PubMed · 11225008
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J W Kosmeder, J M Pezzuto. 2001. Intermediate biomarkers.. https://doi.org/10.1007/978-1-4615-1657-6_2
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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.
The process of tumorigenesis requires cellular transformation, hyperproliferation, invasion, angiogenesis, and metastasis. Several genes that mediate these processes are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). The latter is activated by various carcinogens, inflammatory agents, and tumor promoters. Thus, agents that can suppress NF-kappaB activation have the potential to suppress carcinogenesis. Ursolic acid, a pentacyclic triterpene acid, has been shown to suppress the expression of several genes associated with tumorigenesis, but whether ursolic acid mediates its effects through suppression of NF-kappaB is not understood. In the study described in the present report, we found that ursolic acid suppressed NF-kappaB activation induced by various carcinogens including tumor necrosis factor (TNF), phorbol ester, okadaic acid, H(2)O(2), and cigarette smoke. These effects were not cell type specific. Ursolic acid inhibited DNA binding of NF-kappaB consisting of p50 and p65. Ursolic acid inhibited IkappaBalpha degradation, IkappaBalpha phosphorylation, IkappaBalpha kinase activation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Ursolic acid also inhibited NF-kappaB-dependent reporter gene expression activated by TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor, NF-kappaB-inducing kinase, IkappaBalpha kinase, and p65. The inhibition of NF-kappaB activation correlated with suppression of NF-kappaB-dependent cyclin D1, cyclooxygenase 2, and matrix metalloproteinase 9 expression. Thus, overall, our results indicate that ursolic acid inhibits IkappaBalpha kinase and p65 phosphorylation, leading to the suppression of NF-kappaB activation induced by various carcinogens. These actions of ursolic acid may mediate its antitumorigenic and chemosensitizing effects.
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