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(WAF1/Cip1), cyclin D1 and Ogg1.
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 p21(WAF1/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 p21(WAF1/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.
Kinoshita
Anna
A
First Department of Pathology, Osaka City University Medical School, Abeno-ku, Asahi-machi 1-4-3, Osaka 545-8585, Japan.
Wanibuchi
Hideki
H
Imaoka
Susumu
S
Ogawa
Motome
M
Masuda
Chikayoshi
C
Morimura
Keiichirou
K
Funae
Yoshihiko
Y
Fukushima
Shoji
S
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D017209:Apoptosis; D001189:Aryl Hydrocarbon Hydroxylases; D002273:Carcinogens; D002453:Cell Cycle; D002455:Cell Division; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology; D019938:Cyclin D1 / Q000096:biosynthesis*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis*; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis; D003593:Cytoplasm / Q000187:drug effects / Q000201:enzymology; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology; D017665:Hydroxyl Radical; D007150:Immunohistochemistry; D008099:Liver / Q000187:drug effects* / Q000502:physiology*; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis*; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D010634:Phenobarbital; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013250:Steroid Hydroxylases / Q000096:biosynthesis; D013997:Time Factors
D002273:Carcinogens; C493958:Cdkn1a protein, rat; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004277:DNA, Single-Stranded; D018691:Excitatory Amino Acid Antagonists; D012333:RNA, Messenger; D019938:Cyclin D1; D017665:Hydroxyl Radical; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D019362:Cytochrome P-450 CYP2B1; C025619:steroid 16-beta-hydroxylase; C022657:steroid hormone 6-beta-hydroxylase; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine; D010634:Phenobarbital