Three cytochrome P450 genes, named Le. cyp1, Le. cyp2, and Le. cyp3, were isolated from the basidiomycete Lentinula edodes. Le. cyp1 and Le. cyp2 contained coding regions of 1500 bp and 1497 bp, respectively, but Le. cyp3 was found to be a defective gene. The deduced amino acid sequences of Le. CYP1 (CYP510A1, 500 amino acids) and Le. CYP2 (CYP510A3, 499 amino acids) were highly similar to each other (87% identical) and they had 32-33% identities to that of Coprinus cinereus P450 (CYP502) and 27-28% identities to those of two Aspergillus P450s (CYP64 family). Quantitative RT-PCR analysis of the transcripts of Le. cyp1 and Le. cyp2 genes in the course of fruiting-body development of L. edodes showed that the primordium seems to contain larger amounts of these transcripts. The transcript levels of both of these genes in the stipe of the premature fruiting body were higher than those in the whole pileus and gill tissue.

Cytochrome P450 (CYP) 1A1 plays a major role in the metabolic activation of procarcinogens to carcinogens via aryl hydrocarbon receptor (AhR) pathway. Especially, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known as an agonist of AhR. In estrogen responsive cancers, 17β-estradiol (E2) may influence on AhR dependent expression of CYP1 family via the interaction between estrogen receptor (ER) and AhR. In the present study, the effect of E2/ER on the expression of AhR and CYP1A1 genes was investigated for MCF-7 clonal variant (MCF-7 CV) breast cancer cells expressing ER. In reverse transcription-PCR and Western blot analysis, mRNA expression level of AhR was not altered, but its protein expression level was increased by TCDD or E2. The transcriptional and translational levels of CYP1A1 appeared to be increased by TCDD or E2. The increased expression of AhR and CYP1A1 induced by E2 was restored to the control level by the co-treatment of ICI 182,780, indicating that E2 induced the protein expression levels of AhR and CYP1A1 like TCDD via an ER dependent pathway. In an in vivo xenograft mouse model transplanted with MCF-7 CV cells, the protein expression levels of AhR and CYP1A1 of tumor masses were also increased by E2 or TCDD. Taken together, these results indicate that E2 may promote AhR dependent expression of CYP1A1 via ER dependent pathway in MCF-7 CV cells expressing ER in the absence of TCDD, an agonist of AhR. The relevance of E2 and ER in CYP1A1 activation of estrogen responsive cancers may be targeted for developing more effective cancer treatments.

BACKGROUND

Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast.

METHODS

CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone.

RESULTS

CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles.

CONCLUSIONS

Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds.

CONCLUSIONS

Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.

Rational approaches have been extensively used to investigate the role of active site residues in cytochrome P450 (CYP) functions. However, recent studies using random mutagenesis suggest an important role for non-active site residues in CYP functions. Meta-analysis of the random mutants showed that 75% of the functionally important non-active site residues are present in 20% of the entire protein between helices E and H (E-H) and conserved sequence motif (CSM) between 7 and 11. The CSM approach was developed recently to investigate the functional role of non-active site residues in CYP2B4. Furthermore, we identified and analyzed the CSM in multiple CYP families and subfamilies in the E-H region. Results from CSM analysis showed that CSM 7, 8, 10, and 11 are conserved in CYP1, CYP2, and CYP3 families, while CSM 9 is conserved only in CYP2 family. Analysis of different CYP2 subfamilies showed that CYP2B and CYP2C have similar characteristics in the CSM, while the characteristics of CYP2A and CYP2D subfamilies are different. Finally, we analyzed CSM 7, 8, 10, and 11, which are common in all the CYP families/subfamilies analyzed, in fifteen important drug-metabolizing CYPs. The results showed that while CSM 8 is most conserved among these CYPs, CSM 7, 9, and 10 have significant variations. We suggest that CSM8 has a common role in all the CYPs that have been analyzed, while CSM 7, 10, and 11 may have relatively specific role within the subfamily. We further suggest that these CSM play important role in opening and closing of the substrate access/egress channel by modulating the flexible/plastic region of the protein. Thus, site-directed mutagenesis of these CSM can be used to study structure-function and dynamic/plasticity-function relationships and to design CYP biocatalysts.

Objectives: Benzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression.

Methods: The human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus.

Results: We found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation.

Conclusion: Here, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.

