Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.
Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.
Muguruma
Masako
M
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu City, Tokyo 183-8509, Japan. muguruma@cc.tuat.ac.jp <muguruma@cc.tuat.ac.jp>
Nishimura
Jihei
J
Jin
Meilan
M
Kashida
Yoko
Y
Moto
Mitsuyoshi
M
Takahashi
Miwa
M
Yokouchi
Yusuke
Y
Mitsumori
Kunitoshi
K
D000818:Animals; D034961:Antigens, Nuclear / Q000096:biosynthesis / Q000235:genetics; D001835:Body Weight / Q000187:drug effects; D002273:Carcinogens; D019938:Cyclin D1 / Q000096:biosynthesis / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004032:Diet; D004435:Eating / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D000072200:Ku Autoantigen; D008099:Liver / Q000473:pathology; D008113:Liver Neoplasms / Q000139:chemically induced* / Q000473:pathology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D020411:Oligonucleotide Array Sequence Analysis; D009929:Organ Size / Q000187:drug effects; D010574:Pesticide Synergists / Q000633:toxicity*; D010882:Piperonyl Butoxide / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D034961:Antigens, Nuclear; D002273:Carcinogens; D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010574:Pesticide Synergists; D017382:Reactive Oxygen Species; D019938:Cyclin D1; D003577:Cytochrome P-450 Enzyme System; C000606512:Xrcc5 protein, mouse; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen; D010882:Piperonyl Butoxide