Environ. Sci. Technol. 2007, 41, 7941-7946
Combined Cadmium and Thiuram Show Synergistic Toxicity and Induce Mitochondrial Petite Mutants
assay, previously discussed only in theory, has entered into a stage of experimental possibility. Here we demonstrate the utility of a transcriptome bioassay system to assess the synergic effect of mixed chemicals. Synergistic toxicities had been limited to phenomenological evaluation and have now been a target of transcriptome bioassay.
HITOSHI IWAHASHI,* EMI ISHIDOU, EMIKO KITAGAWA, AND YUKO MOMOSE Human Stress Signal Research Center, National Institute of Advanced Industrial Science and Technology, Onogawa 16-1, Tsukuba, Ibaraki 305-8569, Japan
Experimental Section
A bioassay is a system for monitoring toxicity of chemicals in the environment via the biological responses of experimental organisms. These responses can be detected by analysis of genome-wide changes in mRNA expression levels using DNA microarrays. We applied this system for evaluation of synergistic toxicity by cadmium and thiuram, as this combination showed mutual growth inhibition in yeast. Hierarchical cluster analysis using mRNA expression profiles suggested the response of yeast to this combination is similar to that seen with cadmium treatment alone. Functional characterization of genes induced by this combinational treatment also suggests that the toxicity of cadmium was enhanced. This toxicity was observed as the damage to mitochondrial functions, which were not observed with either cadmium or thiuram treatments alone. The potential toxicity to mitochondria by this combinational treatment was confirmed as the result of deletion of mitochondria with petite colony formation frequency. We could evaluate synergistic toxicity by cadmium and thiuram and show the possible use of transcriptome bioassay for synergistic toxicity.
Introduction At present, more than 22 million materials are registered in the Chemical Abstracts Service database (http://www.cas.org/), and it is estimated that more than 10 000 synthetic compounds are accumulating in our environment (1). Industrial chemicals have given us numerous benefits; however; there is no doubt that they have also damaged the environment and consequently human health. Dispersal of chemical compounds on the earth should be carefully controlled. While many chemicals can be detected in environmental samples, only 10% of these chemicals can be identified by current technologies (1). An inability to detect dioxins in the environment, for example, cannot ensure the safety of the environment. Bioassays, systems for monitoring toxicity in the environment via the biological response of an experimental organism after exposure to environmental samples, are starting to play a role in environmental screening and control (1). Among bioassay systems, transcriptome bioassays have been a focus of constant attention (2). In transcriptome bioassays, biological responses are monitored by analysis of genome-wide changes in mRNA expression levels detected using DNA microarrays (3). With the establishment of microarray technology, the transcriptome bio* Corresponding author phone +81-29-861-8508; fax+81-29-8618508; e-mail:
[email protected]. 10.1021/es071313y CCC: $37.00 Published on Web 10/06/2007
2007 American Chemical Society
Strain and Culture Conditions. Saccharomyces cerevisiae S288C (MATapha SUC2 mal mel gal2 CUP1) was grown in YPD medium (1% Bacto yeast extract, 2% polypeptone, 2% glucose) at 25 °C according to procedure outlined by Kitagawa et al. (4). The concentration of chemicals causing growth inhibition was determined by exposing the yeast cells to various amounts of chemicals. For monitoring yeast cell growth, absorbance at 650 nm of cultures in 96 well microplates was measured after 12 h incubation at 25 °C. Increase in culture density of cells treated with chemical was compared to the density increase of an untreated control culture (100% growth) to calculate the effect of each treatment. For the combination treatment, yeast cultures in YPD medium (A660 ) 1.0) were incubated with 10 µM cadmium and 2.5 µM thiuram for 2 h. At 10 µM cadmium and 2.5 µM thiuram, concentrations