DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae Daisuke Yasokawa, 1, ⁎ Satomi Murata, 2 Yumiko Iwahashi, 3 Emiko Kitagawa, 2 Katsuyuki Kishi, 4 Yukihiro Okumura, 1 and Hitoshi Iwahashi 2 Hokkaido Food Processing Research Center, Department of Food Biotechnology, 589-4 Bunkyodai Midorimachi, Ebetsu, Hokkaido, 0690836, Japan 1 Health Technology Research Center (HTRC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba-Nishi 3A, 16-1, Onogawa, Tsukuba, Ibaraki 3058569, Japan 2 National Food Research Institute (NFRI), National Food Research Institute National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, Ibaraki 3058642, Japan 3 and Japan Pulp and Paper Research Institute, Inc., Tokodai 5-13-11, Tsukuba, Ibaraki, 3002635, Japan 4 Received 4 September 2009; accepted 30 October 2009 Available online 22 November 2009 Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation. © 2009, The Society for Biotechnology, Japan. All rights reserved. [Key words: Zinc pyrithione; DNA microarray; Saccharomyces cerevisiae; Iron; Toxicity; Cytochrome] 1-Hydroxy-2-pyridinethione, or 2-mercaptopyridine N-oxide, also known as pyrithione (PT) under the trade name Omadine, is well known for its biocidal effect. PT is a lipophilic chelator, and its metal complex, zinc pyrithione (Zpt), has been used in anti-dandruff shampoos (1). It is also used as an anti-fouling paint, after The International Convention on the Control of Harmful Anti-fouling Systems on Ships banned organic tin (2, 3). Some studies have been carried out on the physiological and biochemical effects of PT. It interferes with thymidine uptake (4), and is suggested to be an anti- metabolite for vitamin B6 or nicotinic acid (5). Chandler and Segal (6) presented a hypothesis based on the finding that low concentrations of PT inhibited plasma membrane transport and promoted a decrease in cellular ATP levels and protein synthesis. PT was also reported to induce electrical depolarization of the membrane through inhibition of the proton pump (7). In that report, the effect of a range of Zn 2+ concentrations (1 to 5 mM) on the membrane potential was also tested. However, no effect on the membrane potential was observed. Recently, PT has been used as a zinc ionophore to study zinc homeostasis in neurons in relation to Alzheimer's dementia (8). Furthermore, Zpt was found to inhibit cell growth strongly (9, 10). Dinning et al. (11) reported that both PT and Zpt reduced intracellular ATP levels in gram-negative bacteria, and that they are active against the cell membrane. However, its mode of action has not yet been elucidated clearly. Saccharomyces cerevisiae is one of the most studied eukaryotic organisms, possessing a variety of advantageous characteristics as a model organism for bioassays. These include its relatively short life cycle, inexpensive media requirements and ease of handling. Recently, yeast microarrays have been used to monitor gene expression levels as a function of toxin exposure and as a means of determining the mechanisms of toxicity. In this study, we exposed S. cerevisiae to sub-lethal concentrations of Zpt and analyzed the resultant genome-wide mRNA expression profiles. The results indicated that the yeast exhibited severe iron starvation and that Zpt appeared to act as a strong iron chelator or ionophore. This iron starvation caused heme depletion and may lead to decreased levels of ATP. MATERIALS AND METHODS Strains, growth conditions and chemicals The S. cerevisiae strain S288C (Mat alpha SUC2 mal mel gal2 CUP1) was grown as a pre-culture in YPD medium (2% polypeptone, 1% yeast extract, 2% glucose) at 25 °C for 2–3 days. This strain was chosen because the DNA microarray probes were produced using S288C as the template for the PCR reaction. The deletion strains, Δrad50, Δrad51 and Δrad52, which are derived from BY4743 (MATa/MATα his3Δ/his3Δ leu2Δ0/leu2Δ0 ura3Δ0/ ura3Δ0 met15Δ0/MET15 lys2Δ0/LYS2), were purchased from Invitrogen (Carlsbad, CA, USA). Zinc pyrithione was purchased from Wako Pure Chemical Industries (Osaka, Japan). It has been reported that Zpt is degraded rapidly by aqueous photolysis (12–14). Although normal laboratory light did not have a great influence on the result of the acute toxicity bioassay (15), we limited the exposure of the Zpt and Zpt-containing samples to light throughout these experiments as much as possible. Journal of Bioscience and Bioengineering VOL. 109 No. 5, 479 – 486, 2010 www.elsevier.com/locate/jbiosc ⁎ Corresponding author. Tel.: +81 11 387 4118; fax: +81 11 387 4664. E-mail address: duke@foodhokkaido.gr.jp (D. Yasokawa). 1389-1723/$ - see front matter © 2009, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2009.10.025