Activation of aryl hydrocarbon receptor (AhR) alleviates depressive-like behaviors in LPS-induced mice.

The role of the aryl hydrocarbon receptor (AhR) in regulating oxidative stress and immune responses has been increasingly recognized. However, its involvement in depression and the underlying mechanisms remain poorly understood. This study aimed to investigate the effect of 6-formylindolo[3,2-b]carbazole (FICZ), an endogenous AhR ligand, on a lipopolysaccharide (LPS)-induced depression model and the underlying mechanism. After being treated with FICZ (50 mg/kg), male C57BL/6J mice received intraperitoneal injection of LPS and underwent behavioral tests 24 h later. The levels of inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, were measured in the hippocampus and serum using enzyme-linked immunosorbent assay (ELISA). The expression levels of CYP1A1, AhR and NLRP3 were analyzed using qPCR and Western blot. The results showed that, compared with control group, LPS alone significantly down-regulated the expression levels of CYP1A1 mRNA and AhR protein in the hippocampus of mice, reduced glucose preference, prolonged immobility time in forced swimming test, increased IL-6 and IL-1ß levels in the hippocampus, increased serum IL-1ß level, and up-regulated NLRP3 mRNA and protein expression levels in mouse hippocampus, while FICZ significantly reversed the aforementioned effects of LPS. These findings suggest that AhR activation attenuates the inflammatory response associated with depression and modulates the expression of NLRP3. The present study provides novel insights into the role of AhR in the development of depression, and presents AhR as a potential therapeutic target for the treatment of depression.

Amplified and distinctive genotoxicity of titanium dioxide nanoparticles in transformed yeast reporters with human cytochrome P450 (CYP) genes.

Titanium dioxide nanoparticles (nTiO2) have been considered a possible carcinogen to humans, but most existing studies have overlooked the role of human enzymes in assessing the genotoxicity of nTiO2. Here, a toxicogenomics-based in vitro genotoxicity assay using a GFP-fused yeast reporter library was employed to elucidate the genotoxic potential and mechanisms of nTiO2. Moreover, two new GFP-fused yeast reporter libraries containing either human CYP1A1 or CYP1A2 genes were constructed by transformation to investigate the potential modulation of nTiO2 genotoxicity in the presence of human CYP enzymes. This study found a lack of appreciable nTiO2 genotoxicity as indicated by the yeast reporter library in the absence of CYP expression but a significantly elevated indication of genotoxicity in either CYP1A1- or CYP1A2-expressing yeast. The intracellular reactive oxygen species (ROS) measurement indicated significantly higher ROS in yeast expressing either enzyme. The detected mitochondrial DNA damage suggested mitochondria as one of the target sites for oxidative damage by nTiO2 in the presence of either one of the CYP enzymes. The results thus indicated that the genotoxicity of nTiO2 was enhanced by human CYP1A1 or CYP1A2 enzyme and was associated with elevated oxidative stress, which suggested that the similar mechanisms could occur in human cells.

Development of a Normal Porcine Cell Line Growing in a Heme-Supplemented, Serum-Free Condition for Cultured Meat.

A key element for the cost-effective development of cultured meat is a cell line culturable in serum-free conditions to reduce production costs. Heme supplementation in cultured meat mimics the original meat flavor and color. This study introduced a bacterial extract generated from Corynebacterium that was selected for high-heme expression by directed evolution. A normal porcine cell line, PK15, was used to apply the bacterial heme extract as a supplement. Consistent with prior research, we observed the cytotoxicity of PK15 to the heme extract at 10 mM or higher. However, after long-term exposure, PK15 adapted to tolerate up to 40 mM of heme. An RNA-seq analysis of these heme-adapted PK15 cells (PK15H) revealed a set of altered genes, mainly involved in cell proliferation, metabolism, and inflammation. We found that cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), lactoperoxidase (LPO), and glutathione peroxidase 5 (GPX5) were upregulated in the PK15H heme dose dependently. When we reduced serum serially from 2% to serum free, we derived the PK15H subpopulation that was transiently maintained with 5-10 mM heme extract. Altogether, our study reports a porcine cell culturable in high-heme media that can be maintained in serum-free conditions and proposes a marker gene that plays a critical role in this adaptation process.

Computer-aided discovery of novel aryl hydrocarbon receptor ligands to regulate CYP1A1 expression in inflammatory macrophages.

