title: Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes.
abstract: The formation of DNA adducts in human HepG2 cells and human hepatocytes exposed to 14C-labelled 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was examined using Accelerator Mass Spectrometry (AMS). PhIP generated DNA adducts in a linear dose-dependent manner between 100 pM and 20 micro M. Co-treatment with the dietary isothiocyanate, sulforaphane (SFN, 1-10 micro M), or the flavonoid, quercetin (5-20 micro M), significantly reduced the level of PhIP-DNA adducts in a dose-dependent manner. The degree of protection was dependent on PhIP concentration, i.e. after 100 pM PhIP exposure, SFN or quercetin reduced adduct levels to below the limit of detection (0.15 amol PhIP/ micro g DNA) but at higher PhIP exposure (10 nM and 1 micro M), the protection was 60 and 10%, respectively. The involvement of phase I, phase II and DNA repair enzymes in this protection against PhIP-DNA adduct formation was investigated using real-time RT-PCR and enzyme activity assays. In intact HepG2 cells, quercetin inhibited cytochrome P450 (CYP)1A2, the main phase I enzyme responsible for PhIP bioactivation. In contrast, SFN induced phase II detoxification enzymes, UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 mRNA expression. SFN and quercetin showed no effect on DNA repair, neither in terms of the level of PhIP-DNA adducts, when cells were treated with phytochemicals after the carcinogen exposure, nor the regulation of mRNA expression of two DNA repair enzymes, apurinic endonuclease and DNA polymerase beta. This study indicates that dietary isothiocyanates and flavonoids modulate phase I and phase II enzyme expression, hence increasing the rate of detoxification of the dietary carcinogen PhIP in human HepG2 cells but do not affect the rate of PhIP-DNA adduct repair. The formation of PhIP-DNA adducts in human hepatocytes was also dose-dependent with PhIP-concentration and the levels of protection by SFN or quercetin were up to 60% after 10 nM PhIP treatment, but showed large inter-individual variation with no observed protection in some individuals.
authors.affiliation: Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, UK.
authors.forename: James R
authors.initials: JR
authors.lastname: Bacon
authors.forename: Gary
authors.initials: G
authors.lastname: Williamson
authors.forename: R Colin
authors.initials: RC
authors.lastname: Garner
authors.forename: Graham
authors.initials: G
authors.lastname: Lappin
authors.forename: Sophie
authors.initials: S
authors.lastname: Langouët
authors.forename: Yongping
authors.initials: Y
authors.lastname: Bao
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology; D015415:Biomarkers; D002273:Carcinogens; D002460:Cell Line; D045744:Cell Line, Tumor; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics; D018736:DNA Adducts; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D017879:Isothiocyanates / Q000737:chemistry; D013058:Mass Spectrometry; D011794:Quercetin / Q000494:pharmacology*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013454:Sulfoxides; D013861:Thiocyanates / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D016588:Anticarcinogenic Agents; D015415:Biomarkers; D002273:Carcinogens; D018736:DNA Adducts; D007093:Imidazoles; D017879:Isothiocyanates; D012333:RNA, Messenger; D013454:Sulfoxides; D013861:Thiocyanates; D012313:RNA; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D011794:Quercetin; D019388:Cytochrome P-450 CYP1A2; D005982:Glutathione Transferase; D019951:DNA Polymerase beta; C016766:sulforaphane