corresponding to 1/30 of the individual IC50s, the cadmium:thiuram (4:1) IC50 was achieved. To estimate petite colony formation efficiency, we cultured yeast cells for 20 generations with chemicals. After that, cells were grown on YPG plates (1% yeast extract, 2% polypeptone, 2% glycerol, 0.1% glucose, and 2% agar), and the frequency of petite colony formation was determined by counting normal and petite colonies. DNA Microarray Analysis. Each microarray, spotted on a glass slide for hybridization with labeled mRNA probes, represented almost all ORFs of yeast (5809-5819 genes, depending on the lot of microarray slides). Total RNA was extracted by the hot-phenol method, and poly (A) +RNA was purified from approximately 400 µg total RNA using the Oligotex-dT30 mRNA purification kit (TaKaRa, Otsu, Japan) (4). For both the untreated control and chemical-treated samples, a minimum of 4 µg mRNA was labeled with Cy3 or Cy5, respectively (4). Hybridization was carried out at 65 °C for 24-48 h. A Scan Array 4000 laser scanner (GSI Lumonics, Billeria, MA) was used to acquire hybridization signals. Array images were analyzed with Gene Pix 4000 (Inter Medical, Nagoya, Japan) (4). We did at least three times independent experiments for each condition. Thus, the data are based on biological and technical replication. The data obtained in this research were deposited to GEO (Gene Expression Omnibus) (http://www.ncbi.nih.gov/projects/geo) with the accretion number of GSE8718. Cluster Analysis of the mRNA Expression Profiles after Combinational Treatment. Hierarchical cluster analysis was performed with the GeneSpring ver. 7.3.1 software (Silicon Genetics, CA) (5). The clustering algorithm arranges conditions according to their similarity in expression profiles across all the conditions, such that conditions with similar patterns are clustered together as in a taxonomic tree. The setting for the calculation was as follows: The similarity was measured by “correlation”, the separation ratio was 1.0, the minimum distance was 0.001, and 3800 ORFs were used for the calculation. These 3800 ORFs were selected on the basis of previously showing higher than average intensities in other trials (6, 7). This selection is simply to remove low-intensity untrustworthy data (6, 7). VOL. 41, NO. 22, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Percent Growth of Yeast Cells under the Presence of Mixed Chemicals concentration of Cd (µM)
thiuram (µM) 0 0.27 0.55 1.1 2.2 4.4 8.8 18 35 TPN (µM) 0 27 55 110 220 440 880 1800 a
0
0.57
1.1
2.3
4.5
9.1
18
100 100 98 97 97 91 79 71 37
100 97 100 88 86 75 71 53 NG
97 94 94 88 75 64 60 51 NG
94 88 91 77 61 55 53 45 NG
81 81 75 64 54 51 46 37 NG
71 67 58 52 51 46 37 NG NG
63 58 52 46 44 37 NGa NG NG
100 100 97 94 97 88 73 38
100 97 97 94 91 86 70 40
97 97 100 91 91 83 70 40
94 94 94 91 88 88 68 39
81 83 81 79 75 75 64 36
71 72 70 67 67 65 49 NGa
63 63 59 58 52 51 33 NG
NG; No growth was observed.
Reverse Transcriptase Polymerase Chain Reaction (RTPCR). RT-PCR was carried out to confirm the result from microarray experiments. The reaction was performed using the StrataScript First-Strand Synthesis System (Stratagene, CA). PCR reaction was carried out by TaKaRa Ex Taq HS (TaKaRa, Shiga, Japan) with the diluted cDNA solution. Temperature and cycles conditions were as follows: 94 °C for 5 min, 25 or 28 cycle of 98 °C 10 s, 55 °C 30 s, and 72 °C 30 s, and 72 °C for 10 min. Quantification of RT-PCR was using ATTO densitograph system (ATTO, Tokyo, Japan). Gene name (systematic name), forward primer sequences, and reverse primer sequences are as follows: GTT2 (YLL060C) 5′-TCTCAATGGCTGACATCACAGTA-3′ and 5′-CCTCTTGAATAGCTTCATGATTGG-3′, YNL200c 5′-CCTCAATTAATCCTGCTGTTCTTGT-3′ and 5′GTGGCATTATAGATTTGGGCTATCA-3′, YSA1 (YBR111c) 5′-CGTTGAGTTGCACAAGTTTCCT-3′ and 5′-CATAAATGGGACACTATCTTGTTGC-3′, MSC1 (YML128C) 5′-TTAAACAGACTTCTACGAAGGACGA3′ and 5′-TGCTATTAGCTTGTCCATTTTACGG-3′, CAF17 (YJR122W) 5′-CATTTCTCTGCTGCCTTCTCATCT3′ and 5′-GAGAACTCAAGGCTGTTCATTGTTT-3′, YPR098c 5′-TCTTTCTAATGTTTGCGGGATG-3′ and 5′TTTAGCCAAGATCAGGATAGCAGA-3′, ACT1 (YFL039C) 5′- ATTGCCGAAAGAATGCAAAAGG-3′ and 5′- CGCACAAAAGCAGAGATTAGAAACA-3′.
