Elsevier

Toxicology Letters

Volume 173, Issue 2, 10 September 2007, Pages 80-87
Toxicology Letters

Protective effects of kahweol and cafestol against hydrogen peroxide-induced oxidative stress and DNA damage

https://doi.org/10.1016/j.toxlet.2007.06.008Get rights and content

Abstract

There is an increasing evidence that oxidative stress is implicated in the processes of inflammation and carcinogenesis. It has been shown that kahweol and cafestol, coffee-specific diterpenes, exhibit chemoprotective effects. This study investigated the effects of kahweol and cafestol, coffee-specific diterpenes, on the hydrogen peroxide (H2O2)-induced oxidative stress and DNA damage in NIH3T3 cells. When the cells were treated with kahweol or cafestol, cytotoxicity, lipid peroxidation, and reactive oxygen species production induced by H2O2 were markedly reduced in a dose-dependent manner. Moreover, kahweol and cafestol were shown to be highly protected against H2O2-induced oxidative DNA damage as determined by the Comet (single cell gel electrophoresis) assay and the measurement of 8-oxoguanine content in NIH3T3 cells. Kahweol and cafestol also protected hydroxyl radical-induced 2-deoxy-d-ribose degradation by ferric ion-nitrilotriacetic acid and H2O2. In addition, kahweol and cafestol efficiently removed the superoxide anion generated from the xanthine/xanthine oxidase system. These results suggest that kahweol and cafestol are effective in protecting against H2O2-induced oxidative stress and DNA damage, probably via scavenging free oxygen radicals, and that kahweol and cafestol act as antioxidants.

Introduction

Oxygen-containing free radicals are considered to be involved in many important biological reactions, such as cancer, inflammation, cardiovascular diseases, and aging (Lee and Lee, 2006). Normally, the reactive oxygen species (ROS) are scavenged by endogenous antioxidants and cellular enzymatic defense mechanisms, mediated by superoxide dismutase, glutathione peroxidase and catalase (Yu, 1994). However, cellular injury can occur when large acute doses of or chronic exposure to toxic substances overpower the cellular antioxidant defense system (Halliwell, 1996). Several harmful reactions such as DNA degradation, membrane peroxidation and the destruction of endothelial cells have been attributed to oxygen-derived free radicals (Brown et al., 1995). DNA damage by ROS can initiate carcinogenesis (Klaunig and Kamendulis, 2004).

It is increasingly being acknowledged that foods contain non-nutritional constituents, which may possess biological activities compatible with beneficial health effects, such as anti-inflammatory and anti-carcinogenic properties (Karihtala and Soini, 2007). Kahweol and cafestol are two diterpenes (Fig. 1) that are present in considerable quantities in coffee beans, as well as in the final, unfiltered beverage, e.g. in Turkish or Scandinavian style coffees (Gross et al., 1997). They have been shown to exhibit both adverse and chemoprotective properties (De Roos et al., 1999, Cavin et al., 2002). It is documented that kahweol and cafestol increase blood cholesterol in both human and animal models (De Roos et al., 1999). However, animal studies have shown that kahweol and cafestol afford protection against the action of well-known carcinogens, such as 7,12-dimethylbenz[a]anthracene, aflatoxin B1, and 2-amino-l-methyl-6-phenylimidazo[4,5-b]-pyridine (Huber et al., 1997, Cavin et al., 2001, Cavin et al., 2002). In line with these observations, there is epidemiological evidence in humans that the consumption of coffee with a high amount of kahweol and cafestol is associated with a lower rate of colon cancer, one of the most frequent cancers in the Western world (Giovannucci, 1998). The chemoprotective effects of kahweol and cafestol have thus far been primarily related to the beneficial modifications of the xenobiotic metabolism. Such effects include the reduced activation of mutagens/carcinogens, e.g. via the inhibition of cytochrome P450 enzymes (Cavin et al., 2001), as well as their enhanced detoxification, e.g. via the induction of carcinogen-detoxifying enzyme systems (Cavin et al., 2002, Huber et al., 2002). Recently, we reported that kahweol and cafestol exhibit anti-inflammatory properties against the LPS-induced activation of macrophages. These anti-inflammatory effects, at least in part, are associated with the suppression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) gene expression through NF-κB inhibition (Kim et al., 2004a, Kim et al., 2004b, Kim et al., 2006). Excessive prostaglandin E2 and NO production by COX-2 and iNOS, respectively, play a key role in a variety of pathophysiological processes including various forms of inflammation and carcinogenesis (Prescott and Fitzpatrick, 2000, Lala and Chakraborty, 2001). It is well established that ROS are regarded as having carcinogenic potential and antioxidants can block the process of carcinogenesis (Lee and Lee, 2006, Karihtala and Soini, 2007). Newer therapies employing antioxidants for the chemoprevention of inflammatory disease and cancer is urgently needed. However, the mechanisms by which kahweol and cafestol protect against H2O2-induced cytotoxicity are not fully understood. The present study, therefore, was undertaken to determine whether kahweol and cafestol as antioxidative agents can protect against cytotoxic and nuclear DNA damage following H2O2-induced oxidative stress and to assess their ability to scavenge superoxide radicals.

Section snippets

Materials

Chemicals and cell culture materials were obtained from the following sources: kahweol acetate, cafestol acetate, nitrilotriacetic acid, thiobarbituric acid, 1,1,3,3-tetramethoxypropane, xanthine, xanthine oxidase, and 2deoxy-d-ribose from Sigma–Aldrich Chemical Co.; MTT-based colorimetric assay kit and lactate dehydrogenase (LDH) diagnostic kits from Roche Co.; the “Biotrin OxyDNA Assay” kits for the detection of oxidative DNA damage from Biotrin International Ltd.; LumiMax™ Superoxide Anion

Effects of kahweol and cafestol on H2O2-induced cytotoxicity

The protective effects of kahweol and cafestol against H2O2-induced cytotoxic injury of the NIH3T3 cells were quantified by MTT and LDH assay. Kahweol and cafestol, which are non-toxic even at a high concentration (10 μM), afforded protection from cell injury. To more precisely quantify the protective effects of kahweol and cafestol against H2O2, we also performed LDH assays, and the results were consistent with those obtained from the MTT assay. Addition of kahweol and cafestol to the cells

Discussion

H2O2 is particularly attractive as a model oxidant because its cellular actions and its fate have been well studied (Halliwell et al., 2000). H2O2 readily crosses the cellular membranes, and gives rise to the highly reactive hydroxyl radical, which has the ability to react with macromolecules, including DNA, proteins, and lipids, and to ultimately damage a cell (Halliwell et al., 2000). Several previous studies have been carried out using hydrogen peroxide as the oxidant to screen for

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