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

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

Alternatives to Laboratory Animals ◽

10.1177/026119299202000118 ◽

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.

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The Effect of pH on Peroxidase-Mediated Oxidation of and DNA Adduct Formation by Ellipticine

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20061169 ◽

2006 ◽

Vol 71 (8) ◽

pp. 1169-1185 ◽

Cited By ~ 12

Author(s):

Jitka Poljaková ◽

Martin Dračínský ◽

Eva Frei ◽

Jiří Hudeček ◽

Marie Stiborová

Keyword(s):

Horseradish Peroxidase ◽

Dna Adducts ◽

Cytochromes P450 ◽

Antineoplastic Agent ◽

Ph Optimum ◽

Effect Of Ph ◽

Order Of Magnitude ◽

Enzymatic Activation

In order to understand the mechanism of enzymatic activation of an antineoplastic agent ellipticine, we investigated the effect of pH on the efficiency of three model peroxidases (bovine lactoperoxidase, human myeloperoxidase and horseradish peroxidase) in oxidation of ellipticine and in formation ellipticine-DNA adducts. The formation of the major ellipticine metabolite, ellipticine dimer, in which two ellipticine residues are connected through nitrogenN6in the pyrrole ring of one of the ellipticine moieties and carbon C9 of the other ellipticine, and formation of four ellipticine-DNA adducts were analyzed. All three peroxidases oxidize ellipticine to dimer and form ellipticine-DNA adducts, but lactoperoxidase and myeloperoxidase were less efficient in these processes than horseradish peroxidase. More than one order of magnitude higher rates of formation of dimer and amounts of the DNA adducts were found upon horseradish peroxidase than in reactions with lactoperoxidase or myeloperoxidase. An acid pH optimum was found for the formation of ellipticine dimer (pH 6.4), while the highest binding of ellipticine activated by peroxidases to DNA was detectable at pH 8.4. Likewise, the highest binding of 5-(hydroxymethyl)ellipticine, a metabolite of ellipticine generated by cytochrome P450, to DNA was found at pH 8.4. The results presented here are a contribution to the explanation of the reaction mechanism of formation of the major deoxyguanosine adduct in DNA generated from ellipticinein vivoandin vitroby its activation with cytochromes P450 and peroxidases.

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Benzo[a]pyrene-Induced Genotoxicity in Rats is Affected by Co-Exposure to Sudan I by Altering the Expression of Biotransformation Enzymes

International Journal of Molecular Sciences ◽

10.3390/ijms22158062 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8062

Author(s):

Helena Dračínská ◽

Radek Indra ◽

Sandra Jelínková ◽

Věra Černá ◽

Volker Arlt ◽

...

Keyword(s):

Dna Adducts ◽

Azo Dye ◽

Adduct Formation ◽

Dna Adduct ◽

Potent Inducer ◽

Human Carcinogen ◽

Hepatic Microsomes ◽

Sudan I

The environmental pollutant benzo[a]pyrene (BaP) is a human carcinogen that reacts with DNA after metabolic activation catalysed by cytochromes P450 (CYP) 1A1 and 1B1 together with microsomal epoxide hydrolase. The azo dye Sudan I is a potent inducer of CYP1A1/2. Here, Wistar rats were either treated with single doses of BaP (150 mg/kg bw) or Sudan I (50 mg/kg bw) alone or with both compounds in combination to explore BaP-derived DNA adduct formation in vivo. Using 32P-postlabelling, DNA adducts generated by BaP-7,8-dihydrodiol-9,10-epoxide were found in livers of rats treated with BaP alone or co-exposed to Sudan I. During co-exposure to Sudan I prior to BaP treatment, BaP-DNA adduct levels increased 2.1-fold in comparison to BaP treatment alone. Similarly, hepatic microsomes isolated from rats exposed to Sudan I prior to BaP treatment were also the most effective in generating DNA adducts in vitro with the activated metabolites BaP-7,8-dihydrodiol or BaP-9-ol as intermediates. DNA adduct formation correlated with changes in the expression and/or enzyme activities of CYP1A1, 1A2 and 1B1 in hepatic microsomes. Thus, BaP genotoxicity in rats in vivo appears to be related to the enhanced expression and/or activity of hepatic CYP1A1/2 and 1B1 caused by exposure of rats to the studied compounds. Our results indicate that the industrially employed azo dye Sudan I potentiates the genotoxicity of the human carcinogen BaP, and exposure to both substances at the same time seems to be hazardous to humans.

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Pentachlorophenol enhances 9-hydroxybenzo [a] pyrene-induced hepatic DNA adduct formation in vivo and inhibits microsomal epoxide hydrolase and glutathione S-transferase activities in vitro: likely inhibition of epoxide detoxication by pentachlorophenol

Archives of Toxicology ◽

10.1007/s002040050330 ◽

1996 ◽

Vol 70 (11) ◽

pp. 696-703 ◽

Cited By ~ 17

Author(s):

B. Moorthy ◽

Kurt Randerath

Keyword(s):

Epoxide Hydrolase ◽

Adduct Formation ◽

Dna Adduct ◽

Microsomal Epoxide Hydrolase ◽

Glutathione S Transferase

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DNA adduct formation by O-phenylphenol metabolite in vivo and in vitro

Carcinogenesis ◽

10.1093/carcin/13.8.1469 ◽

1992 ◽

Vol 13 (8) ◽

pp. 1469-1473 ◽

Cited By ~ 19

Author(s):

Keiko Ushiyama ◽

Fumiko Nagai ◽

Atsuko Nakagawa ◽

Itsu Kano

Keyword(s):

Adduct Formation ◽

Dna Adduct

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In vitro and in vivo effects of sodium selenite on 7,12-dimethylbenz[a]anthracene-DNA adduct formation in isolated rat mammary epithelial cells

Carcinogenesis ◽

10.1093/carcin/10.5.823 ◽

1989 ◽

Vol 10 (5) ◽

pp. 823-826 ◽

Cited By ~ 6

Author(s):

S. Ejadi ◽

I. D. Bhattacharya ◽

K. Voss ◽

K. Singletary ◽

J. A. Milner

Keyword(s):

Epithelial Cells ◽

Sodium Selenite ◽

Mammary Epithelial Cells ◽

Adduct Formation ◽

Mammary Epithelial ◽

Dna Adduct ◽

In Vivo Effects ◽

Isolated Rat

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

Mutagenesis ◽

10.1093/mutage/gez033 ◽

2019 ◽

Cited By ~ 1

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.

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