CYP1A1 Activity Research Articles

Discovery of a novel AhR–CYP1A1 axis activator for mitigating inflammatory diseases using an in situ functional imaging assay

The aryl hydrocarbon receptor (AhR) plays a crucial role in regulating many physiological processes. Activating the AhR–CYP1A1 axis has emerged as a novel therapeutic strategy against various inflammatory diseases. Here, a practical in situ cell-based fluorometric assay was constructed to screen AhR-CYP1A1 axis modulators, via functional sensing of CYP1A1 activities in live cells. Firstly, a cell-permeable, isoform-specific enzyme-activable fluorogenic substrate for CYP1A1 was rationally constructed for in-situ visualizing the dynamic changes of CYP1A1 function in living systems, which was subsequently used for discovering the efficacious modulators of the AhR–CYP1A1 axis. Following screening of a compound library, LAC-7 was identified as an efficacious activator of the AhR–CYP1A1 axis, which dose-dependently up-regulated the expression levels of both CYP1A1 and AhR in multiple cell lines. LAC-7 also suppressed macrophage M1 polarization and reduced the levels of inflammatory factors in LPS-induced bone marrow-derived macrophages. Animal tests showed that LAC-7 could significantly mitigate DSS-induced ulcerative colitis and LPS-induced acute lung injury in mice, and markedly reduced the levels of multiple inflammatory factors. Collectively, an optimized fluorometric cell-based assay was devised for in situ functional imaging of CYP1A1 activities in living systems, which strongly facilitated the discovery of efficacious modulators of the AhR–CYP1A1 axis as novel anti-inflammatory agents.

Combinatory Effects of Acrylamide and Deoxynivalenol on In Vitro Cell Viability and Cytochrome P450 Enzymes of Human HepaRG Cells.

Acrylamide (AA) can be formed during the thermal processing of carbohydrate-rich foods. Deoxynivalenol (DON), a mycotoxin produced by Fusarium spp., contaminates many cereal-based products. In addition to potential co-exposure through a mixed diet, co-occurrence of AA and DON in thermally processed cereal-based products is also likely, posing the question of combinatory toxicological effects. In the present study, the effects of AA (0.001-3 mM) and DON (0.1-30 µM) on the cytotoxicity, gene transcription, and expression of major cytochrome P450 (CYP) enzymes were investigated in differentiated human hepatic HepaRG cells. In the chosen ratios of AA-DON (10:1; 100:1), cytotoxicity was clearly driven by DON and no overadditive effects were observed. Using quantitative real-time PCR, about twofold enhanced transcript levels of CYP1A1 were observed at low DON concentrations (0.3 and 1 µM), reflected by an increase in CYP1A activity in the EROD assay. In contrast, CYP2E1 and CYP3A4 gene transcription decreased in a concentration-dependent manner after incubation with DON (0.01-0.3 µM). Nevertheless, confocal microscopy showed comparably constant protein levels. The present study provided no indication of an induction of CYP2E1 as a critical step in AA bioactivation by co-occurrence with DON. Taken together, the combination of AA and DON showed no clear physiologically relevant interaction in HepaRG cells.

Isolation and characterisation of two epithelial-like cell lines from the gills of Chrysophrys auratus (Australasian snapper)and Oncorhynchus tshawytscha (Chinook salmon) and their use in aquatic toxicology.

