Toxicology and Applied Pharmacology 256 (2011) 1–7 Contents lists available at ScienceDirect Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p The potential effect of metallothionein 2A − 5 A/G single nucleotide polymorphism on blood cadmium, lead, zinc and copper levels Zeliha Kayaaltı ⁎, Vugar Aliyev, Tülin Söylemezoğlu Ankara University Institute of Forensic Sciences Dikimevi, 06590 Ankara, Turkey a r t i c l e i n f o Article history: Received 28 April 2011 Revised 29 June 2011 Accepted 30 June 2011 Available online 13 July 2011 Keywords: Metallothionein 2A polymorphism Core promoter region PCR-RFLP AAS Metal levels a b s t r a c t Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins. Because of their rich thiol groups, MTs bind to the biologically essential metals and perform these metals' homeostatic regulations; absorb the heavy metals and assist with their transportation and extraction. The aim of this study was to investigate the association between the metallothionein 2A (MT2A) core promoter region − 5 A/G single nucleotide polymorphism (SNP) and Cd, Pb, Zn and Cu levels in the blood samples. MT2A polymorphism was determined by the standard polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique using the 616 blood samples and the genotype frequencies were found as 86.6% homozygote typical (AA), 12.8% heterozygote (AG) and 0.6% homozygote atypical (GG). Metal levels were analyzed by dual atomic absorption spectrophotometer system and the average levels of Cd, Pb, Zn and Cu in the blood samples were 1.69 ± 1.57 ppb, 30.62 ± 14.13 ppb, 0.98 ± 0.49 ppm and 1.04 ± 0.45 ppm, respectively. As a result; highly statistically signiﬁcant associations were detected between the − 5 A/G core promoter region SNP in the MT2A gene and Cd, Pb and Zn levels (p = 0.004, p = 0.012 and p = 0.002, respectively), but no association was found with Cu level (p = 0.595). Individuals with the GG genotype had statistically lower Zn level and higher Cd and Pb levels in the blood samples than individuals with AA and AG genotypes. This study suggests that having the GG genotype individuals may be more sensitive for the metal toxicity and they should be more careful about protecting their health against the toxic effects of the heavy metals. © 2011 Elsevier Inc. All rights reserved. Introduction Metallothioneins (MTs) are metal-binding, low molecular weight proteins, present in virtually all living organism. Due to structural characteristics, i.e. unusually high cysteine content with thiol (SH) groups; they have potent high afﬁnity/high capacity binding properties for various reactive metal ions, in particular cadmium (Cd), zinc (Zn), copper (Cu), lead (Pb) and mercury (Hg) with high redox capabilities (Vasak, 2005). MTs have multiple cellular functions, such as transport, storage and detoxiﬁcation of metals; protection of cells against the deleterious effect of the metals; metabolism of essential metals; free radical scavenger; immune response, genotoxicity and carcinogenicity (Nordberg, 1998). Besides these functions, it has been also showed from in vitro studies that MTs have crucial roles in cell proliferation, apoptosis and differentiation (Cherian, 1994; Nartey et al., 1987). Four major metallothionein isoforms have been invented so far, MT1, MT2A, MT3 and MT4 (Miles et al., 2000). Isoforms distribute in different ratios in human tissues and have differing rates of degradation. MT3 isoform is mainly synthesized in neurons as well as in heart, kidney, stomach, tongue and reproductive tissues (Moffatt ⁎ Corresponding author. Fax: +90 312 3192077. E-mail address: [email protected] (Z. Kayaaltı). 0041-008X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.06.023 and Séguin, 1998). MT4 has been detected only in certain squamous epithelia and the maternal deciduum (Quaife et al., 1994). The most widely expressed isoforms in the body are MT1 and MT2A. MT2A appears to be expressed more in human tissues than MT1. The expression difference between the MT2A and the other MT isoforms is attributed to the enhancer activity in MT2A promoter (Samson and Gedamu, 1998). MT2A promoter region contains seven metal responsive elements (MREs). Exposure to various heavy metals such as Cd, Pb and Hg and in conditions of oxidative stress, MRE-binding transcription factor-1 (MTF-1) is activated and active MTF-1 binds to MRE regions and initializes the gene transcription (Suzuki and Koizumi, 2000). A polymorphism near the 5′ of the MREa especially near the TATA box which has been activated in the transcription process, decreases binding