Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides
Introduction
Liver hypertrophy and liver tumors are major treatment-related events in rodent toxicity or carcinogenicity studies of xenobiotics (Thoolen et al., 2010). Liver hypertrophy can cause increases in liver weights, average sizes of hepatocytes (hepatocellular hypertrophy), and the functional capacity of the liver. Lack of liver hypertrophy and tumors in constitutive androstane receptor (CAR; NR113)-knockout (KO) mice treated with phenobarbital (PB), a prototypical CYP2B inducer, has provided clear evidence that CAR plays an essential role in PB-induced liver hypertrophy and tumor development (Wei et al, 2000, Yamamoto et al, 2004). In contrast, epidemiologic studies of human patients undergoing long-term PB therapy showed no increase in the incidence of hepatic cancer (International Agency for Research on Cancer (IARC), 2001, La Vecchia, Negri, 2014, Lamminpää et al, 2002). Liver hypertrophy had been accepted as a key event of liver tumor development in rodents (Holsapple et al., 2006). However, a recent study showed that CAR activation plays an important role in hepatocarcinogenesis and that hypertrophy is associated with hepatocarcinogenesis (Elcombe et al., 2014). In a flow-chart diagram demonstrating the strategy of the National Toxicology Program for incorporating predictive hepatic transcriptomic datasets, hypertrophy induced by compounds in a 90-day toxicity study was shown to be the primary change associated with the formation of liver tumors. In contrast, at the Third International ESTP Expert Workshop, liver hypertrophy was suggested to have a low predictive value in the identification and elucidation of carcinogenic agents; however, no conclusive evidence was provided (Hall et al., 2012). Our previous study using C3H strain-derived CARKO mice showed that the pathway of CAR-mediated liver hypertrophy is different from that in hepatocarcinogenesis in mice treated with piperonyl butoxide (PBO) or decabromodiphenyl ether, both of which are CYP2B inducers, indicating that liver hypertrophy is not a key event for hepatocarcinogenesis (Sakamoto et al., 2013).
Triazoles, a class of antifungal agents that are widely used as fungicides or pharmaceutical drugs, exert their fungicidal activity by inhibiting 14α-demethylase activity, which is involved in sterol biosynthesis (Ghannoum and Rice, 1999). Various triazoles have been reported to induce liver hypertrophy and tumor development in rodent carcinogenicity studies (INCHEM, 1987, INCHEM, 1992, INCHEM, 1994, INCHEM, 1997, INCHEM, 2001, Regulatory Affairs Department, SDS Biotech K.K., 1997). Three recent studies have indicated that some triazoles induce CAR-mediated liver hypertrophy and/or gene expression in rodents (Currie et al, 2014, Nesnow et al, 2009, Peffer et al, 2007). Aside from these studies, there is no clear evidence regarding the involvement of CAR in rodent liver tumorigenesis by long-term triazole treatment.
Cyproconazole (Cypro), tebuconazole (Teb), and fluconazole (Flu), classified as fungicides (Cypro and Teb) or antifungal medicines (Flu), induce liver hypertrophy and/or liver tumors in rodents (INCHEM, 1994, Regulatory Affairs Department, SDS Biotech K.K., 1997, Sun et al, 2006). We recently conducted a short-term study in which CARKO mice were treated with Cypro, Teb, or Flu for 7 days (Tamura et al., 2013). Our results indicated that these 3 triazoles produced CAR-mediated effects on liver hypertrophy in a dose-dependent manner; interestingly, the proportion of involvement varied for each compound. However, the study did not clarify the involvement of CAR in liver hypertrophy after long-term treatment or in the development of liver tumors induced by these triazoles.
The present study was conducted to investigate the involvement of CAR in the subacute effects of Cypro, Teb, and Flu on liver hypertrophy and liver tumor development induced by these 3 triazoles using CARKO mice and to elucidate the mode of action (MOA) of tumor promotion by these triazoles. The initiation and promotion rodent model was used for the determination of liver tumor development.
Section snippets
Chemicals
Cypro (CAS No. 94361-06-5, purity 96.7%), Teb (CAS No. 107534-96-3, purity 97.3%), and Flu (CAS No. 86386-73-4, purity > 99.5%) were obtained from LKT Laboratories, Inc. (St. Paul, MN, USA). Diethylnitrosamine (DEN, CAS No. 55-18-5, purity 99.9%) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
Animals
Male CARKO mice (Kodama et al, 2004, Yamamoto et al, 2004), in the C3H/HeNCrl background, were provided courtesy of Dr. M. Negishi (NIEHS) and bred at the National Institute of Health
Clinical signs, food consumption, and body and liver weights
No treatment-related clinical signs were detected throughout the treatment period. Final body weights at weeks 4, 13, and 27 are shown in Table 1. In the Cypro and Teb groups, body weights were continuously reduced in mice of both genotypes. At week 27, the degrees of body weight reduction in the Cypro groups of WT and CARKO mice were 8% and 15%, respectively, while those in the Teb groups of WT and CARKO mice were about 13% and 18%, respectively. Flu did not affect body weight. Food
Discussion
Our results indicated that CAR involvement in liver hypertrophy depended on the compound used; that is, liver hypertrophy induced by Cypro and Flu was mediated by CAR, while Teb-induced liver hypertrophy was not dependent on CAR. In particular, Flu appeared to require CAR for induction of liver hypertrophy, as almost no changes in liver hypertrophy were observed in CARKO mice treated with Flu. Additionally, marked increases of Cyp2b10 mRNA expression and CYP2B protein in WT mice were observed
Conclusion
In this study, we investigated whether CAR mediated liver hypertrophy and tumor development induced by 3 triazoles in CARKO mice. Our data demonstrated that CAR was the main mediator of liver hypertrophy induced by Cypro and Flu, but was not involved in Teb-induced liver hypertrophy. In contrast, CAR played an important role in liver tumor development induced by all 3 triazoles. These results supported the currently hypothesized MOA that liver hypertrophy is an associative event involved in
Conflict of interest
The authors declare that there are no conflicts of interest.
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Acknowledgements
This research was commissioned under a grant for the 2013 Cabinet Office Research for Assessment of the Effect of Food on Human Health, Japan (Topic No. 1303). We thank Ms. Tomomi Morikawa, Ms. Yoshimi Komatsu, and Ms. Ayako Saikawa for expert technical assistance in performing the animal experiments and processing histological materials.
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