In vitro gill models are becoming increasingly important in aquatic toxicology, yet the fish gill invitrome is underrepresented, encompassing approximately 0.1% of extant species. Here, we describe the establishment and characterisation of two gill-derived, epithelial-like cell lines isolated from fish species of significant importance to New Zealand: Chrysophrys auratus (Australasian snapper) and Oncorhynchus tshawytscha (Chinook salmon). Designated CAgill1PFR (Chrysophrys auratus, gill 1, Plant & Food Research) and OTgill1PFR (Oncorhynchus tshawytscha, gill 1, Plant & Food Research), these cell lines have each been passaged greater than each 70 times over several years and are considered spontaneously immortalised. Both cell lines required serum for growth and exhibited differential responses to basal media formulations. CAgill1PFR was sensitive to low temperatures (4°C) but replicated at high temperatures (30°C), whereas OTgill1PFR was sensitive to high temperatures but remained viable at low temperatures, mirroring the natural environment of their host species. Immunostaining revealed expression of epithelial cell markers cytokeratin and E-cadherin, alongside positivity for the mesenchymal cell marker, vimentin. CAgill1PFR was more sensitive to the environmental toxin 3,4 dichloroaniline than OTgill1PFR through measurements of metabolic activity, membrane integrity, and lysosomal function. Furthermore, CAgill1PFR produced less CYP1A activity, indicative of ongoing biotransformation processes, in response to beta-naphthoflavone than OTgill1PFR. These cell lines expand the toolbox of resources and emphasise the need for species-specific aquatic toxicology research.

Atlantic salmon gill epithelial cell line ASG-10, an in vitro model for studying effects of microplastics in gills

Microplastics are ubiquitous environmental pollutants frequently detected in aquatic environments. Here we used the Atlantic salmon epithelial gill cell line (ASG-10) to investigate the uptake and effects of polystyrene (PS) microplastic. The ASG-10 cell line has phagocytotic/endocytic capacities and can take up clear PS particles at 0.2 and 1.0 µm, while PS at 10 µm was not taken up. As a response to the uptake, the ASG-10 cells increased their lysosomal activity. Furthermore, no effects on the mitochondria were found, neither on the mitochondrial membrane potential nor the mitochondria morphology (branch length and diameter). Interestingly, even a very high concentration of PS (200 µg/ml) with all tested particle sizes had no effects on cell viability or cell cycle. The environmental toxin Benzo(a)pyrene (B(a)P), a known inducer of CYP1A, is highly hydrophobic and thus sticks to the PS particles. However, co-exposure of B(a)P and PS the particles did not increase the induction of CYP1A activity compared to B(a)P alone. Our study contributes to the understanding of the cellular effects of PS particles using a highly relevant Atlantic salmon gill epithelium in vitro model.

Evaluating the Dynamics of Cyp3a11, MRP2 and MDR1 Modulation by IL-22

Background: IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing. In addition, IL-22 reduces the expression of MRP2 and MDR1, the gene that encodes as Multidrug resistance protein 2 or 1 involve in the drug detoxification. Modulation of these proteins can influence the oral bioavailability of drugs. Aims: The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11, MRP2 and MDR1 expression and protein level or activity in the intestinal epithelium. Methods: In vitro enteroids of the mouse small intestine were cultured in 3D in the presence/absence of IL-22 for 5 days. Cyp3a11, MRP2 and MDR1 transcript expression was evaluated by qPCR followed by protein analysis to define their modulation and activity at varying timepoints after IL-22 treatment cessation. Results: IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. This action of IL-22 are made without any modulation of protein level of Cyp3a11. In the same way, IL-22 reduces the expression and the protein level of MRP2 and MDR1, an effect normalizes after treatment cessation. Conclusions: IL-22 suppresses the expression and the activity of Cyp3a11 without any modulation of the protein level unlike at the level of MRP2 and MDR1 which are reduced. These data suggest that regulation of gene expression, protein or enzyme activity by IL-22 may have variable effects: Cyp3a11 is ATP-independent, whereas MRP2 and MDR1 are ATP-dependent. This direct or indirect enzymatic modulation of these proteins must be taken into account when assessing the role of the drug in the biology of the intestinal mucosa. Dr. Lloyd Sutherland Chair in IBD/GI Research/Weston Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Non-alcoholic fatty liver disease changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which can cause serious complications and gradually increase the mortality rate. However, the effects of NAFLD on drug-metabolizing enzymes and transporters remain unclear, which may cause some confusion regarding patient medication. In this study, a NAFLD rat model was constructed by feeding rats with methionine and choline deficiency diets for 6 weeks, and the mRNA and protein levels of drug-metabolizing enzymes and transporter were analyzed by real-time fluorescent quantitative PCR and Western blot, respectively. The activity of drug-metabolizing enzymes was detected by cocktail methods. In the NAFLD rat model, the mRNA expression of phase I enzymes, phase II enzymes, and transporters decreased. At the protein level, only CYP1A1, CYP1B1, CYP2C11, and CYP2J3 presented a decrease. In addition, the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP3A2, UGT1A1, UGT1A3, UGT1A6, and UGT1A9 decreased. These changes may be caused by the alteration of FXR, HNF4α, LXRα, LXRβ, PXR, and RXR. In conclusion, NAFLD changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats, which may affect drug metabolism and pharmacokinetics. In clinical medication, drug monitoring should be strengthened to avoid potential risks.

