Molecular pathological analysis on the mechanism of liver carcinogenesis in dicyclanil-treated mice

Abstract

To investigate the mechanism of hepatocarcinogenesis due to dicyclanil (DC), an insect growth regulator for sheep, histopathological and molecular biological analyses were performed in the liver of male ICR mice fed on a diet containing 1500 ppm of DC for 2 weeks (Experiment I; Exp. I). In gene expression analyses using a large-scale cDNA microarray and RT-PCR, fluctuations of expressions of metabolism-/oxidation-/reduction-related genes, such as CYP1A, aldehyde dehydrogenase family 1 subfamily A1 (Aldh1a1), and thioredoxin reductase 1 (Txnrd1), were predominantly observed in the liver of the DC-treated group. In Experiment II (Exp. II), small-scale and metabolism/oxidative stress-specific cDNA microarray, real-time RT-PCR, and measurement of NF-κB protein were performed in the mice liver using a two-stage hepatocarcinogenesis model, in which the male ICR mice were fed on a diet containing 1500 ppm of DC for 7 weeks after a single injection of dimethylnitrosamine (DMN). These mice were subjected to two-thirds partial hepatectomy (PH) at week 3. During histopathological examinations, a remarkable increase in γ-glutamyltransferase-positive cells was observed in the DMN + DC + PH group. During the microarray and PCR analyses, the metabolism and oxidative stress-related genes, such as Cyp1a, P450 oxidoreductase (Por), and thioredoxin reductase 1 (Txnrd1); a few DNA damage/repair genes, such as 8-oxoguanine DNA-glycosylase 1 (Ogg1); and growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), were fluctuated in this group, together with a slight increase in the concentration of activated NF-κB. These results suggest that DNA damages due to oxidative stress may be involved in the mechanism of DC-induced hepatocarcinogenesis in mice.

Introduction

Dicyclanil (DC), 4,6-diamino-2-cyclopropylamino-pyrimidine-5-carbonitrile, is a pyrimidine-derived insect growth regulator inhibiting the molting and development of insecticides, and is used in a veterinary field for the prevention of myiasis (fly-strike) in sheep. As a result, small amounts of the parent drug and its metabolites are sometimes detected as residues in the edible tissues of these sheep (WHO, 2000). In an 18-month study conducted on the carcinogenicity of DC, it was observed that the incidence of hepatocellular carcinomas was increased in mice fed on a diet containing 1500 ppm of DC; hepatocellular necrosis was also observed in the groups fed with 100 ppm or more of DC (WHO, 2000). On the other hand, in studies conducted on the genotoxicity of DC, negative results were obtained from the in vivo and in vitro tests, and it was evaluated by the 54th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) that DC is a non-genotoxic carcinogen (WHO, 2000). Additionally, our recent study indicated that negative results were obtained when a single oral administration of DC was performed in the in vivo comet assay using mice and in vivo liver initiation assay using rats, and these results support the evaluation of the JECFA (Moto et al., 2003). Therefore, it was postulated that the DC treatment-induced enhancement of hepatocarcinogenesis resulted from non-genotoxic mechanisms or tumor-promoting actions such as the enhancement of cell proliferation, inhibition of apoptosis, disorder of the cell cycle regulation, secondary genotoxicity, and so on. However, detailed investigations of molecular events in the DC-induced mice liver have not yet been performed.

In the studies conducted to evaluate the mechanism of chemical carcinogenesis, it is extremely important to understand the molecular events during the chemical-induced initiation and promotion phases in the target organs, because gene alterations may vary with the presence or absence of mutated cells and/or pre-neoplastic foci. Recently, large-scale gene expression analyses by the cDNA microarray have been applied in the fields of toxicology and carcinogenicity (Afshari et al., 1999, Irwin et al., 2004). Microarray analyses have been used to investigate the molecular events in the non-genotoxic carcinogens during the various stages of carcinogenesis, and the molecular events on tumor promoting mechanisms have also been reported (Chen et al., 2001, Iida et al., 2003, Kinoshita et al., 2003, Kato et al., 2004). Several non-genotoxic carcinogens are known to show tumor-promoting activities in the two-stage (initiation-promotion) carcinogenesis models (Shirai, 1997, Shibutani et al., 2002). Therefore, the presently discussed carcinogenesis model is believed to be useful in the detection of molecular profiles during the tumor promotion stage.

In the present study, to clarify the enhancement mechanism of hepatocarcinogenesis in DC-induced mice, a large-scale cDNA microarray (3800 genes) was performed to obtain primary information on molecules and/or molecular events associated with the enhancement of hepatocarcinogenesis in mice liver at the early stage of the toxicity and carcinogenicity of DC. Based on the results obtained from this microarray, a low-density and pathway-specific microarray (112 genes) were performed to investigate the details of the molecular events during the tumor promotion phase of DC using the two-stage hepatocarcinogenesis model in mice with partial hepatectomy. In addition, to clarify the mechanism of the tumor promoting effect of DC, further analyses comprising RT-PCR or real-time RT-PCR, histological examinations, and measurements of proteins associated with the pathway were carried out by taking into account the results obtained from the microarray analyses.