The environmental factor aryl hydrocarbon receptor (AhR), a key protein connecting the external environmental signals (e.g., environmental endocrine disruptor TCDD) to internal cellular processes, is involved in the activation of peripheral macrophages and inflammatory response in human body. Thus, there is widespread interest in finding compounds to anti-inflammatory response in macrophages by targeting human AhR. Here, ensemble docking based-virtual screening was first used to screen a library (~200,000 compounds) against human AhR ligand binding domain (LBD) and 25 compounds were identified as potential inhibitors. Then, 9 out of the 25 ligands were found to down-regulate the mRNA expression of CYP1A1 (a downstream gene of AhR signaling) in AhR overexpressing macrophages. The most potent compound AE-411/41415610 was selected for further study and found to reduce both mRNA and protein expressions level of CYP1A1 in mouse peritoneal macrophage. Moreover, protein chip signal pathway analysis indicated that AE-411/41415610 play a role in regulating JAK-STAT and AKT-mTOR pathways. In sum, the discovered hits with novel scaffolds provided a starting point for future design of more effective AhR-targeted lead compounds to regulate CYP1A1 expression of inflammatory peritoneal macrophages.

Biochemical and molecular biomarkers and their association with anthropogenic chemicals in wintering Manx shearwaters (Puffinus puffinus).

Anthropogenic pollution poses a threat to marine conservation by causing chronic toxic effects. Seabirds have contact throughout their lives with pollutants like plastic, metals, polychlorinated biphenyls (PCBs), and organochlorine pesticides such as hexachlorocyclohexanes (HCHs). We assessed 155 Manx shearwaters (Puffinus puffinus) stranded along the Brazilian coast, analyzing associations between organic pollutants, plastic ingestion, biomarkers (transcript levels of aryl hydrocarbon receptor, cytochrome P450-1A-5 [CYP1A5], UDP-glucuronosyl-transferase [UGT1], estrogen receptor alpha-1 [ESR1], and heat shock protein-70 genes) and enzymes activity (ethoxy-resorufin O-deethylase and glutathione S-transferase [GST]). Plastic debris was found in 29 % of the birds. The transcription of UGT1 and CYP1A5 was significantly associated with hexachlorobenzene (HCB) and PCBs levels. ESR1 was associated with HCB and Mirex, and GST was associated with Drins and Mirex. While organic pollutants affected shearwaters more than plastic ingestion, reducing plastic availability remains relevant as xenobiotics are also potentially adsorbed onto plastics.

Computational study on the endocrine-disrupting metabolic activation of Benzophenone-3 catalyzed by cytochrome P450 1A1: A QM/MM approach.

Predicting the metabolic activation mechanism and potential hazardous metabolites of environmental endocrine-disruptors is a challenging and significant task in risk assessment. Here the metabolic activation mechanism of benzophenone-3 catalyzed by P450 1A1 was investigated by using Molecular Dynamics, Quantum Mechanics/Molecular Mechanics and Density Functional Theory approaches. Two elementary reactions involved in the metabolic activation of BP-3 with P450 1A1: electrophilic addition and hydrogen abstraction reactions were both discussed. Further conversion reactions of epoxidation products, ketone products and the formaldehyde formation reaction were investigated in the non-enzymatic environment based on previous experimental reports. Binding affinities analysis of benzophenone-3 and its metabolites to sex hormone binding globulin indirectly demonstrates that they all exhibit endocrine-disrupting property. Toxic analysis shows that the eco-toxicity and bioaccumulation values of the benzophenone-3 metabolites are much lower than those of benzophenone-3. However, the metabolites are found to have skin-sensitization effects. The present study provides a deep insight into the biotransformation process of benzophenone-3 catalyzed by P450 1A1 and alerts us to pay attention to the adverse effects of benzophenone-3 and its metabolites in human livers.

Salmonid smolt caudal fin and liver transcriptome responses to low sulfur marine diesel and high sulfur fuel oil water accommodated fractions for assessing oil spill effects in marine environments.

