Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice
Introduction
Piperonyl butoxide (PBO), α-[2-(2-butoxyethoxy)ethoxy]-4,5-methylenedioxy-2-propyltoluene, is a pesticide synergist that is widely used along with pyrethroids as a grain protector and domestic insecticide. PBO has been reported to act as a hepatocarcinogen in F344 rats fed a diet containing either 1.2% or 2.4% PBO for 2 years (Takahashi et al., 1994). However, this chemical substance is classified as a non-genotoxic carcinogen since negative results were obtained in genotoxicity studies (Beamand et al., 1996). Liver and kidney damage was identified in rats administered 2.4% PBO for 13 weeks (Fujitani et al., 1992); enlarged hepatocytes, anisonucleosis, and single cell necrosis were observed in ICR mice fed a diet containing 0.9% PBO for 20 days (Fujitani et al., 1993). PBO has also been reported to have adverse effects on the reproductive, developmental, and behavioral functions of ICR mice (Tanaka, 1992, Tanaka et al., 1992). Furthermore, in a previous study, PBO administered to rats at 2000 ppm for 4 weeks caused a marked increase in the cytochrome P450 (CYP) 2B1 and CYP1A1/2 levels in their livers (Watanabe et al., 1998). Okamiya et al. (1998) reported that the liver tumor-promoting mechanism of PBO was similar to that of phenobarbital because the former chemical has the ability to induce CYP isoenzymes such as CYP2B1 and inhibit gap junctional intercellular communication. However, there is a lack of data required for a better understanding of the molecular mechanism of PBO-induced hepatocarcinogenesis in mice and rats.
Microarray analysis has been recently used to investigate the molecular events that involve non-genotoxic carcinogens and their tumor-promoting mechanisms (Iida et al., 2003, Kashida et al., 2006, Kinoshita et al., 2003, Moto et al., 2005, Wong and Gill, 2002). Clarifying the techniques of these gene expression analyses would be beneficial from the viewpoint of using them as powerful tools for predicting the toxicological and carcinogenic potentials of newly developed drugs, based on the accumulation of gene expression data.
In the present study, to investigate the possible molecular mechanism underlying the liver tumor-promoting activity of PBO in mice, we used the low-density Mouse Stress & Toxicity PathwayFinder Gene Array (SuperArray Bioscience Corp., Frederick, MD, USA). This array filter contains 96 genes whose expression changes is individual for stress and toxicity, and used to obtain information on the molecular events associated with the enhancement of hepatocellular carcinogenesis in mice subjected to PBO-treatment for 1–8 weeks. In addition, to clarify the mechanism of the tumor-promoting effect of PBO, further analyses comprising real-time reverse transcriptase (RT)-PCR, measurement of reactive oxygen species (ROS), and histological examinations were performed by considering the results obtained from the microarray analysis.
Section snippets
Chemicals, animals, and treatment
PBO (CAS 51-03-6; technical grade; purity >90%) was obtained from ACROS Organics (Morris Plains, NJ, USA). Five-week-old male ICR mice were purchased form Japan SLC, Inc. (Shizuoka, Japan); two or three mice were housed in each polycarbonate cage with paper bedding and under standard conditions (12-h light/dark cycle; 55 ± 5% relative humidity; 22 ± 2 °C, i.e., room temperature). After a 1-week acclimatization period, they were assigned to control or PBO-treatment groups. The mice were fed a basal
Body weights, food consumption, and liver weights
The body weight gain in the mice treated with 6000 ppm PBO was significantly inhibited at weeks 4 and 8 when compared with that in the control group (Table 2). No significant difference in food consumption was observed between the control and PBO-treated groups (data not shown).
The absolute and relative liver weights (liver/body weight × 100) of each group are shown in Table 2. The absolute liver weights of mice administered 6000 ppm PBO were significantly increased at weeks 1, 4, and 8 when
Discussion
It has been reported that the molecular expression levels of genes that might be related to the mode of action of carcinogenicity varies depending on the duration of treatment with non-genotoxic carcinogens (Kashida et al., 2006). Therefore, special attention was paid to these time-course changes in gene expression to clarify the mechanism of liver carcinogenesis induced by PBO. In both microarray and real-time RT-PCR analyses, genes encoding for phases I and II metabolic enzymes – Cyp1A1, 2B9,
Acknowledgments
This work was supported in part by a Grant-in-Aid for the Research Program for Risk Assessment Study on Food Safety Commission, Cabinet Office, Government of Japan. We are grateful to Dr. Minoru Shimoda of the Laboratory of Veterinary Pharmacology, Tokyo University of Agriculture and Technology, for his technical support in creating mouse liver microsome fractions.
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