In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1,☆☆

https://doi.org/10.1016/S0006-291X(03)00435-2Get rights and content

Abstract

Previously, inhibitors of CYP1A1 were rated as candidate chemopreventive agents against cancer mainly according to their effects on the 7-ethoxyresorufin O-deethylation (EROD) of diagnostic probe substrates. Surprisingly, several polyphenols including resveratrol, formerly identified as potent inhibitors by the EROD assay, exhibited no or weak inhibition of procarcinogen activation. We compared the effects of 11 representative natural polyphenols, which normally occur in food, on different activities of CYP1A1, namely epoxidation of 7,8-dihydrodiol-benzo[a]pyrene, the terminal step in the activation leading to the ultimate carcinogenic diolepoxides, hydroxylation of benzo[a]pyrene, and EROD. For the first time, a reconstituted system was used for the determination of IC50 values, consisting of purified enzymes (human CYP1A1 and human NADPH–cytochrome P450 reductase) and dilaurylphosphatidylcholine. The results demonstrate that the inhibitory effects of dietary polyphenols on CYP1A1 activity depend on both the structure of the inhibitor and the type of the reaction and substrate used in the assay. Consequently, a potent EROD inhibition alone is insufficient to count a substance among the chemoprotective agents.

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Materials

Apigenin, myricetin, kaempferol, quercetin, naringenin, hesperetin, naringin, hesperidin, rutin, resveratrol, (−)-epigallo catechin gallate, 7-ethoxyresorufin, resorufin, B[a]P, and DLPC were purchased from Sigma. 7,8-diol-B[a]P and the tetraols RTTC, RTTT, RTCT, and RTCC were purchased from NCI Chemical Carcinogen Repository, Midwest Research Institute, Kansas City, MI, USA. 3-OH-B[a]P was a gift of Dr. A. Seidel (University Mainz, Germany).

Expression, purification, and reconstitution of human CYP1A1 and human P450 reductase

Construction of the modified CYP1A1 cDNA, cloning

Inhibition of different CYP1A1 activities by resveratrol and (−)-epigallo catechin gallate

In preliminary experiments, we determined the apparent Km values for the formation of DE2 from 7,8-diol-B[a]P, the formation of 3-OH-B[a]P from B[a]P, and the EROD reaction. These values were 1, 5.2, and 0.4 μM, respectively. In the present study, inhibitor concentrations were in the order of magnitude of the Km values or about two times higher. Thus, the concentrations of substrates were 2 μM (7,8-diol-B[a]P), 5 μM (B[a]P), and 0.5 μM (ethoxyresorufin).

The effects of resveratrol and (−)-epigallo

Acknowledgements

We are grateful to Dr. F.J. Gonzalez for providing CYP1A1 cDNA and virus for P450 reductase expression (National Cancer Institute, NIH, Bethesda, MD, USA) and to Dr. A. Seidel (Institute for Toxicology, University Mainz, Germany) for a sample of 3-OH-B[a]P. We thank Dr. A. Chernogolov, Dr. H. Honeck, C. Andreé, and A. Sternke (all from Max Delbrueck Center for Molecular Medicine, Berlin-Buch) for preparational work, assistance in HPLC and cell culturing. This work was supported by grants of the

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    ☆☆

    Abbreviations: CYP1A1, human cytochrome P450 1A1; P450 reductase, NADPH cytochrome P450 reductase; B[a]P, benzo[a]pyrene; 7,8-diol-B[a]P, B[a]P-trans-7,8-dihydrodiol; RTTC, r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetrahydro-B[a]P; RTTT, r-7,t-8,t-9,t-10-tetrahydroxy-7,8,9,10-tetrahydro-B[a]P; RTCT, r-7,t-8,c-9,t-10-tetrahydroxy-7,8,9,10-tetrahydro-B[a]P; RTCC, r-7,t-8,c-9,c-10-tetrahydroxy-7,8,9,10-tetrahydro-B[a]P; diolepoxide 2, DE2, (±)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide; diolepoxide 1, DE1, (±)-B[a]P-r-7,t-8-dihydrodiol-c-9,10-epoxide; DLPC, dilaurylphosphatidylcholine; EROD, 7-ethoxyresorufin O-deethylation; AHH, aryl hydrocarbon hydroxylation.

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