Keywords: Aryl hydrocarbon receptor; Benzo(a)Pyrene; Mucin 5AC; Reactive oxygen species; Resveratrol.

We report that the naphthalimide analogue 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (NAP-6) is a highly potent and selective breast cancer targeting molecule. These effects are mediated via the aryl hydrocarbon receptor (AHR) pathway and the subsequent induction of CYP1 metabolising monooxygenases in breast cancer cell line models. Indeed the triple negative breast cancer cell line MDA-MB-468 with a GI₅₀ value of 100 nM is greater than 500-fold more sensitive to NAP-6 compared with other tumour derived cell models. Within 1 h exposure of these cells to NAP-6, CYP1A1 expression increases 25-fold, rising to 250-fold by 24 h. A smaller concurrent increase in CYP1A2 and CYP1B1 is also observed. Within 24 h these cells present with DNA damage as evident by enhanced H2AXγ expression, cell cycle checkpoint activation via increased CHK2 expression, S-phase cell cycle arrest and cell death. Specific small molecule inhibitors of the AHR and CYP1 family ameliorate these events. A positive luciferase reporter assay for NAP-6 induced XRE binding further confirms the role of the AHR in this phenomenon. Non-sensitive cell lines fail to show these biological effects. For the first time we identify 2-(2-aminophenyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione as a new AHR ligand that selectively targets breast cancer.

The endangered red-crowned crane (Grus japonensis) is a protected species in eastern Hokkaido and injured specimens are treated with medication. The present study aimed at understanding the expression profiles of cytochrome P450 (CYP) 1-3 genes in red-crowned crane tissues. We used 14 individuals found dead in the wild in eastern Hokkaido or in Kushiro City Zoo. Nine CYP1-3 genes expressed in the liver of the red-crowned crane were identified by high-throughput sequencing, and phylogenetically classified as CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2U1, CYP2AC1, CYP3A37, and CYP3A80. Based on the quantitative real-time PCR of 13 samples, the rank order of their median expression levels was as follows: CYP3A37 > CYP2AC1 > CYP2C45 > CYP2D49 > CYP2G19 > CYP1A5 > CYP3A80 > CYP2C23. The tissue distribution of the CYP transcripts indicated that many of the CYP1-3 genes examined were mainly expressed in the tissues where drug metabolism occurs, such as the liver, kidneys, and lungs. We found that CYP3A37 was dominant at the transcript level in the liver, indicating it might play a crucial role in liver physiology and xenobiotic metabolism. Similarly, an "orphan" CYP2AC1 was expressed at relatively high levels in the kidneys and liver, suggesting a possible role in renal and liver physiology and xenobiotic metabolism. Our results establish a foundation for future studies on red-crowned cranes aiming to further understand drug sensitivity and develop medication protocols, but also contribute to national and local projects for the conservation of red-crowned crane.

Cassia obtusifolia, belonging to legume family, is important in many fields with high pharmaceutical, economic, and ecological values. These interests of C. obtusifolia triggered in-depth and fundamental genetic and molecular research. Therefore, the stable reference gene is necessary for normalization of the gene expression studies. In this study, 10 candidate reference genes were subjected to expression analysis in 12 different tissues and under different stresses by qRT-PCR. The expression stability was evaluated using geNorm, NormFinder, and BestKeeper software. In conclusion, different suitable reference genes were selected in different tissues and under different stress. CYP1, EF1α2, ACT2, UBQ1 were the most stable reference genes in all samples. The relative expression levels of WRKY gene were detected to confirm the reliability of the selected reference genes. These results provided suitable reference genes that could be used for normalization in C. obtusifolia tissues and under different stress.

Polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (oxy-PAHs), are common environmental pollutants known to cause health effects in humans and wild-life. In particular, vertebrate cardiovascular development and function are sensitive to PACs. However, the interactive effects of PAHs and oxy-PAHs on cardiovascular endpoints have not been well studied. In this study, we used zebrafish embryos (ZFEs) as a model to examine developmental and cardiovascular toxicities induced by the three environmental oxy-PAHs benzo[a]fluorenone (BFLO), 4H-cyclopenta[def]phenanthren-4-one (4H-CPO) and, 6H-benzo[cd]pyren-6-one (6H-BPO), and the PAH benzo[a]pyrene (BaP) either as single exposures or binary oxy-PAH + PAH mixtures. 6H-BPO induced developmental and cardiovascular toxicity, including reduced heartbeat rate and blood flow, at lower doses compared to the other compounds. Exposure to binary mixtures generally caused enhanced toxicity and induction of aryl hydrocarbon receptor (AhR)-regulated gene expression (ahr2 and cyp1a) compared to single compound exposure. This was associated with differential expression of genes involved in cardiovascular development and function including atp2a2, myh6, tbx5 and zerg. AhR-knock-down significantly reduced the cardiovascular toxicity of 6H-BPO and its binary mixture with BaP indicating a significant AhR-dependence of the effects. Measurements of internal concentrations showed that the toxicokinetics of BaP and 6H-BPO were altered in the binary mixture compared to the single compound exposure, and most likely due to CYP1 inhibition by 6H-BPO. Altogether, these data support that similar to interactions between PAHs, mixtures of PAHs and oxy-PAHs may cause increased developmental and cardiovascular toxicity in ZFEs through an AhR-dependent mechanism.

Keywords: Ah receptor-dependent; Benzo[a]pyrene; Cardiovascular toxicity; Mixture effects; Oxy-PAHs; Zebrafish.

Cytochrome P4501B1 is a ubiquitous family protein that is majorly overexpressed in tumors and is responsible for biotransformation-based inactivation of anti-cancer drugs. This inactivation marks the cause of resistance to chemotherapeutics. In the present study, integrated in-silico approaches were utilized to identify selective CYP1B1 inhibitors. To achieve this objective, we initially developed different machine learning models corresponding to two isoforms of the CYP1 family i.e. CYP1A1 and CYP1B1. Subsequently, small molecule databases including ChemBridge, Maybridge, and natural compound library were screened from the selected models of CYP1B1 and CYP1A1. The obtained CYP1B1 inhibitors were further subjected to molecular docking and ADMET analysis. The selectivity of the obtained hits for CYP1B1 over the other isoforms was also judged with molecular docking analysis. Finally, two hits were found to be the most stable which retained key interactions within the active site of CYP1B1 after the molecular dynamics simulations. Novel compound with CYP-D9 and CYP-14 IDs were found to be the most selective CYP1B1 inhibitors which may address the issue of resistance. Moreover, these compounds can be considered as safe agents for further cell-based and animal model studies.Communicated by Ramaswamy H. Sarma.

Keywords: ADMET; CYP1B1; machine learning; molecular docking; molecular dynamics.

As part of a programme to develop anticancer prodrugs which are activated by cytochrome P450 (CYP)1B1, a library of 4,6-diaryl-2-pyridones was synthesised in yields of 6-60% from the corresponding chalcones. A number of these derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing little toxicity towards a non-tumour breast cell line with no CYP expression. Metabolism studies provided evidence supporting the involvement of CYP1 enzymes in the bioactivation of these compounds.

Lung cancer has the second highest incidence and highest mortality compared to all other cancers. Polycyclic aromatic hydrocarbon (PAH) molecules belong to a class of compounds that are present in tobacco smoke, diesel exhausts, smoked foods, as well as particulate matter (PM). PAH-derived reactive metabolites are significant contributors to lung cancer development. The formation of these reactive metabolites entails metabolism of the parent PAHs by cytochrome P4501A1/1B1 (CYP1A1/1B1) and epoxide hydrolase enzymes. These reactive metabolites then react with DNA to form DNA adducts, which contribute to key gene mutations, such as the tumor suppressor gene, p53 and are linked to pulmonary carcinogenesis. PAH exposure also leads to upregulation of CYP1A1 transcription by binding to the aryl hydrocarbon receptor (AHR) and eliciting transcription of the CYP1A1 promoter, which comprises specific xenobiotic-responsive element (XREs). While hepatic and pulmonary CYP1A1/1B1 metabolize PAHs to DNA-reactive metabolites, the hepatic CYP1A2, however, may protect against lung tumor development by suppressing both liver and lung CYP1A1 enzymes. Further analysis of these enzymes has shown that PAH-exposure also induces sustained transcription of CYP1A1, which is independent of the persistence of the parent PAH. CYP1A2 enzyme plays an important role in the sustained induction of hepatic CYP1A1. PAH exposure may further contribute to pulmonary carcinogenesis by producing epigenetic alterations. DNA methylation, histone modification, long interspersed nuclear element (LINE-1) activation, and non-coding RNA, specifically microRNA (miRNA) alterations may all be induced by PAH exposure. The relationship between PAH-induced enzymatic reactive metabolite formation and epigenetic alterations is a key area of research that warrants further exploration. Investigation into the potential interplay between these two mechanisms may lead to further understanding of the mechanisms of PAH carcinogenesis. These mechanisms will be crucial for the development of effective targeted therapies and early diagnostic tools.