afﬁnity of MTF-1 to MREs, and reduces the metallothionein transcription (Koizumi et al., 1999). As a result, a MT2A core promoter region single nucleotide polymorphism decreases the induction of gene transcription where as the metallothionein level is expected to increase, as a response to heavy metal exposure, cellular oxidative stress etc. Cd is a toxic, bioaccumulative, non-essential and highly widespread heavy metal with a variety of known adverse effects on human health even at low concentrations (Pan et al., 2010), and humans are exposed to Cd mainly via the respiratory or gastrointestinal tracts; important non-industrial sources of exposure are cigarette smoke, 2 Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7 food, drinking water and air (Järup and Akesson, 2009). The half-life of Cd in the human body is 10 to 30 years since Cd has tendency to accumulate; thus, with continuous environmental exposure, tissue concentrations of this metal increase throughout life (Goering and Klaassen, 1984). When the Cd is absorbed, it is rapidly transported by blood to liver, where it is bound to MT. The Cd bound to MT is freely ﬁltered at the glomerulus and this complex is very efﬁciently reabsorbed in the renal tubule (Chen et al., 2006). MT-bound Cd is non-toxic in the liver and kidney, however non-MT-bound Cd is toxic and causes a toxic insult to the cell (Chan and Cherian, 1993). The renal accumulation of Cd in the form of MT-bound Cd still remains one of the best-documented mechanisms. Pb is a ubiquitous environmental and occupational contaminant widely distributed around the world. General populations are exposed to lead, especially inner city residents of low socioeconomic status and workers such as smelter workers, battery makers, ceramics makers, ship burners and construction workers (Silbergeld and Weaver, 2007). Blood Pb levels have a median biologic half-life of about a month, mostly reﬂect current exposure (Barbosa et al., 2005), whereas bone Pb levels, with a half-life of approximately 25–30 years, reﬂect accumulated exposure (Nilsson et al., 1991). Pb accumulates in bone which contains 90–95% of the body burden in adults (Hu, 1998) and it is an accumulative toxin that can produce multisystem toxicity even at very low levels of exposure. Interaction with proteins is an important mechanism of lead toxicity, especially interactions with high-afﬁnity metal-binding proteins which have high content of sulfhydryl groups. Therefore, these proteins such as MTs, divalent metal transporter (DMT) and delta-aminolevulinic acid dehydratase (ALAD) are capable of binding bivalent cations (Landrigan et al., 2000). Pb binds to MT under ex vivo conditions (Waalkes et al., 1984) and tissue MT levels can be increased after Pb exposure (Ikebuchi et al., 1986). The adverse effects of Pb are mitigated by MT (Qu et al., 2002; Waalkes et al., 2004), this creates the distinct possibility that humans that poorly express MT may be predisposed to Pb toxicity, although this has not been tested in humans. It is known that both MT null mice and cells are more sensitive to lead toxicity than their wild type equivalents (Qu et al., 2002; Waalkes et al., 1984). In fact, MTnull mice are hypersensitive to Pb-induced nephrocarcinogenesis (Waalkes et al., 2004). The other proposed functions of MT are homeostasis of essential metals such as Zn and Cu, regulating gene expression and tissue regeneration (Cherian and Kang, 2006), all contribute to MT protection against heavy metals toxicity. Zinc is found in nearly 300 speciﬁc enzymes that serves as structural ions in transcription factors during development or protein synthesis (Jacob et al., 1998) and is stored and transferred by metallothioneins. MTs act both as a cellular reservoir for Zn and as a buffering protein in the presence of excessive amount of Zn in order to prevent its toxicity (Kelly et al., 1996). Cu is required for the function of over 30 proteins, including superoxide dismutase, ceruloplasmin, ferroxidases and cytochrome oxidase (Rana, 2008). Although Cu is an essential metal for living systems, excess Cu can be toxic, particularly when associated with a deﬁcit in Cu excretion (Bremner, 1998). Metallothioneins have a crucial role in the normal biological activity of Cu in the body. They bind to Cu and regulate its absorption into the blood stream and also, they protect the cells against the toxicity of Cu under extreme conditions. So far, there have been a lot of studies in the literature regarding metallothionein expression levels, metallothionein polymorphisms in aging (Kayaaltı