The environmental impact of oil spills is a critical concern, particularly pertaining to low sulfur marine diesel (LSMD) and high sulfur fuel oil (HSFO) that are commonly involved in coastal spills. Although transcriptomic biomonitoring of sentinel animals can be a powerful tool for assessing biological effects, conventional methods utilize lethal sampling to examine the liver. As a non-lethal alternative, we have previously shown salmonid caudal fin cyp1a1 is significantly responsive to LSMD-derived toxicants. The present study further investigated the transcriptomic biomonitoring potential of coho salmon smolt caudal fin in comparison to liver tissue in the context of LSMD and HSFO seawater accommodated fraction (seaWAF) exposure in cold-water marine environments. Assessing the toxicity of these seaWAFs involved quantifying polycyclic aromatic hydrocarbon (tPAH50) concentrations and generating gene expression profiles. Initial qPCR analyses revealed significant cyp1a1 response in both liver and caudal fin tissues of both genetic sexes to all seaWAF exposures. RNA-Seq analysis, focusing on the highest LSMD and HSFO seaWAF concentrations (28.4±1.8 and 645.08±146.3 µg/L tPAH50, respectively), revealed distinct tissue-specific and genetic sex-independent transcriptomic responses with an overall enrichment of oxidative stress, cell adhesion, and morphogenesis-related pathways. Remarkably, the caudal fin tissue exhibited transcriptomic response patterns comparable to liver tissue, particularly consistent differential expression of 33 gene transcripts in the liver (independent of sex and oil type) and 44 in the caudal fin. The present work underscores the viability of using the caudal fin as a non-lethal alternative to liver sampling for assessing and tracking oil spill exposure in marine environments.

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.

A novel approach to assess the health risk of aryl hydrocarbon receptor-bound contaminants via inhalation exposure using CYP1A1 expression as a biomarker.

Polycyclic aromatic hydrocarbons (PAHs) and dioxins are potential causes of multiple diseases by activating the aryl hydrocarbon receptor (AhR) pathway. Health risk assessment of chemicals primarily relies on the relative potency factor (RPF), although its accuracy may be limited when solely using EC50 values. The induction of cytochrome P4501A1 (CYP1A1) serves as a biomarker for AhR activation and is an integrator of dioxin-like toxicity. Here, we present a method for evaluating the risks associated with AhR activation using mathematical models of dose-CYP1A1 induction. The dose-effect curves for certain PAHs and dioxins, including Ant, BghiP, 1,2,3,4,7,8-HxCDD, and others, exhibited a non-classical S-shaped form. The toxic equivalent factor (TEF) profiles revealed a broad range of toxic equivalent factor values. The TEFs for PAHs ranged from approximately 0.01 to 6, with higher values being observed when the concentration was less than 10-10 M, with the exceptions of Ace, Phe, and BghiP. Most congeners of dioxins got the lowest TEF value at around 10-10 M, ranging from 0.04 to 1.00. The binding affinity of AhR to ligands did not display a strong correlation with the EC50 of CYP1A1 expression, suggesting that the AhR-mediated effects of PAHs and dioxins are not fixed but instead fluctuate with the dose. Air samples acquired from a parking area were used to compare the proficiency of RPF and our current approach. In the current method, naphthalene and chrysene were the primary contributors of PAHs to AhR-mediated risks in parking lots air samples, respectively. However, the contributions of naphthalene and chrysene could be disregarded in the RPF approach.

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.

AhR agonists screening and identification in indoor dust based on non-target chemical analysis by GC-Q-TOFMS and biological effect evaluation referring to ToxCast/Tox21 database.

Numerous studies reported the concentration of agonists of aryl hydrocarbon receptor (AhR) in indoor dust by target chemical analysis or the biological effects of activating the AhR by indoor extracts, but the major AhR agonists identification in indoor dust were rarely researched. In the present study, the indoor dust samples were collected for 7-ethoxyresorufin O-deethylase (EROD) assay and both non-targeted and targeted chemical analysis for AhR agonists by gas chromatography quadrupole time-of-flight mass spectrometry and gas chromatography-mass spectrometry analysis. Coupled with non-targeted analysis and toxicity Forecaster (ToxCast)/Tox21 database, 104 ToxCast chemicals were screened to be able to induce EROD response. The combination of targeted chemical analyses and biological effects evaluation indicated that PAHs, dibutyl phthalate (DBP) and Cypermethrin might be the important AhR-agonists in different indoor dust and mainly contributed in 1.84%-97.56 % (median: 26.62%) of total observed biological effects through comparing toxic equivalency quotient derived from chemical analysis with biological equivalences derived from bioassay. DBP and cypermethrin seldom reported in the analysis of AhR agonists should raise great concern. In addition, the present results in experiment of synthetic solution of 4 selected AhR-agonists pointed out that some unidentified AhR agonists existed in indoor dust.

