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J Occup Health 2003; 45: 331–334 Journal of Occupational Health Review DNA Microarray Analysis of Altered Gene Expression in Cadmiumexposed Human Cells Shinji KOIZUMI1 and Hirotomo YAMADA2 1 Department of Hazard Assessment and 2Department of Health Effects Research, National Institute of Industrial Health, Japan Abstract: DNA Microarray Analysis of Altered Gene Expression in Cadmium-exposed Human Cells: Shinji K O I Z U M I , et al. Department of Hazard Assessment, National Institute of Industrial Health—Cadmium (Cd) is a heavy metal known to be toxic and carcinogenic, but its mechanism of action remains to be elucidated. Development of the DNA microarray technology has recently made the comprehensive analysis of gene expression possible, and it could be a powerful tool also in toxicological studies. With microarray slides containing 7,000–9,000 genes, we have been studying the gene expression profiles of a human cell line exposed to Cd. By exposure to a non-lethal concentration of Cd, 46 upregulated and 10 downregulated genes whose expression levels changed twofold or greater were observed. The expression of genes related to cellular protection and damage control mechanisms such as those encoding metallothioneins, anti-oxidant proteins and heat shock proteins was simultaneously induced. In addition, altered expression of many genes involved in signaling, metabolism and so on was newly observed. As a whole, a number of genes appear to be coordinately regulated toward survival from Cd toxicity. When cells were exposed to a higher concentration of Cd, more remarkable effects were observed both in the number of affected genes and in the extent of altered expression. These findings will contribute to the understanding of the complicated biological effects of Cd. (J Occup Health 2003; 45: 331–334) Key words: DNA microarray, Cadmium, Gene expression profile, Stress response Known biological effects of Cd Exposures to cadmium (Cd) have occurred both occupationally and environmentally1). This metal is used in such occupations as electroplating and the manufactures of batteries, plastics, paints, alloys and fertilizers. Cd is also generated as a by-product of the mining of lead, copper and zinc. As a most striking example of accidental human exposure, a Japanese population was exposed to Cd through rice contaminated by a runoff from a mine. Chronic exposure to Cd causes disorders in the kidney, calcium metabolism and respiratory system1, 2). Cd has also been reported to be carcinogenic2, 3). It is also known that Cd affects the transcription of a number of genes. Cd induces the human genes with protective functions 4), including those coding for metallothioneins (MTs)5, 6) that chelate Cd ions to make it biologically inert, heat shock proteins (HSPs)7, 8) that play roles in renaturing damaged proteins and hemeoxygenase 19) that plays an anti-oxidative role. Cd also induces protooncogenes10, 11) that might be related to the mechanistic background of carcinogenesis. Its effects on the sex hormone receptor genes12) suggest endocrinedisrupting activity. However, these effects of Cd on gene expression probably represent only a fraction, and there might be a number of unidentified effects on genes relevant to the expression of toxicity and protection against it. By a comprehensive screening of altered gene expression with the new and powerful tool of DNA microarray technology, we have been trying to obtain a general picture of the health effects caused by Cd. DNA Microarray analysis of genes affected by Cd Received Sep 2, 2003; Accepted Sep 24, 2003 Correspondence to: S. Koizumi, Department of Hazard Assessment National Institute of Industrial Health, 21–1, Nagao 6-chome, Tama-ku, Kawasaki 214-8585, Japan First, we examined the genes whose expression is changed after exposure to a low concentration of Cd13). A cultured cell line HeLa S3, that is derived from human cervical carcinoma, was exposed to 5 µM CdSO4 for 6 h. This concentration is higher than the known blood concentrations in occupationally exposed workers2, 14), but did not affect cell viability within the period of 332 J Occup Health, Vol. 45, 2003 Figure. Genes upregulated and downregulated by Cd Genes the expression of which was altered by exposure to 5 µM CdSO4 for 6 h are summarized. Upregulation and downregulation are indicated by upward and downward arrows, respectively. experimental exposure. RNA was prepared from unexposed and Cd-exposed cells by the acid guanidiniumphenol-chloroform (AGPC) method15), and was inspected to be free of degradation by agarose gel electorphoresis followed by ethidium bromide staining, as well as northern blotting with probes specific to a few mRNA species. With these samples, DNA microarray analysis was carried out by Incyte Genomics, Inc. (St. Louis, MO, USA). Messenger RNAs from unexposed and Cdexposed cells were reverse-transcribed into complementary DNAs (cDNAs), that were simultaneously labeled with fluorescent dyes Cy3 and Cy5, respectively. The DNA microarray slide used for the target gene search contained 7,075 spots of human DNA. The mixture of the labeled cDNAs from unexposed and Cd-exposed cells was hybridized with the DNA probes on the array. The fluorescence intensity of the spots was measured for Cy3 and Cy5, and the induction ratio of each gene was calculated based on these signals. For the exposure at a higher concentration of Cd (50 µM), analysis was carried out in the same way with a microarray slide with 9.182 genes. Gene expression altered by a low concentration of Cd The numbers of genes induced and repressed by 5 µM Cd twofold or greater were 46 and 10, respectively, out of the 7,075 genes on the array13). The genes with relatively high fluorescence signals (and therefore considered to be more reliable) are shown in the figure. Some of these genes were functionally related to protection and damage control. Cd also affected genes involved in signaling, which may be relevant to the control of the above and/or other genes. The induction ratio is indicated in parenthesis for each gene mentioned below. (a) Genes relevant to protective functions As expected from previous findings4), several stress response genes were induced13). With regard to MTs, isoform genes including the MT-IL (58.8 