Results Synergistic Effect of Mixed Chemicals. Six compounds were selected for these trials: cadmium chloride (3), capsaicin (8), [bis(dimethyldithiocarbamoyl) disulfide] (thiuram) (9), tetrachloroisophthalonitrile (TPN, or chlorothalonil) (9), sodium arsenite (6), and sodium dodecyl sulfate (SDS) (7). The effects of each of these chemicals on mRNA expression profiles have previously been characterized using yeast DNA microarrays. Among 15 different combinations of compounds tested for their effect on yeast growth, the effect of 14 combinations was additive relative to the effect of each chemical alone. For example, cadmium or TPN alone reduced the growth of yeast cultures to 81% (4.5 µM) or 73% (880 µM) of wild-type levels, respectively. Yeast growth in the presence of both cadmium and TPN (4.5 µM and 880 µM) was 64% of untreated controls, which is an additive effect (Table 1). Only one combination of chemicals, cadmium and thiuram, 7942
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showed a synergistic (greater than additive) reduction in yeast growth: Thiuram (8.8 µM) slowed growth to 79%, whereas cadmium and thiuram together reduced growth to 46% of the control (Table 1). This value is lower than the additive effect of the individual compounds. The synergistic effect of these compounds on growth reduction led us to select this combination for testing the transcriptome bioassay system. In a previous report, while characterizing the effects of cadmium and thiuram individually, we cultured yeast cells with 300 µM cadmium (3) or 75 µM thiuram for 2 h (9). These concentrations corresponded to the IC50 values for yeast cells. To examine their combined effects, we mixed cadmium and thiuram at a ratio of 4:1 (the ratio of individual IC50s) and measured the toxicity of increasing amounts of the dual mixed compound on yeast cells. At 10 µM cadmium and 2.5 µM thiuram, concentrations corresponding to 1/30 of the individual IC50s, the cadmium:thiuram (4:1) IC50 was achieved. We then characterized gene expression profiles at this toxicity concentration using yeast DNA microarrays (3). Characterization of Expression Profiles by the Combination of Cadmium and Thiuram. The mRNA expression profiles were obtained as described previously (3), after treatment with 10 µM cadmium and 2.5 µM thiuram for 2 h at 25 °C. This profile was compared by hierarchical cluster analysis with the expression profiles obtained with other chemicals, including different doses of cadmium and thiuram treatments (http://kasumi.nibh.go.jp/∼iwahashi/) (Figure 1). Hierarchical cluster analysis was performed using the GeneSpring ver. 7.3.1 software (6). The combination treatment data clustered with two kinds of cadmium treatments (Figure 1) but not with thiuram treatment alone. This suggests that the effect of the cadmium:thiuram combination on gene expression was closer to cadmium treatment alone, and that the addition of thiuram may enhance the effect of cadmium. To investigate the mechanism of toxicity by 10 µM cadmium:2.5 µM thiuram treatment, we selected 39 induced genes that have at least 2-fold higher intensity with t-test P-value less than 0.05 in the cadmium:thiuram treatment relative to the control. As the first step, we compared these 39 genes with the genes induced by 20 µM cadmium treatment (351 genes) and with genes from 5.0 µM thiuram treatment (28 genes) using the functional categories according to MIPS (Munich Information Center for Protein Sequences) (http://mips.gsf.de/genre/proj/yeast/index.jsp).
