Alteration of xenobiotic metabolizing enzymes by resveratrol in liver and lung of CD1 mice

Abstract

Conflicting data on the anticancer properties of the polyphenolic natural product resveratrol (RSV) have been reported. Since the inhibition of “bioactivating” Phase-I xenobiotic metabolizing enzymes (XMEs) and/or induction of “detoxifying” Phase-II XMEs have long been considered important cancer chemopreventive strategies, in the current study we investigated the effect of RSV treatment on several Cytochrome P450 (CYP)-dependent oxidations and Phase-II markers in liver and lung subcellular preparations from CD1 male mice. These mice were i.p treated with RSV (25 or 50 mg/Kg b.w.) daily for one or for seven consecutive days. Using either specific probes for different CYPs, or the regio- and stereo-selective metabolism of testosterone, we found that most of the Phase-I XMEs were significantly suppressed (up to ∼61% loss for the CYP3A1/2-linked 6 β-hydroxylation of testosterone in liver and up to ∼97% loss for 2 α-hydroxylase in lung) following RSV treatment for 7 days at 50 mg/kg b.w. Glutathione S-transferase was significantly inhibited, particularly in lung (∼76% loss of activity) after single administration of 25 mg/kg b.w. A different response for the UDP-glucuronosyl transferase was observed, where a significant induction was seen (∼83%) in the liver and a significant reduction was observed in the lung (up to ∼83% loss) following treatment with 25 mg/kg b.w. for seven days. These data indicate that murine XMEs are altered by RSV, and that this alteration is dependent on the RSV dose, duration and way of administration. These results could provide mechanistic explanations for the conflicting chemopreventive results reported for RSV.

Introduction

A large number of natural products are under scrutiny for their therapeutic potential, both in terms of disease prevention and treatment. A remarkable representative of this group of compounds is resveratrol (RSV, 3,4′,5-trihydroxystilbene). RSV is a polyphenolic compound synthesized by a wide variety of plant species, including grapes, and is present in red wine (Pervaiz, 2003). The relatively high concentration of RSV in wine (Siemann and Creasy, 1992), and its documented cardio-protective effect (Sato et al., 2002), formed the basis for the so-called “French paradox” (Kopp, 1998). Subsequently, a large number of in vitro and in vivo studies identified multiple beneficial effects for this compound (Fremont, 2000).

Most of the initial work on RSV was centred on its effects on metabolic pathways regulating cardiovascular biology, such as lipid metabolism and platelet function, atherosclerosis, arthritis or autoimmune disorders. Jang et al. (1997) documented the ability of RSV, in vitro, to inhibit the carcinogenic process at multiple stages. Consequently, several in vivo studies showed that the systemic administration of RSV was associated with inhibition of the initiation and growth of many tumours including breast, prostate, colon, lung, esophagus, liver, non-melanoma skin cancer, leukemia, neuroblastoma and fibrosarcoma, in a wide variety of rodent cancer models (Athar et al., 2007). The efficacy of low doses, for example 200 μg/kg b.w. daily in a rat model of colon carcinogenesis, suggested that the concentration of RSV in dietary sources, such as red wine, could be therapeutic in some cases (Tessitore et al., 2000). At higher, but pharmacologically acceptable doses, the protective effects of RSV were frequently observed. For example, a daily dose of 40 mg/kg b.w. RSV increased the survival of mice with subcutaneous neuroblastomas from 0% to 70% (Chen et al., 2004).

Since the discovery of the cancer chemopreventive properties of RSV in animal models, many investigators directed their attention to understanding the molecular mechanisms underlying its observed biological effects. These included the suppression of cellular proliferation via inhibition of key steps in the signal transduction pathways (Mgbonyebi et al., 1998, Haworth and Avkiran, 2001, Tou and Urbizo, 2001, Pozo-Guisado et al., 2002) and cyclin-dependent kinases (cdks) (Ragione et al., 1998), promotion of cellular differentiation (Mizutani et al., 1998), scavenging/suppression of intracellular reactive oxygen intermediates (ROI) (Manna et al., 2000, Lee and Lee, 2006), induction of apoptotic cell death, both by directly triggering apoptosis-promoting signalling cascades, and by blocking antiapoptotic mechanisms (Fulda and Debatim, 2006, Clement et al., 1998, Huang et al., 1999, Hsieh and Wu, 1999), anti-inflammatory activity via down-regulation of proinflammatory cytokines (Rotondo et al., 1998, Wadsworth and Koop, 1999) and inhibition of androgen receptor function and estrogenic activity (Lu and Serrero, 1999, Mitchell et al., 1999).

Although most in vivo studies seem to support a chemopreventive effect of RSV, there are notable exceptions in which no benefit has been observed. For example, despite promising in vitro results, i.p. administration of 1–5 mg/kg b.w. RSV daily for 23 consecutive days failed to affect the growth or metastasis of breast cancer in mice (Bove et al., 2002). In two 8-weeks-long rat feeding experiments, with 50 and 300 mg/kg b.w. of RSV, no cancer-preventive effects were seen, which was explained to be probably due to the formation of various RSV conjugates which reduced its bioavailability (Wenzel et al., 2005). In N-nitroso-bis(2-oxopropyl)amine treated hamsters, 10 ppm RSV supplemented diet for three weeks failed to reduce pancreatic cancer lesions (Kuroiwa et al., 2006). RSV consumed ad libitum in the diet at doses of 4, 20 or 90 mg/kg b.w., did not modify tumorigenesis in Apc(Min/+) mice (Ziegler et al., 2004). Short RSV treatment (10–100 mg/kg b.w.) of prepubertal female rats affected endocrine functions and, especially, accelerated MNU-induced mammary carcinoma development (Sato et al., 2003). In addition, a recent study indicated that RSV was not likely to be useful in melanoma treatment in athymic mice (Niles et al., 2006).

Xenobiotic metabolizing enzyme (XME) modulation by phytochemicals is believed to be one of the most promising chemopreventive strategies (Talalay, 2000). A substantial body of evidence supports the concept that compounds able to inhibit Phase-I “bioactivating” enzymes and/or induce the Phase-II “detoxifying” enzymes may reduce the formation of reactive intermediates able to interact with DNA, thus lowering the production of genotoxins. In this context, with the aim to evaluate whether the chemopreventive activity of RSV stems from XME modulation, and to better define its metabolic/toxicological profile, the effects of RSV supplementation on microsomal Phase-I and Phase-II murine metabolism were investigated in different tissues of CD1 mice. The potential effect of XME manipulation by RSV in the field of cancer chemoprevention is discussed.