et al., 2011) and various diseases (Brüwer et al., 2001; Ebert et al., 2000) or relations of metallothionein levels with different inducers (Liu et al., 2000), such as heavy metals, oxidative stress, interleukins etc. Furthermore, the effect of the metallothionein gene polymorphism on metal levels in placenta tissues (Tekin et al., 2011) and autopsy kidney tissues (Kayaalti et al., 2010) were researched. The aim of the preset study was to investigate relationships between the MT2A core promoter region (around the gene transcrip- tional start site) polymorphism and Cd, Pb, Zn and Cu levels in individuals blood samples. To our knowledge, this is the ﬁrst study to investigate the effect of MT2A − 5A/G core promoter region single nucleotide polymorphism (SNP) (rs28366003; GeneID: 4502; accession no: NM_005953) on Cd, Pb, Zn and Cu levels in the blood samples. Material and methods Study subjects. In this study, blood samples were obtained from 616 genetically unrelated and healthy individuals (mean age 45.71 ± 12.30 years), including 314 males (mean age 47.14 ± 13.10 years; range 18–67 years) and 302 females (mean age 44.23 ± 11.46 years; range 18–65 years). The exclusion criteria for the subjects included a medical history of renal failure, carcinoma and diagnosed hepatic and cardiovascular diseases that may be related with the possible heavy metal accumulation resulting from environmental and occupational exposure. All the participating volunteers did not smoke. Blood samples were provided from volunteers referred to Ankara Occupational Diseases Hospital for routine control. Informed consent was obtained from each individual who was selected randomly control group sample among from Turkish population. The geographical distributions of the subjects were from the Central Anatolia region and other regions representing the Turkish population from all regions of the country. A small questionnaire for gathering the demographic and ethnic information was also given to the individuals and the individuals stating themselves as Turkish were included in the study. The study design was approved by the institutional ethics committee (approval No: 152-4826 in 2009). Samplings were performed in accordance with the principles of The Declaration of Helsinki. Blood samples were kept at 4 °C while they were in active use. DNA extraction and genotyping of MT2A −5 A/G polymorphism. Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulant whole-blood samples using the QIAamp blood DNA mini-kit (Qiagen, Hilden, Germany) according to the method recommended by the manufacturer. DNA concentration was determined using the PicoGreen dsDNA quantitation kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions. DNAs were stored at −20 °C until the polymerase chain reaction (PCR) analysis. MT2A −5 A/G SNP was genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP) method (Kayaalti and Söylemezoğlu, 2010). In order to screen for −5 A/G polymorphism of MT2A, 241 bp fragment was ampliﬁed by PCR using the following primers: forward: 5′-CGC CTG GAG CCG CAA GTG AC-3′; and reverse: 5′-TGG GCA TCC CCA GCC TCT TA-3′. Ampliﬁcation was carried out on a Techne Tc 512 PCR System in a 50 μl reaction mixture containing 200 μM of dNTPs, 10 pmol each of forward (F) and reverse (R) primers, 1 U Hot Star Taq DNA polymerase (Qiagen), 1 X PCR buffer (Qiagen) and 100 ng genomic DNA. The PCR cycling conditions consisted of an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and ﬁnal extension step at 72 °C for 10 min. Then the PCR product (241 bp) was digested with BsgI (New England Biolabs, Hertfordshire, UK) and incubated at 37 °C for 1.5–2 h. The undigested polymerase chain reaction product and digested products were separated on a 2% agarose gel electrophoresis, visualized by ethidium bromide staining under an ultraviolet illuminator, scanned and photographed using Syngene Monitoring System. Digestion of PCR product by BsgI yields 144 bp, 56 bp and 41 bp fragments for the presence of A allele; 185 bp and 56 bp for the presence of G allele. Digested and undigested PCR products on the agarose gel electrophoresis are indicated in Fig. 1. Results of RFLP for each variant in 30 randomly selected samples were conﬁrmed by DNA sequencing method using the Big-Dye Terminator Cycle Sequencing Ready Reaction kit on an ABI Prism 3100 Genetic Analyzer. The automated DNA sequencing Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7 Fig. 1. Representative gel image of undigested and digested PCR products (M: 100 bp ladder); Lane 1: undigested PCR product (241 bp); Lanes 2–4: digested PCR products with BsgI; Lane 2: homozygote typical genotype (AA) (144, 56 and 41 bp); Lane 3: heterozygote genotype (AG) (185, 144, 56 and 41 bp); and Lane 4: homozygote atypical genotype (GG) (185 and 56 bp). was employed to conﬁrm the authenticity of the ampliﬁed PCR products. Determination of metal levels. A microwave system (CEM Mars Xpress) was utilized for digestion of the samples with concentrated nitric acid solution. The analysis was carried out with a dual atomic absorption spectrophotometer (AAS) system (Varian 240). 