AP-1 and SP1 trans-activate the expression of hepatic CYP1A1 and CYP2A6 in the bioactivation of AFB1 in chicken.

Dietary exposure to aflatoxin B1 (AFB1) is harmful to the health and performance of domestic animals. The hepatic cytochrome P450s (CYPs), CYP1A1 and CYP2A6, are the primary enzymes responsible for the bioactivation of AFB1 to the highly toxic exo-AFB1-8,9-epoxide (AFBO) in chicks. However, the transcriptional regulation mechanism of these CYP genes in the liver of chicks in AFB1 metabolism remains unknown. Dual-luciferase reporter assay, bioinformatics and site-directed mutation results indicated that specificity protein 1 (SP1) and activator protein-1 (AP-1) motifs were located in the core region -1,063/-948, -606/-541 of the CYP1A1 promoter as well as -636/-595, -503/-462, -147/-1 of the CYP2A6 promoter. Furthermore, overexpression and decoy oligodeoxynucleotide technologies demonstrated that SP1 and AP-1 were pivotal transcriptional activators regulating the promoter activity of CYP1A1 and CYP2A6. Moreover, bioactivation of AFB1 to AFBO could be increased by upregulation of CYP1A1 and CYP2A6 expression, which was trans-activated owing to the upregulalion of AP-1, rather than SP1, stimulated by AFB1-induced reactive oxygen species. Additionally, nano-selenium could reduce ROS, downregulate AP-1 expression and then decrease the expression of CYP1A1 and CYP2A6, thus alleviating the toxicity of AFB1. In conclusion, AP-1 and SP1 played important roles in the transactivation of CYP1A1 and CYP2A6 expression and further bioactivated AFB1 to AFBO in chicken liver, which could provide novel targets for the remediation of aflatoxicosis in chicks.

On the Possible Effect of Phytic Acid (Myo-Inositol Hexaphosphoric Acid, IP6) on Cytochromes P450 and Systems of Xenobiotic Metabolism in Different Hepatic Models.

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug-drug interactions involving IP6 are low.

Circadian rhythms in CYP2A5 expression underlie the time-dependent effect of tegafur on breast cancer

The chemotherapeutic agent tegafur, a prodrug that prolongs the half-life of fluorouracil (5-FU), exerts antitumor effects against various cancers. Since tegafur is metabolized to 5-FU by CYP2A6 in the liver, the expression of CYP2A6 determines the effect of tegafur. Here, we report that the expression rhythm of Cyp2a5, a homolog of human CYP2A6, in female mice causes dosing time-dependent differences in tegafur metabolism. In the livers of female mice, CYP2A5 expression showed a circadian rhythm, peaking during the dark period. This rhythm is regulated by RORA, a core clock component, and abrogation of the CYP2A5 activity abolished the time-dependent difference in the rate of tegafur metabolism in female mice. Furthermore, administration of tegafur to mice transplanted with 4T1 breast cancer cells during the dark period suppressed increases in tumor size compared to female mice treated during the light period. Our findings reveal a novel relationship between 5-FU prodrugs and circadian clock machinery, potentially influencing antitumor effects, and contributing to the development of time-aware chemotherapy regimens for breast cancer.