x), MT-IE (6.5 x), MT-IB (5.3 x) and MT-III (3.8 x) genes were induced. Since the MT-IL gene is considered to be nonfunctional16), and the expression of the MT-III gene is almost restricted to brain and unaffected by Cd17, 18), the apparent expression of these genes is possibly due to cross-hybridization between isoform sequences closely related to each other. Similar artifacts can often occur in microarray experiments, which must always be carefully inspected. It should also be noted that not all the MT isoform genes were present on the microarray slide used, that is, the absence of certain isoforms in the figure does not always mean that they are not expressed in HeLa cells. It has been suggested that Cd exposure generates reactive oxygen species (ROS) through indirect Shinji KOIZUMI, et al.: Gene Expression Affected by Cadmium mechanisms such as depletion of antioxidant molecules19). Consistent with this, several genes that have anti-oxidative functions20) were upregulated, including those coding for the heavy and light chains of ferritin (protein limiting the availability of Fe for oxygen radical formation; 3.4x and 2.1x, respectively), Mn SOD (Mn superoxide dismutase: enzyme reducing superoxide generated in mitochondria; 2.5x) and γGCS (γ-glutamylcysteine synthetase: key enzyme of glutathione biosynthesis; 2.0x). (b) Genes involved in damage control A series of HSP genes, which code for chaperones and co-chaperones required to renature damaged proteins21–23), were upregulated. These include the genes encoding HSP70–1 (11.6x), HSP70–6 (7.7x), HSP70–5 (7.1x), HSP110α (4.4x), HSP40 (2.8x), Hop (HSP70/HSP90 organizing protein; 2.4x), annexin I (2.1x), HSP90β (2.0x) and HSP60–1 (2.0x). It was found that several genes involved in the ubiquitin system24) were induced: the genes coding for ubiquitin B (signal protein for proteolysis; 2.2x), Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4: ubiquitin-protein ligase; 2.1x), Mif1 (involved in ubiquitin-dependent proteolysis; 2.2x), and Cacy BP (calcyclin binding protein: involved in ubiquitindependent proteolysis; 2.1x). These genes are thought to play a role in the degradation of proteins unable to be renatured. Interestingly, it has been reported that the ubiquitin system is important for resistance to Cd in yeast25). Expression of the ataxin-2 (2.9x), reported to be involved in apotosis26), was also enhanced. (c) Genes involved in signaling We also found that Cd affects a group of genes that code for proteins related to signal transduction13). This includes the genes coding for RXRγ (retinoid X receptor γ : nuclear receptor; 2.4x), IL-1α (interleukin-1 α: cytokine; 2.1x), C-193 (cytokine inducible nuclear protein; 2.1x), importin β2 (nuclear transport receptor; 2.0x), vabl2 (v-abelson murine leukemia oncogene homolog 2 product: homolog of tyrosine kinase; 2.0x) , RAI3 (retinoic acid induced 3: G-protein; 2.0x) and rit42 (reduced in tumor 42kd: stress-inducible protein causing growth arrest; 2.0x) that were upregulated, and NKG2D (neutral killer group 2D: surface receptor of natural killer cells; 0.34x) that was downregulated. These genes may be relevant to the regulation of the Cd-inducible genes identified in this study and/or other Cd-regulated genes (Figure). Although at present it is unclear with what regulatory pathways the signaling proteins are concerned, these findings will be useful for the elucidation of Cd signaling. (d) Other genes In addition, the altered expression of genes that code for several metabolic enzymes and other proteins was 333 detected 13) . These genes were those coding for spermidine/spermine acetyltransferase (key enzyme of polyamine catabolism; 4.4x), keratin 17 (epithelial structural protein; 2.5x), ATA1 (amino acid transporter system A1: transporter of amino acids; 2.3x), 1,4α-glucan branching enzyme 1 (glycogen branching enzyme; 2.3x), bactercidal/permeability-increasing protein (antibacterial, endotoxin-neutralising protein; 2.3x), cytosolic malic enzyme (enzyme producing NADH; 2.2x) and lysine hydroxylase 2 (enzyme catalyzing hydroxylation of Lys in collagen; 2.1x) that were upregulated, and MATIIα (methionine adenosyltransferase IIα: enzyme synthesizing S-adenosylmethionine; 0.37x) that was downregulated. These changes may reflect the detrimental effects of Cd or recovery processes from them. Gene expression profile after exposure to a high concentration of Cd We are also trying to list up genes affected by a higher concentration of Cd. RNA extracted from HeLa cells exposed to 50 µM CdSO4 for 6 h was subjected to DNA microarray analysis as well. Although the outcome is now under inspection and analysis (Yamada and Koizumi, to be published), its outline is as follows. Out of 9,182 genes on a human DNA array, 82 genes were upregulated and 75 genes were downregulated twofold or greater. (i) As compared with the low dose exposure, further activation of genes involved in protection and damage control was observed. The number of induced genes was increased, and many of the genes activated by 5 µM Cd showed higher induction ratios. (ii) Gene expression appeared to be directed toward growth inhibition, protein degradation and apoptosis. (iii) Expression of a lot of genes involved in signaling and transcriptional activation was altered. Seven genes whose expression had been observed to increase in this study were further inspected for their induction by northern blotting. All of these genes were confirmed to be Cd-inducible, demonstrating high reliability of the microarray analysis and data handling. Usually higher induction ratios were obtained in the northern blot analysis. Summary Our studies successfully detected known Cd-inducible genes such as the MT and HSP genes, demonstrating the DNA microarray screening functioned well. In addition, the expression of many genes relevant to recovery from damage and signaling was newly found to be modulated by Cd. It appeared that a number of cellular functions were synchronously directed to survival, to resist the detrimental effects caused by Cd. For many of these changes in gene expression, their biological significance remains ambiguous. However, they are expected to serve as important clues to depict a general picture of Cd 334 J Occup Health, Vol. 45, 2003 response. 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