1 ml whole blood samples were dissolved in 10 ml of nitric acid, after which all samples were transferred to teﬂon tubes and digested in microwave at 200 °C for 20 min. Digested sample solutions were diluted before being introduced to a graphite furnace. AAS equipped with a graphite furnace and Zeeman background correction system was used for Cd and Pb determination. By contrast with serum samples were diluted 20 fold with deionized water and nitric acit to analysis by ﬂame atomic absorption spectrometry for Zn and Cu. The AAS method was tested by studying the certiﬁed reference materials (Seronorm™ Trace Elements Whole Blood Level-2; Ref Number: 201605) with the certiﬁed values. Statistical analysis. The Statistical Package for Social Sciences (SPSS) version 16.0 software for Windows was used for the statistical analysis. The frequencies of MT2A alleles and genotypes were obtained by direct count, and the departure from the Hardy–Weinberg equilibrium was evaluated by the χ2 test and power analysis was made using the F-Test with a target signiﬁcance level of 0.05. In the exploratory analysis, data showed a non-normal distribution (by Shapiro-Wilk statistic); therefore, the Mann–Whitney and Kruskal–Wallis non-parametric tests were used for the comparison of groups in terms of metric variables and data were measured as median, minimum and maximum ranges. Categorical variables were compared by the χ2 test. The Spearman correlation coefﬁcient was used for determination of the association between two variables, and p b 0.05 was considered as statistically signiﬁcant. Results In the ﬁrst part of the study, the DNAs of 616 blood samples from individuals were isolated and MT2A promoter region was ampliﬁed with PCR method. The − 5 A/G single nucleotide polymorphism in the core promoter region of MT2A was determined using RFLP technique in agarose gel electrophoresis (Fig. 1). The genotype frequencies in 616 samples were as follows; 86.6% homozygote typical (AA), 12.8% heterozygote (AG) and 0.6% homozygote atypical (GG). The frequency of A allele was found as 92.9% and of G allele as 7.1%. The genotype and allele frequencies were consistent with Hardy–Weinberg equilibrium. Hardy–Weinberg exact test p-value was 0.53. A one-way design with 3 groups has sample sizes of 533, 79 and 4. The null hypothesis was that the standard deviation of the group means was 0.0 and the 3 alternative standard deviation of the group means is 1.9. The total sample of 616 subjects achieved a power of 85.1%. MT2A core promoter region polymorphism was also statistically evaluated for signiﬁcance with gender and age; and no signiﬁcant association was assessed (p = 0.916; p = 0.947, respectively). In the second part of the study, it was investigated whether MT2A promoter region polymorphism has an effect on toxic metal and trace element levels in the blood samples. Cd and Pb levels were measured by Graphite Furnace Atomic Spectrometry; Cu and Zn were measured by Flame Atomic Absorption Spectrometry. When the metal levels in the blood samples were evaluated among the female and male groups, male group had higher Zn levels and lower Cu levels than female group and these results were statistically signiﬁcant (p b 0.05). However, there was no signiﬁcant difference between males and females in terms of Cd and Pb levels (p N 0.05) (Table 1). In addition to these, the correlation analyses among the metals were calculated and the statistically positive correlation between the Pb and Cu (r = 0.183; p = 0.001); the negative correlation between the Pb and Zn (r = −166; p = 0.001) were found. No signiﬁcant correlation was found among the other metals (p N 0.05). The average levels of Cd, Pb, Zn and Cu in the blood samples were 1.69 ± 1.57 ppb, 30.62 ± 14.13 ppb, 0.98 ± 0.49 ppm and 1.04 ± 0.45 ppm, respectively. As a result; a highly signiﬁcant associations were detected between the −5 A/G SNP in the MT2A gene and Cd, Pb and Zn levels (p= 0.004, p = 0.012 and 0.002, respectively), but no association was found with Cu