Influence of Different Plant Extracts on CYP-Mediated Skatole and Indole Degradation in Pigs.

One of the primary substances responsible for the unpleasant odor in boar meat is skatole. Enzymes belonging to the cytochrome P450 (CYP) family play a pivotal role in the hepatic clearance of skatole. This study aimed to investigate the impact of oregano essential oil (OEO), Schisandra chinensis extract (SC), and garlic essential oil (GEO) on hepatic CYP2E1 and CYP2A activity in pigs. In three consecutive trials, cannulated castrated male pigs were provided with a diet containing 0.2-0.3% of one of these plant extracts. Following a 14-day feeding period, the animals were slaughtered, and liver and fat samples were collected. The findings indicate that the activities of CYP2E1 were unaffected by any treatment. However, GEO treatment demonstrated a significant reduction in CYP2A activity (p < 0.05). Pigs treated with GEO also exhibited a notable increase in skatole concentrations in both plasma and adipose tissue. In contrast, animals fed SC displayed elevated skatole concentrations in plasma but not in fat tissue. OEO did not influence skatole concentrations in either blood or fat. Furthermore, the study revealed that a supplementation of 6 g GEO per animal per day induced a significant increase in skatole concentrations in blood plasma within 24 h.

A56 EVALUATING THE DYNAMICS OF CYP3A11 AND REG3G MODULATION BY IL-22

Abstract Background IL-22 is a cytokine produced by a number of immune cell populations, including type 3 innate lymphoid cells. We have shown previously that IL-22 suppresses the expression Cyp3a11 in the mouse intestine, a gene that encodes a drug metabolizing enzyme that accounts for roughly 70% of total drug metabolism. In addition, IL-22 induces the expression of Reg3g, the gene that encodes an antimicrobial C-type lectin that has bactericidal activity against Gram+ bacteria in the small intestine. Modulation of these proteins can influence both the oral bioavailability of drugs and microbial composition. Aims The aim of this study was to define the existence of reversible modulation of IL-22 on Cyp3a11 and Reg3g expression in the intestinal epithelium. Methods In vitro enteroids from each part of the mouse small intestine were cultured in 3D in the presence or absence of IL-22 for 5 days. Cyp3a11 and Reg3g transcript expression was evaluated by qPCR followed by protein analysis to define their modulation at varying timepoints after IL-22 treatment cessation. Results IL-22 reduced Cyp3a11 expression, an effect that normalized the day after treatment cessation. However, 4 days after treatment cessation there was rebound hyperexpression of Cyp3a11 in duodenal enteroids, which normalized the following day. IL-22 reduced the expression and activity of Cyp3a11 which normalized soon after stopping IL-22 treatment. In contrast, IL-22 induced the expression of Reg3g, an effect that lasted up to 5 days after treatment cessation. The effect of IL-22 on Cyp3a11 expression and activity was rapidly reversed in contrast to the effect on Reg3g which was more prolonged. Conclusions IL-22 suppresses the expression of Cyp3a11 and induces the expression of Reg3g. The reversible nature of these effects is different between both, with the increased expression of Reg3g 5 days after IL-22 treatment cessation. These data suggest that the regulation of gene expression, protein or enzyme activity by IL-22 may exhibit varying temporal effects and should be considered when assessing its role in intestinal mucosal biology. Funding Agencies This work was funded by the Dr. Lloyd Sutherland Chair in IBD/GI Research and the Weston Foundation.

Chronic Exposure to E-Cigarettes Elevates CYP2A5 Activity, Protein Expression, and Cotinine-Induced Production of Reactive Oxygen Species in Mice.

