Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice

Abstract

Piperonyl butoxide (PBO), α-[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.

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.