Keywords: Cytochrome P450 (CYP1) enzyme; Epigenetics; Lung cancer; Polycyclic aromatic hydrocarbon (PAH); Pulmonary carcinogenesis.

Chlorothalonil (CT) is a commonly used fungicide and its excessive application seriously threatens aquatic life and human health. Resveratrol (RSV) is a natural polyphenol and can be used as a therapeutic and preventive agent for the treatment of various diseases. To explore the toxic mechanism of CT exposure on fish kidney cell, as well as the alleviation effect of RSV, we established CT poisoning and/or RSV treatment fish kidney cell models. Ctenopharyngodon idellus kidney (CIK) cell line was treated with CT (5 μg/L) and/or RSV (10 μM) for 48 h. The results showed that CT exposure activated cytochromeP450s (CYPs) including CYP1A1, CYP1B1 and CYP1C, caused malondialdehyde (MDA) accumulation, inhibited glutathione (GSH) levels and glutathione peroxidase (GPX) activities, increased the expression of miR-15a and downregulated BCL2 and TNFα-induced protein 3 (TNFAIP3, A20), triggered mitochondrial pathway mediated apoptosis and receptor interacting serine/threonine kinase (RIP)-dependent necroptosis in CIK cells. However, cell death under CT exposure could be relieved by RSV treatment through inhibiting the expression of CYP1 family genes and restoring miR-15a/BCL2-A20 axis disorders. Overall, we conclude that RSV could relieve CT-induced apoptosis and necroptosis through miR-15a/Bcl2-A20 axis in CIK cells. These results enrich the toxicological mechanisms of the CT and confirm that RSV can be used as a potential antidote for CT poisoning.

Keywords: Apoptosis; Chlorothalonil; Necroptosis; Resveratrol; miR-15a/BCL2-A20.

It was explored that CYP1 family of cytochromes P450 were over-expressed in several types of cancer. Our study aimed to characterize anti-proliferative activity and metabolism of the natural flavonoid diosmetin in the human hepatoma cell HepG2, expressing CYP1 family. Diosinduced cell apoptosis could be reversed due to p53 blockade and the cellular P53 and CYP1A1/CYP1A2 proteins levels were examined. P53 and CYP1A1/CYP1A2 proteins were upregulated by Dios; when PFT-α was added into cells, the P53 levels were down-regulated accompanied with up-regulated CYP1A1/CYP1A2. Meanwhile, when cells were co-treated with Dios and PFT-α, P53 was down-regulated and CYP1A1/CYP1A2 up-regulated controlled with that of Dios treated cells. The data reveal the new evidence that cytochrome P450 CYP1A regulation by P53 enzyme plays an important role in Diosmetin anti-cancer activity of HepG2 cells.

Cytochrome P450 (CYP450) is a hemoprotein superfamily, among which CYP1, CYP2 and CYP3 play a major role in the metabolism of vast array of xenobiotics and endobiotics. This paper reports on three CYP enzyme variants (CYP1A2, CYP2E1 and CYP3A4) in three species of earthworm (Eisenia fetida, Metaphire guillelmi and Amynthas carnosus). The relative expression levels and localization of the three associated proteins were investigated at three life-cycle points (juvenile, sub-adult and adult), through comparison of anterior and posterior body tissue and between specific organs (body wall, intestine and reproductive tissues) using western blot analysis. This study confirmed the presence of CYP3A4, CYP1A2 and CYP2E1 in all three species of earthworm tested. The levels of expression varied with earthworm species, age, and body location. These differences in occurrence of the three CYP enzymes appeared to reflect the ecological niche (the spatial and temporal location and functional relationship of each individual or population in populations or communities), and the likelihood of contact with soil contaminants of the respective species. These results may help to explain why earthworms are capable of adapting to very different and extensively polluted soil environments and provide important data for subsequent ecotoxicology and ecological adaptability studies.