Coumarin 7'-hydroxylase activity, a specific marker of CYP2A5 activity, and the protein level were measured in liver microsomes of male mice after chronic exposure to e-cigarettes (e-cigs) (2.4% nicotine). After exposure for 240 minutes per day for 5 days, the activity and the protein level in preproenkephalin (ppENK)-heterozygous [ppENK (+/-)] mice were significantly elevated (P <0.05) compared with the untreated control. This elevation was not due to deletion of the ppENK gene because the activity did not differ among untreated ppENK (+/-), ppENK (-/-), and wild-type ppENK (+/+) controls. Hence, the elevation can reasonably be attributed to nicotine exposure. The production of reactive oxygen species (ROS) upon incubation of the hepatic microsomes of these mice with cotinine was higher in microsomes from the e-cig-treated mice compared with the untreated controls (P < 0.01). Liquid chromatography mass spectrometry assay showed three oxidation products of cotinine, viz trans 3'-hydroxycotinine (3'-HC), 5'-hydroxycotinine (5'-HC), and cotinine N-oxide (CNO) in the plasma of these mice. The result identifies these three oxidation reactions as the source of the observed ROS and also shows that, in nicotine-treated mice, the appropriate "nicotine metabolite ratio" is (3'-HC + 5'-HC + CNO)/cotinine. The results suggest intriguing possibilities that 1) this metabolite ratio may correlate with plasma nicotine clearance and hence impact nicotine's psychoactive effects and 2) chronic e-cig treatment causes ROS-induced oxidative stress, which may play a major role in the regulation of CYP2A5 expression. Our present results clearly show that both the activity and the protein level of CYP2A5 are elevated by repeated exposure to nicotine. SIGNIFICANCE STATEMENT: Nicotine, the psychoactive ingredient of tobacco, is eliminated as the oxidation products of cotinine in reactions catalyzed by the enzymes CYP2A5 in mice and CYP2A6 in humans. This study shows that repeated exposure to e-cigarettes elevates the level of CYP2A5 and the formation of reactive oxygen species. The results suggest an intriguing possibility that CYP2A5 may be upregulated by chronic nicotine exposure due to oxidative stress caused by the oxidation of cotinine in this preclinical model of human smokers.

Crizotinib inhibits the metabolism of tramadol by non-competitive suppressing the activities of CYP2D1 and CYP3A2.

To investigate the interaction between tramadol and representative tyrosine kinase inhibitors, and to study the inhibition mode of drug-interaction. Liver microsomal catalyzing assay was developed. Sprague-Dawley rats were administrated tramadol with or without selected tyrosine kinase inhibitors. Samples were prepared and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for analysis. Besides, liver, kidney, and small intestine were collected and morphology was examined by hematoxyline-eosin (H&E) staining. Meanwhile, liver microsomes were prepared and carbon monoxide differential ultraviolet radiation (UV) spectrophotometric quantification was performed. Among the screened inhibitors, crizotinib takes the highest potency in suppressing the metabolism of tramadol in rat/human liver microsome, following non-competitive inhibitory mechanism. In vivo, when crizotinib was co-administered, the AUC value of tramadol increased compared with the control group. Besides, no obvious pathological changes were observed, including cell morphology, size, arrangement, nuclear morphology with the levels of alanine transaminase (ALT) and aspartate transaminase (AST) increased after multiple administration of crizotinib. Meanwhile, the activities of CYP2D1 and CYP3A2 as well as the total cytochrome P450 abundance were found to be decreased in rat liver of combinational group. Crizotinib can inhibit the metabolism of tramadol. Therefore, this recipe should be vigilant to prevent adverse reactions.

Pharmacokinetic and pharmacodynamic drug-drug interaction of Nomilin with atorvastatin in hyperlipidemic mice