Bioluminescence imaging of Cyp1a1-luciferase reporter mice demonstrates prolonged activation of the aryl hydrocarbon receptor in the lung.

Aryl hydrocarbon receptor (AHR) signalling integrates biological processes that sense and respond to environmental, dietary, and metabolic challenges to ensure tissue homeostasis. AHR is a transcription factor that is inactive in the cytosol but upon encounter with ligand translocates to the nucleus and drives the expression of AHR targets, including genes of the cytochrome P4501 family of enzymes such as Cyp1a1. To dynamically visualise AHR activity in vivo, we generated reporter mice in which firefly luciferase (Fluc) was non-disruptively targeted into the endogenous Cyp1a1 locus. Exposure of these animals to FICZ, 3-MC or to dietary I3C induced strong bioluminescence signal and Cyp1a1 expression in many organs including liver, lung and intestine. Longitudinal studies revealed that AHR activity was surprisingly long-lived in the lung, with sustained Cyp1a1 expression evident in discrete populations of cells including columnar epithelia around bronchioles. Our data link diet to lung physiology and also reveal the power of bespoke Cyp1a1-Fluc reporters to longitudinally monitor AHR activity in vivo.

A tryptophan-derived uremic metabolite/Ahr/Pdk4 axis governs skeletal muscle mitochondrial energetics in chronic kidney disease.

Chronic kidney disease (CKD) causes accumulation of uremic metabolites that negatively affect skeletal muscle. Tryptophan-derived uremic metabolites are agonists of the aryl hydrocarbon receptor (AHR), which has been shown to be activated in CKD. This study investigated the role of the AHR in skeletal muscle pathology of CKD. Compared with controls with normal kidney function, AHR-dependent gene expression (CYP1A1 and CYP1B1) was significantly upregulated in skeletal muscle of patients with CKD, and the magnitude of AHR activation was inversely correlated with mitochondrial respiration. In mice with CKD, muscle mitochondrial oxidative phosphorylation (OXPHOS) was markedly impaired and strongly correlated with the serum level of tryptophan-derived uremic metabolites and AHR activation. Muscle-specific deletion of the AHR substantially improved mitochondrial OXPHOS in male mice with the greatest uremic toxicity (CKD + probenecid) and abolished the relationship between uremic metabolites and OXPHOS. The uremic metabolite/AHR/mitochondrial axis in skeletal muscle was verified using muscle-specific AHR knockdown in C57BL/6J mice harboring a high-affinity AHR allele, as well as ectopic viral expression of constitutively active mutant AHR in mice with normal renal function. Notably, OXPHOS changes in AHRmKO mice were present only when mitochondria were fueled by carbohydrates. Further analyses revealed that AHR activation in mice led to significantly increased pyruvate dehydrogenase kinase 4 (Pdk4) expression and phosphorylation of pyruvate dehydrogenase enzyme. These findings establish a uremic metabolite/AHR/Pdk4 axis in skeletal muscle that governs mitochondrial deficits in carbohydrate oxidation during CKD.

Anti-pollutant effect of oleic acid against urban particulate matter is mediated via regulation of AhR- and TRPV1-mediated signaling in vitro.

Urban Particulate Matter (UPM) induces skin aging and inflammatory responses by regulating skin cells through the transient receptor potential vanilloid 1 (TRPV1). Although oleic acid, an unsaturated free fatty acid (FFA), has some functional activities, its effect on UPM-induced skin damage has not been elucidated. Here, we investigated signaling pathways on how oleic acid is involved in attenuating UPM induced cell damage. UPM treatment increased XRE-promoter luciferase activity and increased translocation of AhR to the nucleus, resulting in the upregulation of CYP1A1 gene. However, oleic acid treatment attenuated the UPM effects on AhR signaling. Furthermore, while UPM induced activation of TRPV1 and MAPKs signaling which activated the downstream molecules NFκB and AP-1, these effects were reduced by cotreatment with oleic acid. UPM-dependent generation of reactive oxygen species (ROS) and reduction of cellular proliferation were also attenuated by the treatment of oleic acid. These data reveal that cell damage induced by UPM treatment occurs through AhR signaling and TRPV1 activation which in turn activates ERK and JNK, ultimately inducing NFκB and AP-1 activation. These effects were reduced by the cotreatment of oleic acid on HaCaT cells. These suggest that oleic acid reduces UPM-induced cell damage through inhibiting both the AhR signaling and activation of TRPV1 and its downstream molecules, leading to a reduction of pro-inflammatory cytokine and recovery of cell proliferation.