FIGURE 1. Comparison of gene expression profiles in cadmium:thiumam-treated cells with those in cadmium-, thiuram-, and other individual stress-treated cells. The expression profiles in the combination treatment were compared with those by other stress conditions (see http://kasumi.nibh.jp/∼iwahashi/). The hierarchical clustering was performed using GeneSpring. The samples exposed to chemical stresses were treated with the conditions shown in this figure for 2 h.
TABLE 2. Localization of Gene Products Induced by the Cadmium:Thiuran Combination: Cadmium and Thiuram Treatments combination
c
20 µM cadmium
10 µM thiuram
localization
total count
counta
(%)b
impactc
count
(%)
impact
count
(%)
impact
extracellular bud cell wall cell periphery plasma membrane integral membrane/endomembranes cytoplasm cytoskeleton ER golgi transport vesicles nucleus mitochondria peroxisome endosome vacuole microsomes lipid particles punctate composite ambiguous known localization unknown localization total
54 149 42 216 186 176 2906 204 557 132 139 2157 1056 52 57 280 5 27 141 237 5209 1516 6725
0 0 0 1 2 2 28 0 6 0 0 16 14 0 0 2 0 1 1 1 37 2 39
0.0 0.0 0.0 0.5 1.1 1.1 1.0 0.0 1.1 0.0 0.0 0.7 1.3 0.0 0.0 0.7 0.0 3.7 0.7 0.4 0.7 0.1
0.0 0.0 0.0 2.4 4.9 4.9 68.3 0.0 14.6 0.0 0.0 39.0 34.1 0.0 0.0 4.9 0.0 2.4 2.4 2.4 90.2 4.9
5 2 4 12 16 18 184 1 47 4 3 92 57 4 3 27 0 3 10 15 289 62 351
9.3 1.3 9.5 5.6 8.6 10.2 6.3 0.5 8.4 3.0 2.2 4.3 5.4 7.7 5.3 9.6 0.0 11.1 7.1 6.3 5.5 4.1
1.4 0.6 1.1 3.4 4.5 5.1 51.8 0.3 13.2 1.1 0.8 25.9 16.1 1.1 0.8 7.6 0.0 0.8 2.8 4.2 81.4 17.5
0 1 0 0 1 0 12 0 2 0 0 10 4 1 0 1 0 0 0 2 19 9 28
0.0 0.7 0.0 0.0 0.5 0.0 0.4 0.0 0.4 0.0 0.0 0.5 0.4 1.9 0.0 0.4 0.0 0.0 0.0 0.8 0.4 0.6
0.0 3.4 0.0 0.0 3.4 0.0 41.4 0.0 6.9 0.0 0.0 34.5 13.8 3.4 0.0 3.4 0.0 0.0 0.0 6.9 65.5 31.0
a Count; Number of genes selected in the category. b %,: (number of induced genes in the category/number of genes in the category) × 100. Impact (number of induced genes in the category/number of genes slected as induced genes) × 100.
The results show that the selected genes were scattered and were not concentrated in specific functional categories (data not shown). At the next step, we characterized localization of the products encoded by the induced genes according to MIPS localization information (Table 2). The highly localized organelles were nucleus (impact of 39.0) and mitochondria (34.1) in addition to cytoplasm (68.3). This trend was also observed with 20 µM cadmium (nucleus; 25.9, mitochondria;
16.1, cytoplasm; 51.8) and 5.0 µM thiuram (34.5, 13.8, 41.4). Interestingly, the impacts ((number of induced genes in the category/number of genes slected as induced genes) × 100) of these localizations were higher with the cadmium: thiuram combination, especially to the mitochondria. This result suggests that the effect to mitochondria, nucleus, and cytoplasm are increased by cadmium:thiuram combination and that mitochondrial function is significantly affected. VOL. 41, NO. 22, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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a
YSA1 CAF17
ADH6
ATG22
FET5
MSC1
P-value; P-values from t-test were modified by multiple testing correction by “Benjamini and Hochberg FalseDiscovery Rate”.