Atorvastatin (Atv) is widely used to lower cholesterol levels and treat hyperlipidemia in clinical application. Nomilin (Nom) is a kind of limonoids, which is found and isolated from the citrus herbs of Rutaceae family, which are widely used as patent medicines, functional foods, and nutritional supplements in many countries. In previous studies, Nom has the effect of anti-obesity and curing other metabolic diseases. Nevertheless, in recent years, the drug-drug interaction (DDI) caused by the administration of drugs with synergistic effects have raised worldwide concerns. To investigate the DDI of Nom and Atv in vivo, the pharmacokinetic studies were performed with using C57BL/6 mice. The plasma concentrations of Nom and Atv were measured after oral administration of different drug combinations by a simple and sensitive UHPLC-MS/MS method. The experimental mice were randomly divided into five groups, including control group, model group, administered Nom individually group, administered Atv individually group and co-administered of Nom and Atv group. The lipid levels including total cholesterol (TC), triglycerides (TG), high density lipoproteins-cholesterol (HDL-C), low density lipoproteins-cholesterol (LDL-C) were measured for pharmacodynamic study. The hepatic microsomal Cytochrome P450 (CYP1A2, CYP2E1 and CYP3A11) activities were probed using cocktail assay. The gene and protein expressions of CYP3A11 were detected via qPCR and Western blot method. The results shown that the area under the plasma concentration-time curve (AUC) of Atv in administered Atv individually group was 69.30 ± 15.45 ng/mL × h, while that of combined Nom with Atv group was 42.37 ± 10.15 ng/mL × h (p＜0.05). The degree of reduction in lipid levels of mice treated with co-administration of Atv and Nom was less than that of mice treated with Atv alone. In addition, Nom could cause an increased hepatic microsomal CYP3A11 activity significantly, and induce the gene levels and protein expressions of CYP3A11 elevated in mice livers. In conclusion, Nom could up-regulate CYP3A11 activity, thereby impacting on the pharmacokinetic profile and pharmacodynamic effect of Atv. The findings provide more insight for the use risk of these two drugs to treat hyperlipidemia diseases.

Crosstalk between aryl hydrocarbon receptor and Wnt/β-catenin signaling pathway: Possible culprit of di (2-ethylhexyl) phthalate-mediated cardiotoxicity in zebrafish larvae

Typical plasticizer di (2-ethylhexyl) phthalate (DEHP) has been demonstrated to induce cardiotoxicity in zebrafish, but the potential molecular mechanisms involved have not been fully elucidated. Aryl hydrocarbon receptor (AhR), an essential protein for inducing developmental abnormalities, has been demonstrated to be activated by DEHP in other species, but whether the AhR signaling pathway also contributes to DEHP-mediated cardiac developmental toxicity in zebrafish remains unclear. Firstly, molecular docking simulations initially confirmed the possibility that DEHP has AhR agonistic activity. To further confirm this conjecture, this work analyzed the changes of cardiac-related indexes in zebrafish stressed by DEHP at individual, protein, and gene levels. The results showed that DEHP mediated cardiac phenotypic developmental defects, increased CYP1A1 activity, and oxidative stress as well as significant changes in the expression levels of key proteins and genes of AhR, Wnt/β-catenin, and Nrf2-Keap1 signaling pathways. Notably, the addition of AhR inhibitors effectively alleviated the above negative effects, indicating that the AhR signaling pathway and its crosstalk with the Wnt/β-catenin signaling pathway is an essential pathway for DEHP-mediated cardiac developmental toxicity. Overall, this work enriches the molecular mechanism of DEHP-mediated cardiac developmental defects in zebrafish and provides a reliable biomarker for future environmental risk assessment of DEHP.

CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4

CYP2A5, an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1-null and Cry2-null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting, respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression. In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics.

Glutaredoxin-1 alleviates acetaminophen-induced liver injury by decreasing its toxic metabolites