Of the three enzymes in the human cytochrome P450 family 1, <em>CYP1</em>A2 is an important enzyme mediating metabolism of xenobiotics including drugs in the liver, while <em>CYP1</em>A1 and <em>CYP1</em>B1 are expressed in extrahepatic tissues. Currently used CYP substrates, such as 7-ethoxycoumarin and 7-ethoxyresorufin, are oxidized by all individual <em>CYP1</em> forms. The main aim of this study was to find profluorescent coumarin substrates that are more selective for the individual <em>CYP1</em> forms. Eleven 3-phenylcoumarin derivatives were synthetized, their enzyme kinetic parameters were determined, and their interactions in the active sites of <em>CYP1</em> enzymes were analyzed by docking and molecular dynamic simulations. All coumarin derivatives and 7-ethoxyresorufin and 7-pentoxyresorufin were oxidized by at least one <em>CYP1</em> enzyme. 3-(3-Methoxyphenyl)-6-methoxycoumarin (<b>19</b>) was 7-O-demethylated by similar high efficiency [21-30 ML/(min·mol CYP)] by all <em>CYP1</em> forms and displayed similar binding in the enzyme active sites. 3-(3-Fluoro-4-acetoxyphenyl)coumarin (<b>14</b>) was selectively 7-O-demethylated by <em>CYP1</em>A1, but with low efficiency [0.16 ML/(min mol)]. This was explained by better orientation and stronger H-bond interactions in the active site of <em>CYP1</em>A1 than that of <em>CYP1</em>A2 and <em>CYP1</em>B1. 3-(4-Acetoxyphenyl)-6-chlorocoumarin (<b>20</b>) was 7-O-demethylated most efficiently by <em>CYP1</em>B1 [53 ML/(min·mol CYP)], followed by <em>CYP1</em>A1 [16 ML/(min·mol CYP)] and <em>CYP1</em>A2 [0.6 ML/(min·mol CYP)]. Variations in stabilities of complexes between <b>20</b> and the individual CYP enzymes explained these differences. Compounds <b>14</b>, <b>19</b>, and <b>20</b> are candidates to replace traditional substrates in measuring activity of human <em>CYP1</em> enzymes.

Inhibitors of CYP1 enzymes may play vital roles in the prevention of cancer and overcoming chemo-resistance to anticancer drugs. In this letter, we report synthesis of twenty-three pyrrole based heterocyclic chalcones which were screened for inhibition of CYP1 isoforms. Compound 3n potently inhibited CYP1B1 with an IC50 of ∼0.2μM in Sacchrosomes™ and CYP1B1-expressing live human cells. However, compound 3j which inhibited both CYP1A1 and CYP1B1 with an IC50 of ∼0.9µM, using the same systems, also potently antagonized B[a]P-mediated induction of AhR signaling in yeast (IC50, 1.5µM), fully protected human cells from B[a]P toxicity and completely reversed cisplatin resistance in human cells that overexpress CYP1B1 by restoring cisplatin's cytotoxicity. Molecular modeling studies were performed to rationalize the observed potency and selectivity of enzyme inhibition by compounds 3j and 3n.

Cytochrome P450 (CYP) enzymes play important roles in the detoxification and bioactivation of environmental contaminants and are involved in the responses to pollution in fish. In this study, we cloned four new CYP1 genes, CYP1A, CYP1B, CYP1C1, and CYP1C2, from black rockfish (Sebastes schlegelii), a dominant and economically important fish species in Korea. This species is at a significant risk of exposure to petrohydrocarbons, such as benzo[a]pyrene(B[a]P), due to frequent oil spills along the Korean coast. Quantitative PCR analysis of CYP1 gene transcription in 12 organs of the fish revealed tissue-specific expression patterns. CYP1A was significantly expressed in the liver, heart, kidneys, and muscle, and CYP1B was significantly expressed in the gills, muscle, and heart. CYP1C1 and CYP1C2 showed similar tissue expression patterns, with the highest levels in the muscle. Furthermore, exposure to an aryl hydrocarbon receptor (AHR) agonist, B[a]P, at 2, 20, and 200 mg/kg body weight showed significant dysregulation of the CYP1A, CYP1B, CYP1C1, and CYP1C2 expression levels in the gills, liver, kidneys, and spleen. The mRNA expression levels of CYP1A and CYP1B were upregulated by 450- and 17-fold, respectively, in the spleen. Compared with their levels in the control, CYP1C1 increased by 45-fold, while CYP1C2 remained unchanged in the gills, indicating differential effects of the polycyclic aromatic hydrocarbon on CYP1 expression in different fish organs. The results suggested that expression profiles of inducible CYP1 enzymes in S. schlegelii might be used as indicators for assessing aquatic contamination by AHR agonists. Determination of the basal and induced expression levels, as well as substrate specificity, of the four CYP1 enzymes may contribute to a better understanding of their roles in the metabolization of toxicants or drugs.

