Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus

Abstract

A heat-labile protein has been identified in fruit bodies of the edible mushroom, Agaricus bisporus, which protects Raji cells (a human lymphoma cell line) against H₂O₂-induced oxidative damage to cellular DNA. This protein has been purified following salt fractionation, combined with ion-exchange, hydrophobic interaction and adsorption chromatography. Based on catalytic and electrophoretic properties, and inhibition studies using tropolone, the protein was identified as tyrosinase. The genoprotective effect of A. bisporus tyrosinase, determined using the single-cell gel electrophoresis (“Comet”) assay, has been shown to be dependent upon the enzymic hydroxylation of tyrosine to L-DOPA and subsequent conversion of this metabolite to dopaquinone. The possible role of dopaquinone, and other L-DOPA oxidation products, in enhancing the cellular antioxidant defence mechanisms is discussed.

Introduction

Low levels of reactive oxygen species (ROS) serve useful roles in signal transduction and in the modulation of gene expression but are extremely harmful to DNA and other biological macromolecules at higher concentrations [1], [2], [3]. During the past two decades, the mechanisms involved in ROS-induced damage to DNA have been studied extensively since such damage is related to the etiology of cancer and various age-related diseases [4], [5], [6]. Injury to DNA is caused by the accumulation of ROS resulting from overproduction and/or inefficient intracellular antioxidant defence systems [7].

Efforts to develop dietary supplements and preventative treatments for offsetting the detrimental effects of ROS have focused on identifying antioxidants from a variety of natural products. Traditionally, fruits, vegetables and green tea are seen as rich sources of natural low-molecular weight antioxidants including ascorbic acid, α-tocopherol, carotenoids, flavonoids and polyphenols. More recently, mushrooms have been investigated as a potential source of natural antioxidants. This special group of fungi has long been acknowledged in Eastern cultures as possessing a wide range of medicinal properties [8], and modern techniques have identified numerous bioactive mushroom components which are variously reported to exhibit anti-cancer, anti-tumour, anti-viral, immunomodulatory, hypocholesterolaemic and hepatoprotective activities [9], [10], [11], [12], [13], [14]. We have shown that several mushroom species also display antioxidant power in the Ferric Reducing Antioxidant Power (FRAP) assay [15], and polysaccharoproteins from several mushroom species were reported to scavenge active oxygen species [16]. A thermostable antioxidant protein isolated from the Shiitake mushroom, Lentinula edodes, also suppressed the oxidative degradation of 2-deoxyribose and ΦX174 DNA by hydroxyl radical [17].

More recently, we have used the single cell gel electrophoresis method, also called the “comet assay” [18], [19], to screen extracts of the fruit bodies of eight mushroom species for ability to protect against oxidative damage to cellular DNA [20]. Both cold water extracts of Agaricus bisporus and hot water (100 °C) extracts of Ganoderma lucidum protected against H₂O_2-induced damage to cellular DNA in an in vitro cell culture system. Cold-water extracts of A. bisporus also protected the DNA of lymphocytes taken from rats administered these extracts by intraperitoneal injection [20]. The nature of the genoprotective effect was not identified but did not involve direct neutralisation of the oxidant. In this paper, we show for the first time that the genoprotective effect exhibited by A. bisporus is associated with the enzymic generation and further conversion of L-DOPA, and that this is catalysed by a heat-labile tyrosinase which was isolated from fruit bodies of the mushroom.