glutathione S-transferase chaperone and cysteine protease methylglyoxal reductase (NADPH-dependent) high affinity iron transporter questionable protein protein involved in directing meiotic recombination putative alcohol dehydrogenase multicopy oxidase protein of unknown function breakdown of autophagic vesicles inside the vacuole similarity to R.capsulatus halidohydrolase NADPH-dependent alcohol dehydrogenase protein of unknown function sugar-nucleotide hydrolase CCR4-associated factor mitocho ndria cytoplas m cytoplas m nucleus plasma membra ne unknown ER mitochondria cytoplasm nucleus vacuole mitochondria integral membrane vacuole cytoplasm cytoplasm cytoplasm nucleus cytoplasm mitochondria nucleus mitochondria GTT2 HSP31 GRE2 FTR1
5.1 3.1 3.2 1.7 2.0 2.4 2.3 1.7 1.6 1.8 1.0 1.2 1.7 1.4 1.7 0.7 1.3 0.8 0.9 2.0 1.2 1.0 0.8 1.2 1.1 1.8 0.6 1.1 1.5 1.4 8.2 6.4 5.8 3.5 3.2 2.9 2.6 2.6 2.5 2.2 2.2 2.1 2.1 2.1 2.0 YLL060c YDR533c YOL151w YER145c YOL150c YML128c YNL134c YFL041w YNL200c YCL038c YNR064c YMR318c YDR391c YBR111c YJR122w
0.046 0.046 0.033 0.045 0.048 0.044 0.047 0.048 0.040 0.046 0.045 0.048 0.046 0.045 0.044
DESCRIPTION from MIPS localization common name 20 µM Cd
P-valuea
induction fold
5 µM thiuram
induction fold
10 µM Cd-2.5 µM thiuram
induction fold
Cadmium, a worldwide contaminant, is especially known in Japan for its role in Itai-Itai disease (13) and its uptake in rice (13). It is generally accepted that cadmium in humans leads to nephrotoxicity (14), hepatotoxicity (15), serious damage to the nervous system (16), and a high frequency of chromatid aberrations (17). Thiuram is a widely used fungicide that protects against diseases in vegetables, crops, seeds, and in paddy fields (4). Using the Ames test with Salmonella typhimurium TA100 and TA98, thiuram showed direct mutagenicity and toxicity (18, 19). In Japan, concentrations of cadmium and thiuram in effluents are controlled by law to levels below 0.1 ppm for
systematic name
Discussion
TABLE 3. Extraction of Specifically Induced Genes by the the Cadmium:Thiuran Combination
Finally, we tried to further characterize the genes induced by cadmium:thiuram combination compared to cadmium or thiuram treatment alone. Table 3 list the genes that were selected as the genes induced by cadmium:thiuram combination showing higher fold change values over the cadmium and thiuram. For making the list, we employed multiple testing correction of Benjamini and Hochberg False Discovery Rate. The most highly induced gene was GTT2 followed by HSP31, GRE2, and FTR1. These genes were also highly induced by 20 µM cadmium treatment. This is agreement with the cluster analysis that suggested the similar toxicity of the combination treatment with cadmium treatment alone. GTT2 is the gene encoding a glutathione S-transferase protein (10). This enzyme is localized in mitochondria and has roles in conjugating glutathione to toxic substances (10, 11). This suggests the accumulation of toxic substances in the mitochondria. Furthermore, among these 15 genes there were 5 other genes whose products were localized in mitochondria. We tried to confirm the investigated expression of these genes by RT-PCR (Figure 2). We could observe the induction of these genes by the combinational treatment, and we confirmed their higher induction over cadmium and thiuram alone, except for the YSA1 gene. YPR098c was selected as the positive control because this gene was induced more than 2-fold in all the treatments of the combination (2.1 fold), cadmium (2.5 fold), and thiuram (2.4 fold) treatments. ACT1 was selected as the stable gene because this gene was widely accepted for housekeeping genes, and the expression levels were one of the stable genes (within 10th place) in our experiments. ACT1 showed the average fold of 1.05 and the SD value of 0.22. The Combination of Cadmium and Thiuram-Induced Petit Colony Formation. It is well-known that S. cerevisiae often loses mitochondria and forms petite colonies after treatment with chemicals that affect mitochondrial DNA or functions (12). Petite colonies form because of a deletion of mitochondrial DNA, and these deletion mutants can be distinguished by growth on a nonfermentable carbon source. To measure the effect of cadmium and thiuram on deletion of mitochondrial DNA, we compared petite colony formation efficiency after treatment with 20 µM cadmium, 5 µM thiuram or 10 µM cadmium, and 2.5 µM thiuram for 20 generations. Following treatment, cells were grown on YPG plates, and the frequency of petite colony formation was determined by counting (Figure 3). Generally, petite colonies form spontaneously at a low frequency. The control, cadmium treatment, and thiuram treatment showed less than 5% petite colony formation efficiency, with the cadmium treatment showing a slightly higher efficiency. As suggested by the previous analyses, only the combination treatment showed a significant increase in the efficiency of petite colony formation (to 65%), again indicating the specific effect of the cadmium:thiuram combination on mitochondrial functions. Our data strongly suggest that some fraction of the synergistic toxicity by the combination of cadmium and thiuram occurs in the mitochondria.
