological environment.

Stage Dependent Enantioselective Metabolism of Bifenthrin in Embryos of Zebrafish (Danio rerio) and Japanese Medaka (Oryzias latipes).

Bifenthrin (BF) is a widely used pyrethroid that has been frequently detected in surface waters. Previous studies indicated that BF had antiestrogenic activity in zebrafish embryos but estrogenic activity in posthatch fish. To determine whether age-related differences in metabolism contribute to the endocrine effects in developing fish, embryos from zebrafish and Japanese medaka were exposed to BF before and after liver development. Since the commercial mixture of BF is an isomer-enriched product containing two enantiomers (1R-cis-BF and 1S-cis-BF), enantioselective metabolism was also evaluated. The estrogenic metabolite, 4-hydroxybifenthrin (4-OH-BF) was identified in zebrafish embryos, and formation was higher in animals after liver development (>48 hpf). Treatments with β-glucuronidase indicated that 4-OH-BF underwent conjugation in embryos. Formation was reduced by cotreatment of the cytochrome P450 (CYP450) inhibitor, ketoconazole. Formation of 4-OH-BF was greater when treated with 1R-cis-BF compared to the S-enantiomer. However, metabolites were not observed in medaka embryos. These data indicate enantioselective oxidation of BF to an estrogenic metabolite occurs in zebrafish embryos and, since it is increased after liver development, may partially explain estrogenic activity observed in older animals. The lack of activity in medaka suggests species-specific effects with BF metabolism and may influence risk assessment strategies in wildlife.

Enantioselective metabolism of phenylpyrazole insecticides by rat liver microsomal CYP3A1, CYP2E1 and CYP2D2

The stereoselective difference of chiral pesticide enantiomers is an important factor of risk evaluation and the subject has received wide attention. In the present work, enantioselective metabolism of chiral phenylpyrazole insecticides including fipronil, ethiprole and flufiprole in rat liver microsomes was investigated in vitro. The result showed remarkable enantioselectivity for fipronil and ethiprole with the EF values of 0.11–0.58. The metabolite fipronil-sulfone was formed with the degradation of fipronil. R-Ethiprole to S-ethiprole transformation was observed, but not S-ethiprole to R-ethiprole. No enantioselective metabolism was observed for flufiprole with the EF values of 0.49–0.51. The enzymatic assays showed that the inhibition ratio of R-fipronil and S-ethiprole was 1.5–2.1times that of the corresponding enantiomers on CYP2E1 and CYP2D2 activity, leading to the enantioselective metabolism. The result of the homology modeling and molecular docking further revealed that S-fipronil (−7.56 kcal mol−1) and R-ethiprole (−6.45 kcal mol−1) performed better binding with CYP2E1 and CYP2D2, respectively. The results provided useful data for the risk evaluation of chiral phenylpyrazole insecticides on ecological safety and human health.

Benoxacor is enantioselectively metabolized by rat liver subcellular fractions

This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.

Enantioselective disposition and metabolic products of isofenphos-methyl in rats and the hepatotoxic effects

Isofenphos-methyl (IFP), a chiral organophosphorus pesticide, is one of the main chemicals used to control underground insects and nematodes. Recently, the use of IFP on vegetables and fruits has been prohibited due to its high toxicity. In this study, we investigated the enantioselective distribution and metabolism of IFP and its metabolites, namely, isofenphos-methyl oxon (IFPO) and isocarbophos oxon (ICPO), in male Sprague Dawley (SD) rats. Forty eight hours (48 h) after exposure, ICPO was the main detectable compound in blood (up to 75%) and urine (up to 77%), and we found that (S)-ICPO was significantly more stable than (R)-ICPO (p < 0.05). Therefore, (S)-ICPO was proposed as a suitable candidate biomarker for the biomonitoring of IFP in human urine and blood. After 48 h exposure, 21.2–41.0%, 4.1–15.1%, and 8.6–18.7% of dosed IFP was detected in the liver of racemic, R and S enantiomer-exposed rats, respectively, and R-IFP and R-IFPO showed a faster degradation (p < 0.05). Our results showed that after one week of consecutive exposure to IFP, ICPO was accumulated in the liver of rats in both racemic and enantiopure groups (no difference between the groups, p > 0.05). We found that cytochrome P450 (CYP) (i.e. CYP2C11, CYP2D2 and CYP3A2 enzymes and carboxylesterases) is responsible for the enantioselective metabolism of IFP in liver. In addition, rats exposed to (S)-IFP exhibited hepatic lipid peroxidation, liver inflammation and hepatic fibrosis. This study provides useful information and a reference for the biomonitoring and risk assessment of IFP and organophosphorus pesticide exposure.