Keywords: Benzo[a]pyrene; Cytochrome P450 enzymes; Sebastes schlegelii; Tissue-specific expression patterns.

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.

Keywords: A549 cells; P2X7 receptor; cancer; cytoskeleton; phlorotannins; pollution.

Polychlorinated biphenyls (PCBs) are ubiquitously detected and have been linked to metabolic diseases. Gut microbiome is recognized as a critical regulator of disease susceptibility, however, little is known how PCBs and gut microbiome interact to modulate hepatic xenobiotic and intermediary metabolism. We hypothesized the gut microbiome regulates PCB-mediated changes in the metabolic fingerprints and hepatic transcriptome. Ninety-day-old female conventional (CV) and germ-free (GF) mice were orally exposed to the Fox River Mixture (synthetic PCB mixture, 6 or 30 mg/kg) or corn oil (vehicle control, 10 ml/kg), once daily for 3 consecutive days. RNA-Seq was conducted in liver, and endogenous metabolites were measured in liver and serum by LC-MS. Prototypical target genes of AhR, PXR, and CAR were more readily up-regulated by PCBs in CV conditions, indicating PCBs, to the hepatic transcriptome, act partly through the gut microbiome. In a gut microbiome-dependent manner, xenobiotic and steroid metabolism pathways were up-regulated, whereas response to misfolded proteins-related pathways were down-regulated by PCBs. At the high PCB dose, NADP and arginine appear to interact with drug-metabolizing enzymes (i.e. Cyp1-3 family), which are highly correlated with Ruminiclostridium and Roseburia, providing a novel explanation of gut-liver interaction from PCB-exposure. Utilizing the LINCS L1000 database, therapeutics targeting anti-inflammatory and ER stress pathways are predicted to be remedies that can mitigate PCB toxicity. Our findings demonstrate that habitation of the gut microbiota drives PCB-mediated hepatic responses. Our study adds knowledge of physiological response differences from PCB exposure and considerations for further investigations for gut microbiome-dependent therapeutics.

2'-Hydroxyflavanone (2'OHFva), 3'OHFva, 4'OHFva, and 6OHFva, the major oxidative products of flavanone by human cytochrome P450 (P450, CYP) enzymes, were studied in regard to further oxidation by human CYP1A1, 1A2, 1B1.1, 1B1.3, and 2A6. The products formed were analyzed with LC-MS/MS and characterized by their positive ion fragmentations on mass spectrometry. Several di-hydroxylated flavanone (diOHFva) and di-hydroxylated flavone (diOHFvo) products, detected by analyzing parent ions at m/z 257 and 255, respectively, were found following incubation of these four hydroxylated flavanones with P450s. The m/z 257 products were produced at higher levels than the latter with four substrates examined. The structures of the m/z 257 products were characterized by LC-MS/MS product ion spectra, and the results suggest that 3'OHFva and 4'OHFva are further oxidized mainly at B-ring by P450s while 6OHFva oxidation was at A-ring. Different diOHFvo products (m/z 255) were also characterized by LC-MS/MS, and the results suggested that most of these diOHFvo products were formed through oxidation or desaturation of the diOHFva products (m/z 257) by P450s. Only when 4'OHFva (m/z 241) was used as a substrate, formation of 4'OHFvo (m/z 239) was detected, indicating that diOHFvo might also be formed through oxidation of 4'OHFvo by P450s. Finally, our results indicated that CYP1 family enzymes were more active than CYP2A6 in catalyzing the oxidation of these four hydroxylated flavanones, and these findings were supported by molecular docking studies of these chemicals with active sites of P450 enzymes.

Keywords: Hydroxy flavanones; LC-MS/MS; cytochrome P450; human; oxidation.