FIGURE 2. A reverse transcriptase-polymerase chain reaction (RTPCR) was carried out to confirm the result from microarray experiments. Genes which were up-regulated by the combinational treatment of Cd and thiuram, and whose gene products are known to be localized in mitochondrion, were selected. In the right table, relative induction values by RT-PCR (DNA microarray) were demonstrated.
in the cell is possible. The chelated cadmium can be more readily transported to mitochondria than cadmium alone (22). Interestingly, mitochondrial DNA deletions were reported to occur in proximal tube cells (23). It was suggested that in cadmium poisoning, this was the primary event for the pathogenesis of osteomalacia in Itai-Itai disease (23). At present there is no evidence that thiuram or dithiocarbamate pollution was present in the region of Itai-Itai disease cases; however, we cannot yet discount the possibility of a combination effect that could enhance cadmium toxicity. It should be noted that rice is widely polluted by cadmium in Japan (13) and that thiuram was used as fungicide to prevent rice blast (4). They had chance to be found in paddy fields. The combination effects of chemical pollutants are the focus of great attention, as described in reports of the synergistic effects of chemical mixtures (24). While these findings contribute to our understanding of environmental control and safety, these reports limit the effect of toxin combinations to measurements of growth or individual enzyme activity (24). In reality, the results of these carefully designed experiments are far from simulating the complex mechanisms of synergic effects of chemical combinations on cellular changes in gene expression. By utilizing a transcriptome bioassay, we determined that one of the probable causes for the synergic effect of cadmium and thiuram toxicity is a broad impairment of mitochondrial function. Our findings experimentally demonstrate the utility of the transcriptome bioassay for the evaluation of synergistic toxicity by chemicals.
Acknowledgments Authors thank Dr. Randeep Rakwal for improving the manuscript.
Literature Cited
FIGURE 3. Petite colony formation efficiency after treatment with cadmium, thiuram, and their combinations. Yeast cells were grown for 20 generations under the conditions indicated. The petite colonies were counted on YPG agar plates. The efficiency was shown by mean values with standard deviations from three independent experiments. cadmium and 0.06 ppm for thiuram (1). We applied these concentrations to yeast to assess the combination effect, but we did not observe increased efficiency of petite colony formation (Figure 3), suggesting that the concentrations of cadmium and thiuram less than those values in effluent may not result in deletion of mitochondria of yeast cells. The combination effect of cadmium and thiuram is not the first example of synergy. Danielsson (20) showed that dithiocarbamates including thiuram increased the concentration of both Cd and Hg in brain and most other maternal organs. Especially thiuram was suggested to strongly increase Cd concentrations in whole fetuses (around 17-fold at 4 h) and all fetal organs measured. Mutoh et al. (21) showed that dithiocarbamate induces the synthesis of cadystins, a family of heavy metal chelating isopeptides in the fission yeast Schizosaccharomyces pombe. Kamenosono et al. showed that dithiocarbamates, which can be applied for removing cadmium from the kidney, function as chelating agents for cadmium. Chelated cadmium then exits the kidney through organic anion transport and active transport systems (22). So far, we have no evidence whether dithiocarbamate directly or indirectly chelates cadmium, but the chelating of cadmium
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Received for review June 4, 2007. Revised manuscript received August 28, 2007. Accepted September 4, 2007. ES071313Y