level (p= 0.595) (Table 2; Figs. 2 and 3). Individuals with the GG genotype had statistically lower Zn levels and higher Cd and Pb levels than individuals with AA and AG genotypes. Our study results also have shown that the Cd and Pb concentrations were signiﬁcantly increased with G allele (G+) when compared with without G allele (G-) individuals, but Zn levels were signiﬁcantly decreased. Discussion Metals are a major category of globally-distributed environmental pollutants. Although many adverse health effects of heavy metals such as Cd and Pb have been known for a long time, we are all exposed either voluntarily or involuntarily to heavy metals which are still used for human industry and products (Al-Saleh et al., 2011). Therefore, exposure to heavy metals continues to increase in some parts of the world, in particular in less developed countries. Heavy metals in particular Cd and Pb have ability to accumulate over the various tissues and impact on health even at low levels of exposure due to excreted only a small fraction of inhaled or ingested amounts by human body. Their acute, chronic or sub-chronic toxicity can lead to neurotoxic, carcinogenic, mutagenic or teratogenic effects. (Järup, 2003). MTs are considered as an important defense protein for the detoxiﬁcation of non-essential and excessive essential metals. The protective role of the MTs is related to the induction of the protein in Table 1 Distribution of Cd, Pb, Zn, Cu levels in blood samples between the female and males. Metals Sex Mean ± SD Median Min Max p Cd (ppb) Female (n = 302) Male (n = 314) Total (n = 616) Female (n = 302) Male (n = 314) Total (n = 616) Female (n = 302) Male (n = 314) Total (n = 616) Female (n = 302) Male (n = 314) Total (n = 616) 1.66 ± 1.46 1.73 ± 1.68 1.69 ± 1.57 30.85 ± 14.57 30.40 ± 13.72 30.62 ± 14.13 0.90 ± 0.43 1.07 ± 0.53 0.98 ± 0.49 1.21 ± 0.50 0.87 ± 0.31 1.04 ± 0.45 1.28 1.20 1.25 27.81 28.58 28.10 1.01 1.16 1.08 1.18 0.91 1.00 0.10 0.20 0.10 8.29 5.47 5.47 0.06 0.06 0.06 0.11 0.11 0.11 15.70 12.20 15.70 77.64 75.23 77.64 2.15 2.36 2.36 3.01 1.79 3.01 0.565 Pb (ppb) Zn (ppm) Cu (ppm) ⁎ p b 0.01. 0.697 0.001⁎ 0.001⁎ 4 Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7 Table 2 MT2A − 5 A/G core promoter region polymorphism and Cd, Pb, Zn and Cu concentrations of blood samples. Polymorphism Metals Cd (ppb) Pb (ppb) Mean ± SD Median Min Max Mean ± SD Zn (ppm) MT2A GENOTYPES N Median Min Max AA AG GG p G- (AA) G+ (AG + GG) p 533 (86.6%) 1.60 ± 1.44 1.20 79 (12.8%) 2.09 ± 1.85 1.40 4 (0.6%) 5.98 ± 4.38 5.92 0.004⁎⁎ 0.10 15.70 30.13 ± 13.91 27.49 0.30 9.84 32.94 ± 14.92 29.34 1.07 11.00 50.42 ± 11.48 51.27 0.012⁎ 5.47 75.23 1.01 ± 0.48 1.09 12.17 77.64 0.84 ± 0.50 0.94 35.57 63.56 039 ± 0.33 0.28 0.002⁎⁎ 533 (86.6%) 1.60 ± 1.44 1.20 83 (13.4%) 2.28 ± 2.16 1.40 0.001⁎⁎ 0.10 15.70 30.13 ± 13.91 27.49 0.30 11.00 33.78 ± 15.19 30.49 0.029⁎ 5.47 75.23 1.01 ± 0.48 1.09 12.17 77.64 0.82 ± 0.50 0.88 0.001⁎⁎ Cu (ppm) Mean ± SD Median Min Max Mean ± SD Median Min 0.06 2.36 1.04 ± 0.44 0.06 1.84 1.02 ± 0.52 0.15 0.88 0.91 ± 0.37 0.596 0.06 2.36 1.04 ± 0.44 0.06 1.84 1.01 ± 0.51 0.522 Max 1.01 0.94 1.00 0.11 2.80 0.11 3.01 0.39 1.26 1.01 0.94 0.11 2.80 0.11 3.01 ⁎ p b 0.05. ⁎⁎ p b 0.01. an oxidative stress situation or by the presence of toxic factors such as heavy metals (Dabrio et al., 2002). The association between the inducers and metallothionein gene expression levels in different tissues has been reported by several researches. According to their ﬁndings, there has been statistically signiﬁcant association between the metal levels and metallothionein expression in the various tissues. (Liu et al., 2000; Yoshida et al., 1998). The early induction of MT by metals makes this protein a potential biomarker useful to assess the ecotoxicological signiﬁcance of non-essential (Cd, Pb) and essential, but potentially toxic excessive (Cu) essential metals (Amiard et al., 2006). The most expressed isoform of MT in humans is MT2A. The expression difference between MT2A and other metallothionein isoforms is attributed to the enhancer activity in MT2A promoter where the gene transcription initializes (Samson and Gedamu, 1998). A mutation on core promoter region (including the TATA box region) of a gene has stronger effect on expression comparing to the other gene regions. For the initiation of gene expression, basic transcription factors bind to the TATA box-containing core region of the promoter (Robert et al., 1996). Therefore, in the present study, for investigation of SNP in MT2A, -5 A/G core region of the promoter was chosen due to