Emissions from plastic incineration induce inflammation, oxidative stress, and impaired bioenergetics in primary human respiratory epithelial cells.

Inhalation exposure to plastic incineration emissions (PIEs) is a problem of increasing human relevance, as plastic production and waste creation have drastically increased since mainstream integration during the 20th century. We investigated the effects of PIEs on human nasal epithelial cells (HNECs) to understand if such exposures cause damage and dysfunction to respiratory epithelia. Primary HNECs from male and female donors were cultured at air-liquid interface (ALI), and 16HBE cells were cultured on coverslips. Smoke condensates were generated from incineration of plastic at flaming (640°C) and smoldering (500°C) temperatures, and cells were subsequently exposed to these materials at 5-50 µg/cm2 concentrations. HNECs were assessed for mitochondrial dysfunction and 16HBE cells for glutathione oxidation in real-time analyses. HNEC culture supernatants and total RNA were collected at 4-h postexposure for cytokine and gene expression analysis, and results show that PIEs can acutely induce inflammation, oxidative stress, and mitochondrial dysfunction in HNECs, and that incineration temperature modifies biological responses. Specifically, condensates from flaming and smoldering PIEs significantly increased HNEC secretion of cytokines IL-8, IL-1ß, and IL-13, as well as expression of xenobiotic metabolism pathways and genes such as CYP1A1 and CYP1B1 at 5 and 20 µg/cm2 concentrations. Only 50 µg/cm2 flaming PIEs significantly increased glutathione oxidation in 16HBEs, and decreased respiration and ATP production in HNEC mitochondria. Impact Statement: Our data reveal the impact of incineration temperatures on biological outcomes associated with PIE exposures, emphasizing the importance of temperature as a factor when evaluating respiratory disease associated with PIEs exposure.

Blockade of aryl hydrocarbon receptor restricts omeprazole-induced chronic kidney disease.

Chronic kidney disease (CKD) is the 16th leading cause of mortality worldwide. Clinical studies have raised that long-term use of omeprazole (OME) is associated with the morbidity of CKD. OME is commonly used in clinical practice to treat peptic ulcers and gastroesophageal reflux disease. However, the mechanism underlying renal failure following OME treatment remains mostly unknown and the rodent model of OME-induced CKD is yet to be established. We described the process of renal injury after exposure to OME in mice; the early renal injury markers were increased in renal tubular epithelial cells (RTECs). And after long-term OME treatment, the OME-induced CKD mice model was established. Herein, aryl hydrocarbon receptor (AHR) translocation appeared after exposure to OME in HK-2 cells. Then for both in vivo and in vitro, we found that Ahr-knockout (KO) and AHR small interfering RNA (siRNA) substantially alleviated the OME-induced renal function impairment and tubular cell damage. Furthermore, our data demonstrate that antagonists of AHR and CYP1A1 could attenuate OME-induced tubular cell impairment in HK-2 cells. Taken together, these data indicate that OME induces CKD through the activation of the AHR-CYP axis in RTECs. Our findings suggest that blocking the AHR-CYP1A1 pathway acts as a potential strategy for the treatment of CKD caused by OME. KEY MESSAGES: We provide an omeprazole-induced chronic kidney disease (CKD) mice model. AHR activation and translocation process was involved in renal tubular damage and promoted the occurrence of CKD. The process of omeprazole nephrotoxicity can be ameliorated by blockade of the AHR-CYP1A1 axis.

Use of Lentivirus-Based Method for Establishing TK6 Human Cell Lines Expressing Cytochrome P450 and its Applications in Genotoxicity Testing.