Excessive N-acetyl-p-benzoquinone imine (NAPQI) formation is a starting event that triggers oxidative stress and subsequent hepatocyte necrosis in acetaminophen (APAP) overdose caused acute liver failure (ALF). S-glutathionylation is a reversible redox post-translational modification and a prospective mechanism of APAP hepatotoxicity. Glutaredoxin-1 (Glrx1), a glutathione-specific thioltransferase, is a primary enzyme to catalyze deglutathionylation. The objective of this study was to explored whether and how Glrx1 is associated with the development of ALF induced by APAP. The Glrx1 knockout mice (Glrx1−/−) and liver-specific overexpression of Glrx1 (AAV8-Glrx1) mice were produced and underwent APAP-induced ALF. Pirfenidone (PFD), a potential inducer of Glrx1, was administrated preceding APAP to assess its protective effects. Our results revealed that the hepatic total protein S-glutathionylation (PSSG) increased and the Glrx1 level reduced in mice after APAP toxicity. Glrx1−/− mice were more sensitive to APAP overdose, with higher oxidative stress and more toxic metabolites of APAP. This was attributed to Glrx1 deficiency increasing the total hepatic PSSG and the S-glutathionylation of cytochrome p450 3a11 (Cyp3a11), which likely increased the activity of Cyp3a11. Conversely, AAV8-Glrx1 mice were defended against liver damage caused by APAP overdose by inhibiting the S-glutathionylation and activity of Cyp3a11, which reduced the toxic metabolites of APAP and oxidative stress. PFD precede administration upregulated Glrx1 expression and alleviated APAP-induced ALF by decreasing oxidative stress. We have identified the function of Glrx1 mediated PSSG in liver injury caused by APAP overdose. Increasing Glrx1 expression may be investigated for the medical treatment of APAP-caused hepatic injury.

Wogonin alleviates BaP-induced DNA damage and oxidative stress in human airway epithelial cells by dual inhibiting CYP1A1 activity and expression.

Benzo[a]pyrene (BaP) is a common air pollutant that has been reported to cause oxidative stress and carcinogenesis. Wogonin, a flavonoid compound extracted from the roots of Scutellaria baicalensis, has been found to possess a variety of pharmacological activities, including anti-inflammatory and anti-cancer effects. The purpose of this study was to examine the ability of wogonin to alleviate the cytotoxicity induced by BaP in human airway epithelial cells and explore the corresponding mechanism. Our study found that wogonin treatment inhibited DNA damage and reactive oxygen species overproduction induced by BaP in human airway epithelial cells. In vitro enzyme assays showed that wogonin significantly inhibited the enzymatic activity of CYP1A1. In addition, wogonin decreased the basal level of CYP1A1 and inhibited the CYP1A1 overexpression induced by BaP, whereas overexpression of CYP1A1 partially reversed the effect of wogonin on BaP-induced DNA damage. Meanwhile, a CYP1A1 inhibitor and CYP1A1 knockdown also showed these same effects. Further studies showed that wogonin regulates CYP1A1 expression by inhibiting CDK7 and CDK9 activity. The use of CDK7 or CDK9 inhibitors decreased BaP-induced cytotoxicity and CYP1A1 expression. Finally, we found that the methoxy group of wogonin was crucial for its inhibitory activity. In conclusion, our data indicated that wogonin could effectively relieve BaP induced cytotoxicity, and its mechanism was related to the dual inhibition of CYP1A1 activity and expression.

A fish perspective on SARS-CoV-2: Toxicity of benzalkonium chloride on Danio rerio

SARS-CoV-2 outbreak led to an increased marketing of disinfectants, creating a potential environmental problem. For instance, pre-pandemic environmental levels of the disinfectant benzalkonium chloride (BAC) ranging from 0.5 to 5 mgL−1 in effluents were expected to further increase threatening aquatic life. Our aim was to characterize potential adverse effects after an acute exposure of zebrafish to different concentrations of BAC. An increase in the overall swimming activity, thigmotaxis behavior, and erratic movements were observed. An increase in CYP1A1 and catalase activities, but inhibitions of CY1A2, GSTs and GPx activities were also noticed. BAC is metabolized by CYP1A1, increasing the production of H2O2, thereby activating the antioxidant enzyme CAT. Data also showed an increase of AChE activity. Our study highlights adverse embryonic, behavioral, and metabolic effects of noteworthy environmental significance, especially considering that the use and release of BAC is most likely to increase in a near future.

Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract.

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC50) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC40 value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC50 value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.