scholarly journals Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells

Mutagenesis ◽  
2019 ◽  
Author(s):  
Lindsay Reed ◽  
Ian W H Jarvis ◽  
David H Phillips ◽  
Volker M Arlt
Keyword(s):  
Cytochrome P450 ◽  
Mouse Model ◽  
Metabolic Activation ◽  
Adduct Formation ◽  
Dna Adduct ◽  
Environmental Carcinogen ◽  
P450 Oxidoreductase ◽  
Cyp Enzymes ◽  
Cytochrome P450 Oxidoreductase

Abstract The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

scholarly journals Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase

2019 ◽  
Author(s):  
Maria Natalia Rojas Velazquez ◽  
Shaheena Parween ◽  
Sameer S Udhane ◽  
Amit V Pandey
Keyword(s):  
Cytochrome P450 ◽  
Drug Metabolism ◽  
Recombinant Proteins ◽  
Correct Diagnosis ◽  
Detailed Knowledge ◽  
Drug Metabolizing Enzymes ◽  
Metabolizing Enzymes ◽  
P450 Oxidoreductase ◽  
Cytochrome P450 Oxidoreductase

AbstractA broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

scholarly journals The impact of p53 function on the metabolic activation of the carcinogenic air pollutant 3-nitrobenzanthrone and its metabolites 3-aminobenzanthrone and N-hydroxy-3-aminobenzanthrone in human cells

Mutagenesis ◽  
2018 ◽  
Vol 33 (4) ◽  
pp. 311-321 ◽  
Author(s):  
Laura E Wohak ◽  
Ann-Christin Baranski ◽  
Annette M Krais ◽  
Heinz H Schmeiser ◽  
David H Phillips ◽  
...  
Keyword(s):  
P53 Protein ◽  
Mutant Cell ◽  
Metabolic Activation ◽  
Adduct Formation ◽  
Human Cells ◽  
Air Pollutant ◽  
Dna Adduct ◽  
Tumour Suppressor ◽  
Environmental Carcinogen ◽  
The Impact

Abstract The tumour suppressor p53, encoded by TP53, is a key player in a wide network of signalling pathways. We investigated its role in the bioactivation of the environmental carcinogen 3-nitrobenzanthrone (3-NBA)found in diesel exhaust and its metabolites 3-aminobenzanthrone (3-ABA) and N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA) in a panel of isogenic human colorectal HCT116 cells differing only with respect to their TP53 status [i.e. TP53(+/+), TP53(+/−), TP53(−/−), TP53(R248W/+) or TP53(R248W/−)]. As a measure of metabolic competence, DNA adduct formation was determined using 32P-postlabelling. Wild-type (WT) p53 did not affect the bioactivation of 3-NBA; no difference in DNA adduct formation was observed in TP53(+/+), TP53(+/−) and TP53(−/−) cells. Bioactivation of both metabolites 3-ABA and N-OH-3-ABA on the other hand was WT-TP53 dependent. Lower 3-ABA- and N-OH-3-ABA-DNA adduct levels were found in TP53(+/−) and TP53(−/−) cells compared to TP53(+/+) cells, and p53’s impact was attributed to differences in cytochrome P450 (CYP) 1A1 expression for 3-ABA whereas for N-OH-3-ABA, an impact of this tumour suppressor on sulphotransferase (SULT) 1A1/3 expression was detected. Mutant R248W-p53 protein function was similar to or exceeded the ability of WT-p53 in activating 3-NBA and its metabolites, measured as DNA adducts. However, identification of the xenobiotic-metabolising enzyme(s) (XMEs), through which mutant-p53 regulates these responses, proved difficult to decipher. For example, although both mutant cell lines exhibited higher CYP1A1 induction after 3-NBA treatment compared to TP53(+/+) cells, 3-NBA-derived DNA adduct levels were only higher in TP53(R248W/−) cells but not in TP53(R248W/+) cells. Our results show that p53’s influence on carcinogen activation depends on the agent studied and thereby on the XMEs that mediate the bioactivation of that particular compound. The phenomenon of p53 regulating CYP1A1 expression in human cells is consistent with other recent findings; however, this is the first study highlighting the impact of p53 on sulphotransferase-mediated (i.e. SULT1A1) carcinogen metabolism in human cells.

BaP Metabolism and DNA-adduct Formation in Cultured Human Lymphocytes Treated In Vitro with BaP and (-)-BaP-7,8-dihydrodiol

1992 ◽  
Vol 20 (1) ◽  
pp. 126-137
Author(s):  
Sofia Pavanello ◽  
Nicola Zanesi ◽  
Angelo Gino Levis
Keyword(s):  
Dna Adducts ◽  
Human Lymphocyte ◽  
Culture Medium ◽  
Human Lymphocytes ◽  
Metabolic Activation ◽  
Adduct Formation ◽  
Dna Adduct ◽  
Repair Mechanism ◽  
Dna 2

