Induction of Nrf2-mediated cellular defenses and alteration of phase I activities as mechanisms of chemoprotective effects of coffee in the liver

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Abstract

Coffee consumption has been associated with a significant decrease in the risk of developing chronic diseases such as Parkinson disease, diabetes type-2 and several types of cancers (e.g. colon, liver). In the present study, a coffee-dependent induction of enzymes involved in xenobiotic detoxification processes was observed in rat liver and primary hepatocytes. In addition, coffee was found to induce the mRNA and protein expression of enzymes involved in cellular antioxidant defenses. These inductions were correlated with the activation of the Nrf2 transcription factor as shown using an ARE-reporter luciferase assay. The induction of detoxifying enzymes GSTs and AKR is compatible with a protection against both genotoxicity and cytotoxicity of aflatoxin B1 (AFB1). This hypothesis was confirmed in in vitro and ex vivo test systems, where coffee reduced both AFB1-DNA and protein adducts. Interestingly, coffee was also found to inhibit cytochrome CYP1A1/2, indicating that other mechanisms different from a stimulation of detoxification may also play a significant role in the chemoprotective effects of coffee. Further investigations in either human liver cell line and primary hepatocytes indicated that the chemoprotective effects of coffee against AFB1 genotoxicity are likely to be of relevance for humans. These data strongly suggest that coffee may protect against the adverse effects of AFB1. In addition, the coffee-mediated stimulation of the Nrf2-ARE pathway resulting in increased endogenous defense mechanisms against electrophilic but also oxidative insults further support that coffee may be associated with a protection against various types of chemical stresses.

Introduction

The diet is believed to play various roles, either beneficial or detrimental, in the aetiology of human cancers. Dietary chemoprotective agents are very diverse in chemical structure and they may act at one or several steps of the carcinogenic process. Like all plant based foods and beverages, coffee is a complex mixture, containing more than a thousand different chemical entities. It may therefore be speculate that several of these compounds may possess biological activities compatible with chemoprotection (Clarke and Vitzthum, 2001, Dorea and da Costa, 2005). The potential relationship between coffee and cancer has attracted considerable attention during the last decades. Overall, there has been no evidence that moderate (3–5 cups a day) coffee consumption represents a significant risk for the development of cancer in humans (Higdon and Frei, 2006, Schilter et al., 2001). In contrast, a number of epidemiological studies provided supportive evidence for a potential coffee-dependent protection against specific forms of cancer in the colon and the liver (Giovannucci, 1998, Larsson and Wolk, 2007, La Vecchia, 2005, Tavani and La Vecchia, 2004).

The International Agency for Cancer Research (IARC) reported a significant inverse association between coffee consumption and the incidence of colon cancer (IARC, 1991). Such a trend was confirmed in a meta-analysis (Giovannucci, 1998) integrating the data of many good quality case-control studies. However, the author of this meta-analysis and of a recent review (Tavani and La Vecchia, 2004) could not definitively conclude on the beneficial effect of coffee on colorectal cancer because of the limited support from large prospective studies (Michels et al., 2005). With regards to liver cancer, several studies including at least two prospective trials showed significant inverse association between coffee consumption and the risk of developing hepatocellular carcinoma (Gelatti et al., 2005, Inoue et al., 2005, Shimazu et al., 2005). This protective effect of coffee was further supported by a recent meta-analysis (Larsson and Wolk, 2007). Support on the potential chemoprotective effect of coffee on various organs is also provided by studies conducted in animal models. Long-term administration of instant coffee to rodents resulted in a reduced incidence of spontaneous tumours at different organ sites (Stalder et al., 1990).

A number of studies performed either in animal models or in culture systems have consistently shown that certain coffee components exert biological effects compatible with chemoprotection. The coffee specific lipidic diterpenes cafestol and kahweol (C+K) were shown to reduce the formation of DNA adducts of several genotoxic carcinogens (Cavin et al., 1998, Cavin et al., 2003, Huber et al., 1997, Majer et al., 2005). C+K have been suggested to act as blocking agents, potentially reducing procarcinogens activation and/or stimulating multiple phase II detoxifying enzymes (Cavin et al., 1998, Cavin et al., 2001, Huber et al., 2002a). In addition to C+K, caffeine and coffee polyphenols (chlorogenic acids, CGAs) have also been considered as good candidates. Several studies provided evidences for the protective effects of CGA against carcinogenesis in different animal models and significant increases in intestinal detoxifying enzyme activities has been found in mice fed CGAs (Matsunaga et al., 2002, Kitts and Wijewickreme, 1994, Feng et al., 2005). Therefore, it is likely that several coffee constituents may be involved in the chemopreventive effects of coffee.

In the present study, we analyzed the pattern of isoform-specific expression of the detoxifying enzymes glutathione S-transferases (GSTs) in rats fed a diet containing coffee. Our findings on GSTs together with the induction of other enzymes and proteins involved in the cellular endogenous antioxidant defenses led to the hypothesis that coffee might induce the Nrf2-mediated gene expression pathway. This was further confirmed using in vitro culture systems in which coffee was shown to induce a wide number of genes containing an antioxidant regulatory element (ARE) in their promoter region. The ARE-motif is recognized by the Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) which is involved in both the basal expression and induction of many genes encoding for detoxification, cytoprotective and antioxidant enzymes (Kensler et al., 2007, Lee and Johnson, 2004). Furthermore, we investigated whether this increased Nrf2 activity was associated with chemoprotective effects by assessing a reduction in the formation of DNA and protein adducts of aflatoxin B1 (AFB1). The involvement of phase I metabolism in the protective effects of coffee against AFB1 was also addressed using human liver THLE cells transfected with specific cytochromes P450 subunits and by determination of CYP450 activity with model substrates. Finally, the species specificity of the protective effects of coffee found in rat hepatocytes were compared with those derived from human livers.

Section snippets

Treatment of animals

Five week old male Sprague–Dawley rats (Iffa Credo S.A., l’Arbresle, France) were used. They were provided with laboratory chow (Nafag 890, Nähr- and Futtermittel A.G., Gossau, Switzerland) and water ad libitum throughout the studies, which were performed under certified good laboratory practices (GLP). Following a few days of acclimatisation on basal diet, the animals were randomly assigned to three treatment groups (A--C). Group A received the control diet. Groups B and C received basal diet

Results

The potential induction of glutathione-S-transferases (GST) and aldo-keto reductase (AKR7A1) by soluble coffee was investigated in rats after 15 days of treatment. Representative results are provided in Fig. 1a. In control animals, little signals specific for GSTs and AKR proteins were detectable. However, dose-dependent increases of GSTA4, GSTP1 and AKR7A1 signals were clearly observed in rats fed coffee at 1% and 5%, whereas for GSTA5 a marked increase was mainly found in rats fed 5% coffee.

Discussion

The majority of studies on the health effects of coffee consumption in humans have focused on potential adverse effects. Concerns about potential health risk of coffee and caffeine raised by human epidemiological studies were observed at high intakes of coffee which are often associated with unhealthy human behaviours such as cigarette smoking, low physical exercise, stress, etc. However, coffee consumption has been associated more recently with a significant decrease in the risk of developing

Acknowledgment

The authors thank the Biological Resources Center of Rennes (CHU Pontchaillou) for providing isolated human hepatocytes.

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