Flow conditions have been shown to be important in improving longevity and functionality of primary hepatocytes, but the impact of flow on HepaRG cells is largely unknown. We studied the expression of genes encoding CYP enzymes and transporter proteins and CYP1 and CYP3A4 activity during 8 weeks of culture in HepaRG cells cultured under static conditions (conventional 24-/96-well plate culture with common bicarbonate/CO₂ buffering) and under flow conditions in an organ-on-chip (OOC) device. Since the OOC-device is a closed system, bicarbonate/CO₂ buffering was not possible, requiring application of another buffering agent, such as HEPES. In order to disentangle the effects of HEPES from the effects of flow, we also applied HEPES-supplemented medium in static cultures and studied gene expression and CYP activity. We found that cells cultured under flow conditions in the OOC-device, as well as cells cultured under static conditions with HEPES-supplemented medium, showed more stable gene expression levels. Furthermore, only cells cultured in the OOC-device showed relatively high baseline CYP1 activity, and their gene expression levels of selected CYPs and transporters were most similar to gene expression levels in human primary hepatocytes. However, there was a decrease in baseline CYP3A4 activity under flow conditions compared to HepaRG cells cultured under static conditions. Altogether, the present study shows that HepaRG cells cultured in the OOC-device were more stable than in static cultures, being a promising in vitro model to study hepatoxicity of chemicals upon chronic exposure.

Polycyclic aromatic hydrocarbons (PAHs) are environmental toxicants primarily produced during incomplete combustion; some are carcinogens. PAHs can be safely metabolized or, paradoxically, bioactivated via specific cytochrome P450 (CYP) enzymes to more reactive metabolites, some of which can damage DNA and proteins. Among the CYP isoforms implicated in PAH metabolism, CYP1A enzymes have been reported to both sensitize and protect from PAH toxicity. To clarify the role of CYP1A in PAH toxicity, we generated transgenic Caenorhabditis elegans that express CYP1A at a basal (but not inducible) level. Because this species does not normally express any CYP1 family enzyme, this approach permitted a test of the role of basally expressed CYP1A in PAH toxicity. We exposed C. elegans at different life stages to either the PAH benzo[a]pyrene (BaP) alone, or a real-world mixture dominated by PAHs extracted from the sediment of a highly-contaminated site on the Elizabeth River (VA, USA). This site, the former Atlantic Wood Industries, was declared a Superfund site due to coal tar creosote contamination that caused very high levels (in the [mg/mL] range) of high molecular weight PAHs within the sediments. We demonstrate that CYP1A protects against BaP-induced growth delay, reproductive toxicity, and reduction of steady state ATP levels. Lack of sensitivity of a DNA repair (Nucleotide Excision Repair)-deficient strain suggested that CYP1A did not produce significant levels of DNA-reactive metabolites from BaP. The protective effects of CYP1A in Elizabeth River sediment extract (ERSE)-exposed nematodes were less pronounced than those seen in BaP-exposed nematodes; CYP1A expression protected against ERSE-induced reduction of steady-state ATP levels, but not other outcomes of exposure to sediment extracts. Overall, we find that in C. elegans, a basal level of CYP1A activity is protective against the examined PAH exposures.

BACKGROUND

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. The interindividual genetic variations in drug metabolizing enzymes and DNA repair genes influence the efficacy and toxicity of numerous chemotherapeutic drugs affecting the treatment outcome.

OBJECTIVE

The aim of the study was to investigate the impact of drug metabolizing CYP1, GSTP1 and DNA repair (XRCC1) genes polymorphisms on the toxicity and response to chemotherapy in childhood ALL.

METHODS

Ninety seven ALL pediatric patients were genotyped for CYP1A1, GSTP1 ILe105Val and XRCC1 Arg194Tryp single nucleotide polymorphisms (SNPs) using PCR-RFLP.

RESULTS

No statistically significant differences were observed between the wild and variant (homozygous and heterozygous) genotypes of the polymorphisms studied in CYP1A1, GSTP1 or XRCC1 genes regarding age, total leukocyte count, immunophenotyping, cytogenetic or risk group. The SNPs in CYP1A1, GSTP1 and XRCC1 genes did not show significant association with complete remission (CR) rate, overall survival (OS) or event free survival (EFS). However, XRCC1 Arg194Trp SNP was associated with higher drug toxicity; carriers of variant genotypes (CT and TT) had a significantly higher frequency of myelosuppression compared to those with the wild CC genotype (21/43[48.8%]) compared to (14/54[25.9%]) (p=0.020). The analysis of the combined effect of studied SNPs did not show any significant association with patient outcome.

CONCLUSIONS

Our study reported a significant association between the DNA repair gene polymorphism and myelosuppression in childhood ALL patients. Adjustment of the dose of chemotherapeutic agents according to XRCC1 Arg194Trp polymorphism may improve outcome in cases with risk of toxicity.