Nine samples of human lymphocytes from six healthy subjects were treated in vitro for 24 hours with [3H]-benzo[a]pyrene (BaP) and unlabelled (-)-BaP-7,8-dihydrodiol; 1μg/ml and 0.5μg/ml, respectively. The separation by HPLC of BaP-DNA adducts showed a significant formation of syn-BaPDE-dGuo adducts in five out of the nine human lymphocyte samples treated in vitro with [3H]-BaP. BaP-(7,10/8,9) tetrol and BaP-(7/8,9,10) tetrol derived from (+)-anti-BaPDE were the predominant isomers extracted from the culture medium of all the lymphocyte samples treated with (-)-BaP-7,8-dihydrodiol (mean ratio of anti-BaPDE/syn-BaPDE tetrols 5.6 + 1.2), suggesting that the metabolic activation of (-)-BaP-7,8-dihydrodiol occurred by its epoxidation to (+)-anti-BaPDE much more frequently (4–8 times) than by epoxidation to the (-)-syn-BaPDE form. In contrast to the predominant formation of anti-BaPDE tetrols, the analysis by HPLC of DNA adducts in the five subjects examined also revealed the significant formation of syn-BaPDE-DNA adducts (mean adduct ratio of (+)-anti-/(-)-syn-BaPDE-DNA = 2.6 ± 0.7). In six human lymphocyte samples treated in vitro with anti-BaPDE and syn-BaPDE, the anti-BaPDE bound to DNA 2–5 times more efficiently than did the syn-BaPDE. However, after 24 hours, the level of anti-BaPDE-DNA adducts significantly decreased almost to the level of the syn-BaPDE-DNA adducts, suggesting that a stereoselective repair mechanism could have preferentially removed the anti-BaPDE-DNA adducts.

scholarly journals Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-Related Variants of NADPH-Cytochrome P450 Oxidoreductase Measured in a Robust In Vitro System

2012 ◽  
Vol 40 (4) ◽  
pp. 754-760 ◽  
Author(s):  
Daniela Moutinho ◽  
Christopher C. Marohnic ◽  
Satya P. Panda ◽  
José Rueff ◽  
Bettie Sue Masters ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Cyp3a4 Activity ◽  
In Vitro System ◽  
P450 Oxidoreductase ◽  
Nadph Cytochrome ◽  
Cytochrome P450 Oxidoreductase

scholarly journals Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase

2018 ◽  
Vol 129 (4) ◽  
pp. 756-768 ◽  
Author(s):  
Pan-Fen Wang ◽  
Alicia Neiner ◽  
Evan D. Kharasch
Keyword(s):  
Cytochrome P450 ◽  
Genetic Variants ◽  
Cytochrome B5 ◽  
Intrinsic Clearance ◽  
Cell System ◽  
Therapeutic Effects ◽  
Wild Type ◽  
P450 Oxidoreductase ◽  
Cytochrome P450 Oxidoreductase

Abstract Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. Methods CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography–mass spectrometry. Michaelis–Menten kinetic parameters were determined. Results For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Conclusions Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

scholarly journals Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2

2008 ◽  
Vol 1 (2) ◽  
pp. 160-168 ◽  
Author(s):  
Dagmar Aimová ◽  
Jitka Poljaková ◽  
Věra Kotrbová ◽  
Michaela Moserová ◽  
Eva Frei ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Dna Adducts ◽  
Cytochromes P450 ◽  
Antineoplastic Agent ◽  
Metabolic Activation ◽  
Experimental Models ◽  
Adduct Formation ◽  
Dna Adduct

Ellipticine and benzo(a)pyrene increase their own metabolic activation via modulation of expression and enzymatic activity of cytochromes P450 1A1 and 1A2Two compounds known to covalently bind to DNA after their activation with cytochromes P450 (CYPs), carcinogenic benzo(a)pyrene (BaP) and an antineoplastic agent ellipticine, were investigated for their potential to induce CYP and NADPH:CYP reductase (POR) enzymes in rodent livers, the main target organ for DNA adduct formation. Two animal models were used in the study: (i) rats as animals mimicking the fate of ellipticine in humans and (ii) mice, especially wild-type (WT) and hepatic POR null (HRN™) mouse lines. Ellipticine and BaP induce expression of CYP1A enzymes in livers of experimental models, which leads to increase in their enzymatic activity. In addition, both compounds are capable of generating DNA adducts, predominantly in livers of studied organisms. As determined by32P postlabelling analysis, levels of ellipticine-derived DNA adducts formedin vivoin the livers of HRN™ mice were reduced (by up to 65%) relative to levels in WT mice, indicating that POR mediated CYP enzyme activity is important for the activation of ellipticine. In contrast to these results, 6.4 fold higher DNA binding of BaP was observed in the livers of HRN™ mice than in WT mice. This finding suggests a detoxication role of CYP1A in BaP metabolismin vivo. Inin vitroexperiments, DNA adduct formation in calf thymus DNA was up to 25 fold higher in incubations of ellipticine or BaP with microsomes from pretreated animals than with controls. This stimulation effect was attributed to induction of CYP1A1/2 enzymes, which are responsible for oxidative activation of both compounds to the metabolites generating major DNA adductsin vitro. Taken together, these results demonstrate that by inducing CYP1A1/2, ellipticine and BaP modulate their own enzymatic metabolic activation and detoxication, thereby modulating their either pharmacological (ellipticine) and/or genotoxic potential (both compounds).

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