possible loss of functionality of metal homeostasis. There have been a few studies related to MT2A gene polymorphisms. So far, the MT2A − 5 A/G SNP has been studied our population (Kayaalti and Söylemezoğlu, 2010), Japanese population (Hayashi et al., 2006; Kita et al., 2006) and Midwestern U.S. Black and White female population (McElroy et al., 2010), and in this study, almost similar genotype frequencies were found for white populations. The genotype frequencies in our previous population study and present study were as follows; 87.0% and 86.6% for AA, 12.3% and 12.8% for AG, and 0.7% and 0.6% for GG genotypes. Statistical methods used in current study have an enough power to analyze the statistical difference between the groups and we calculated statistically well powered (85.1%). MT polymorphisms and their associations with risk for a variety of diseases, such as amyotrophic lateral sclerosis (Hayashi et al., 2006), diabetes type II and atherosclerosis disease (Giacconi et al., 2005) have been studied. However, the impact of MT gene polymorphisms on metal metabolism has not yet been investigated well enough and a lot of questions remain on the role of MT, the relatively high presence of MT genes in the human genome, and the functional relation of MT polymorphisms with regard to storage, homeostasis, and detoxiﬁcation of metals (Gundacker et al., 2009). There are only a few studies in the literature. In a study performed by Kita and co-workers, it was found that the − 5 A/G SNP in the MT2A reduced cadmium-induced transcription of the metallothionein-2A gene in the HEK293 cells (Kita et al., 2006). In other two previous studies conducted by our group related to this SNP in MT2A gene and metal levels, no statistical association was found between the − 5 A/G SNP in the MT2A gene and the Zn and Cu levels in the autopsy kidney tissues, but considerably high accumulation of Cd was monitored for individuals having AG and GG genotypes compared with individuals having AA genotype (Kayaalti et al., 2010). As a result of our other study, maternal blood Cd levels were statistically higher for mothers with AG genotype Fig. 2. The bar charts of the association between the MT2A − 5 A/G SNP and non-essential metal levels (Cd and Pb) in the blood samples. a) The bar chart for Cd levels (n = 616; p = 0.004); b) The bar chart for Pb levels (n = 616; p = 0.012). Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7 5 Fig. 3. The bar charts of the association between the MT2A − 5 A/G SNP and essential metal levels (Zn and Cu) in the blood samples. a) The bar chart for Zn levels (n = 616; p = 0.002); b) The bar chart for Cu levels (n = 616; p = 0.596). compared to AA genotype (pb 0.05). In contrast, placental Cd levels were signiﬁcantly higher in mothers with AA genotype rather than AG genotype (p b 0.05) (Tekin et al., 2011). In the another research, Gundacker and co-workers studied the association between the different SNP (rs10636) in MT2A (+838 untranslated region polymorphism) and Pb levels and, they found a negative association between MT2A +838 C/G variants and blood lead contents (Gundacker et al., 2009). In the present study, we studied that the potential effect of the −5 A/G core promoter region SNP in MT2A gene on the Cd, Pb, Zn and Cu levels in the blood samples of individuals larger than our previous studies mentioned above. The reason for choosing the blood as study sample was the blood metal levels may be useful for evaluating current exposure, and in long-term, low-level blood exposures can also be used for estimating the body burden. Hence, the determination of the blood metal levels may be a signiﬁcant index for human health. The design of this study is different from previous investigations and, to our knowledge, this is the ﬁrst study to investigate the effects of the − 5 A/G SNP in the MT2A gene on Cd, Pb, Zn and Pb element levels in the human blood samples. In the study, there has been no exceeding Cd or Pb concentrations assessed in accordance with the literature, since in Central Anatolia region of Turkey where the samples were collected from, there is no speciﬁc source of Cd and Pb exposures such as cadmium-nickel battery industry, cosmetics, ceramics and paint as well as a speciﬁc environmental contamination. According to present study results, a highly signiﬁcant association was detected between the −5 A/G SNP in the MT2A gene and Cd, Pb and Zn levels. Individuals with the GG genotype had statistically higher Cd levels than individuals with AA and AG genotypes that these results were found to be similar