Journal Article

Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21<sup>WAF1/Cip1</sup>, cyclin D1 and Ogg1

Anna Kinoshita, Hideki Wanibuchi, Susumu Imaoka, Motome Ogawa, Chikayoshi Masuda, Keiichirou Morimura, Yoshihiko Funae and Shoji Fukushima

in Carcinogenesis

Volume 23, issue 2, pages 341-349
Published in print February 2002 | ISSN: 0143-3334
Published online February 2002 | e-ISSN: 1460-2180 | DOI:
Formation of 8-hydroxydeoxyguanosine and cell-cycle arrest in the rat liver via generation of oxidative stress by phenobarbital: association with expression profiles of p21WAF1/Cip1, cyclin D1 and Ogg1

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To evaluate the risk of exposure to so-called non-genotoxic chemicals and elucidate mechanisms underlying their promoting activity on rat liver carcinogenesis the formation of 8-hydroxy-2′-deoxyguanosine (8-OHdG), cytochrome P-450 (P-450) and hydroxyl radicals induction, DNA repair and alteration to cellular proliferation and apoptosis in the rat liver were investigated during 2 weeks of phenobarbital (PB) administration at a dose of 0.05%. Significant increase of hydroxyl radical levels by day 4 of PB exposure accompanied the accumulation of 8-OHdG in the nucleus and P-450 isoenzymes CYP2B1/2 and CYP3A2 in the cytoplasm of hepatocytes. Conspicuous elevation of 8-OHdG and apoptosis in the liver tissue were associated with reduction of the proliferating cell nuclear antigen (PCNA) index after 8 days of PB application. Thereafter, 8-OHdG levels decreased with an increase in mRNA expression for the 8-OHdG repair enzyme, DNA glycosylase 1 (Ogg1). Analysis with LightCycler quantitative 2-step RT–PCR demonstrated induction of cyclin D1 (CD1) and p21WAF1/Cip1 mRNA expression on days 4 and 6, respectively, preceding marked elevation of PCNA and apoptotic indices. These results suggest that similar to genotoxic, non-genotoxic chemicals might induce reversible alteration to nuclear 8-OHdG in the rat liver after several days of continuous application; however, by a different mechanism. Increased 8-OHdG formation is caused by developing oxidative stress or apoptotic degradation of DNA and coordinated with enhanced expression of CD1 mRNA and cell proliferation, subsequent increase of p21WAF1/Cip1 mRNA expression, cell-cycle arrest and apoptosis, while activation of 8-OHdG repair mechanisms contributes to protection of tissue against reactive oxygen species-induced cell death.

Keywords: CYP2B1/2 and CYP3A2, 2B1/2 and 3A2 isoenzymes of cytochrome P-450, respectively; CD1, cyclin D1; CDK, cyclin-dependent kinase; DAB, 3,3′-diaminobenzidine; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DMPO-OH, spin adduct of DMPO and hydroxyl radicals; ESR, electron spin resonance; GAPDH, glyceraldehyde-3-P dehydrogenase; HPLC, high-performance liquid chromatography; LC-RT–PCR, LightCycler RT–PCR; MnO, manganese oxide marker; NADPH, nicotinamide adenine dinucleotide phosphate reduced form; Ogg1, 8-oxoguanine DNA glycosylase 1; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; P-450, cytochrome P-450; PB, phenobarbital; ROS, reactive oxygen species; ssDNA, single-strand DNA.

Journal Article.  7024 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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