The human lymphoblastoid cell line TK6 stands out as the most widely employed human cell line in genotoxicity testing, as recommended by various testing guidelines for in vitro assessments. Nevertheless, like many testing cell lines, TK6 lacks functional phase I drug-metabolizing enzymes crucial for chemical genotoxicity evaluations. This protocol introduces a lentivirus-based methodology for establishing a panel of TK6-derived cell lines, each expressing one of 14 cytochrome P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7). The utilization of a lentiviral expression system ensures stable transduction, offering notable advantages such as sustained transgene expression, high transduction efficiency, positive selection feasibility, and user-friendly application. Additionally, we present a detailed procedure for validating the enhanced expression of each CYP in the established cell lines through real-time PCR, western blotting, and mass spectrometry analysis. Lastly, we exemplify the application of these CYP-expressing TK6 cell lines in genotoxicity testing, employing a flow-cytometry-based in vitro micronucleus test. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Lentivirus production and transduction for TK6 cells Support Protocol: Selecting a single clone of CYP-expressing TK6 cells Basic Protocol 2: Validation of CYP expression in TK6 cell lines Basic Protocol 3: Application of transduced cell lines in flow-cytometry-based micronucleus assay.

Establishment and characterization of cytochrome P450 1A1 CRISPR/Cas9 Knockout Bovine Foetal Hepatocyte Cell Line (BFH12).

The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.

Ganoderic acid A suppresses autophagy by regulating the circFLNA/miR-486-3p/CYP1A1/XRCC1 axis to strengthen the sensitivity of lung cancer cells to cisplatin.

OBJECTIVES: Reportedly, ganoderic acid A (GA-A) increases the sensitivity of hepatocellular carcinoma cells to cisplatin (DDP) chemotherapy. Therefore, this study aims to fathom the influence of GA-A on lung cancer cells. METHODS: After the construction of A549/DDP cells through exposure to DDP, the effects of GA-A on A549 and A549/DDP cells were revealed by cellular functional assays, western blot and quantitative reverse transcription PCR (qRT-PCR). The DDP-resistant lung cancer tumor was established in vivo, followed by further validation of the mechanism of GA-A. RESULTS: GA-A suppressed the viability, migration, and invasion while downregulating Beclin and autophagy marker LC3II/LC3I levels and upregulating P62 levels in A549 and A549/DDP cells. These effects were reversed by circFLNA overexpression. Also, GA-A reinforced the sensitivity of A549/DDP cells to DDP, elevated the apoptosis and regulated the circFLNA/miR-486-3p/cytochrome P450 family 1 subfamily A member 1 (CYP1A1)/X-ray repair cross-complementing 1 (XRCC1) axis. The reversal effects of circFLNA overexpression on GA-A-induced viability and apoptosis of A549/DDP cells could all be counteracted in the presence of 3MA. GA-A inhibited lung cancer tumor growth and blocked autophagy. CONCLUSION: GA-A suppresses autophagy by regulating the circFLNA/miR-486-3p/CYP1A1/XRCC1 axis to strengthen the sensitivity of lung cancer cells to DDP.

Oil spill in an amazon blackwater environment: Biochemical and physiological responses of local fish species.

The accidental spill of petroleum asphalt cement (PAC) in São Raimundo (SR Harbor, located on the Rio Negro (Manaus, Amazonas, Brazil) was monitored through the analysis of polyciclic aromatic hydrocarbons (PAHs) in water and a set of biomarkers in fishes (exposure biomarkes: PAHs-type metabolites concentrations in bile; the activities of ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in liver. Effect biomarkers: lipid peroxidation concentration (LPO) in liver, acetylcholinesterase activity in brain, and genotoxic DNA damage in erythrocytes). Two fish species, Acarichthys heckelii and Satanoperca jurupari, were collected 10, 45, and 90 days after the PAC spill in São Raimundo. At the same time, fish were collected from the Tupé Sustainable Development Reserve (Tupé) which served as a reference area. The sampling periods were related to the rising waters of the natural flood pulse of the Rio Negro. Higher concentrations of PAHs in water were observed at 10 and 45 days and returned to the values of TP 90 days after the PAC spill, a period in which harbor waters rose about 0.2 m. Unlike the PAHs in water, biomarker responses in both fish species significantly increased following the PAC spill in SR. Hepatic ethoxyresorufin-O-deethylase (EROD), PAH-like metabolites in bile, and erythrocyte DNA damage increases, together with inhibition of acetylcholinesterase (AChE) activity in the brain were the most evident responses for both fish species. The calculated pyrolytic index showed mixed sources of PAHs (petrogenic and pyrolytic). The applied PCA-FA indicated important relationships between dissolved organic carbon (DOC) and PAHs concentrations in water, where DOC and PAHs concentrations contributed to biomarkers responses for both fish species in all collection periods.