to our previous studies (Kayaalti et al., 2010; Tekin et al., 2011). Resulting data resemble the ﬁndings in a Japanese population reported by Kita et al. (2006). Distribution of MT2A −5 G/G genotype was observed at very low frequency (only four individuals) and G allele was atypical allele in this gene polymorphism. Therefore, in order to avoid overstatement of study results, AG and GG genotypes were gathered and labeled as G +; the other genotype, AA was labeled as G -. When this study was compared with our previous studies, relatively large sample size (approximately 5.5 times) was used and while the autopsy kidney samples were analyzed in previous study, blood samples were analyzed in this study. Moreover, levels of two toxic metals (Cd and Pb) and two trace elements (Zn and Cu) in blood samples were measured and correlation coefﬁcients among the all analyzed metals were calculated. Also, present study showed new data about low Zn and high Pb in having G + genotypes individuals and not only for AA, AG and GG genotypes but also for having G + and G − genotypes were statistically signiﬁcant with Pb and Zn levels of blood samples (p b 0.05).Although, we determined the statistically signiﬁcant association between the MT2A core promoter region −5 A/G SNP and blood Pb, but Gundacker and co-workers found that inverse association between MT2A untranslated region + 838 C/G variants and blood lead levels (Gundacker et al., 2009). The reason of the conﬂicting results might be because of the different MT2A polymorphisms studied. Polymorphisms can affect the function of the gene or not, depend on the where the SNP occurs. Therefore, in the present study, the core promoter region −5 A/G polymorphism was especially chosen for the investigation due to almost complete loss of functionality in metal toxicokinetics and thus might exert a stronger biological effect. Also, we detected that statistically positive correlation between the Pb and Cu; the negative correlation between the Pb and Zn. The binding afﬁnity of MTs for metal ions is metal-dependent and bivalent cations compete for binding to MTs. In a study performed by Waalkes and co-workers, the order of binding afﬁnity of MTs was Cd N Pb N Cu N Hg N Zn N Ag N Ni N Co (Waalkes et al., 1984). MTs capable of binding to Cd and Pb are much more than Zn. Thus, in the present study, the correlations among the metals can be explained by the different binding afﬁnity. MT2A −5 GG genotype individuals may be more sensitive for the metal toxicity. Because individuals having this genotype express the low MT and low expressed MT previously binds to Cd and Pb comparing with Zn. Absorption of the Cd and Pb is associated with a decrease in Zn, which is an important component of biomembranes and an essential cofactor in a variety of enzymes, as a result of the antagonistic relationships between these elements (Memon et al., 2007). In this study, a total of 533 individuals were identiﬁed to possess AA genotypes and only 4 individuals were determined to have GG genotypes for MT2A polymorphism. Average Cd and Pb levels of GG genotypes individuals were 3.74 and 1.67 times higher than those of AA genotypes, respectively. Even though, maximum Cd and Pb levels of having AA genotype individuals were small higher than having GG genotype, comparing the minimum values of these metals, it was 6 Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7 found that individuals with GG genotype were 10.7 times higher for Cd and 6.50 times higher for Pb than individuals with AA genotypes. The discrepancy between the minimum and maximum levels of the metals was expected for having AA genotype individuals due to large number individuals with AA and small number individuals with GG genotypes (n = 533 vs. n = 4). Moreover, although MT2A is one of the major factors for metal transport, storage and homeostasis; it is not the only metal transporter protein in human sensitivity to heavy metals. Some of the individuals with AA genotype may have a different polymorphic character for the other metal transporter protein. One of these transporters is DMT, which plays important roles in intestinal Fe absorption, accumulation and transport. DMT1 has an important biological function not only for dietary Fe uptake in the duodenum, but also carriers other divalent metals such as Zn, Mn, Co, Cd, Cu, Ni and Pb (Garrick et al., 2006). In conclusion, we suggest that the effect of the MT2A SNP on heavy metal accumulation should be considered in a future occupational health issue. 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