The potential effect of metallothionein 2A 5 A.G single nucleotide polymorphism

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: kayaalti@ankara.edu.tr (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. In this study, when the average Cd and Pb levels in the
blood samples is considered, especially workers who exposed to
heavy metals with the MT2A GG genotype should be more careful
about protecting their health against the toxic effects of the heavy
metals. In the near future, further studies may be focus on the effect of
the other metal transporter gene variants such as divalent metal
transporter 1 (DMT1) and hemochromatosis (HFE) polymorphisms
on toxic metal burden and trace element homeostasis in blood or
various tissues.
Conflict of interest
The authors do not have any conflict of interest.
Acknowledgments
This work was financially supported by T. R. Prime Ministry State
Planning Organization and Research Fund of Ankara University (Grant
number 2003K1201902).
References
Al-Saleh, I., Shinwari, N., Mashhour, A., Mohamed, Gel D., Rabah, A., 2011. Heavy metals
(lead, cadmium and mercury) in maternal, cord blood and placenta of healthy
women. Int. J. Hyg. Environ. Health 214, 79–101.
Amiard, J.C., Amiard-Triquet, C., Barka, S., Pelerin, J., Rainbow, P.S., 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as
biomarkers. Aquat. Toxicol. 76, 160–202.
Barbosa Jr., F., Tanus-Santos, J.E., Gerlach, R.F., Parsons, P.J., 2005. A critical review of
biomarkers used for monitoring human exposure to lead: advantages, limitations,
and future needs. Environ. Health Perspect. 113, 1669–1674.
Bremner, I., 1998. Manifestations of copper excess. Am. J. Clin. Nutr. 67, 1069S–1073S.
Brüwer, M., Schmid, K.W., Metz, K.A., Krieglstein, C.F., Senninger, N., Schürmann, G.,
2001. Increased expression of metallothionein in inflammatory bowel disease.
Inflamm. Res. 50, 289–293.
Chan, H.M., Cherian, M.G., 1993. Mobilization of hepatic cadmium in pregnant rats.
Toxicol. Appl. Pharmacol. 120, 308–314.
Chen, L., Jin, T., Huang, B., Chang, X., Lei, L., Nordberg, G.F., Nordberg, M., 2006. Plasma
metallothionein antibody and cadmium-induced renal dysfunction in an occupational population in China. Toxicol. Sci. 91, 104–112.
Cherian, M.G., 1994. The significance of the nuclear and cytoplasmic localization of
metallothionein in human liver and tumor cells. Environ. Health Perspect. 102, 131–135.
Cherian, M.G., Kang, Y.J., 2006. Metallothionein and liver cell regeneration. Exp. Biol.
Med. 231, 138–144.
Dabrio, M., Rodríguez, A.R., Bordin, G., Bebianno, M.J., De Ley, M., Sestáková, I., Vasák, M.,
Nordberg, M., 2002. Recent developments in quantification methods for metallothionein. J. Inorg. Biochem. 88, 123–134.
Ebert, M.P., Günther, T., Hoffmann, J., Yu, J., Miehlke, S., Schulz, H.U., Roessner, A., Korc,
M., Malfertheiner, P., 2000. Expression of metallothionein II in intestinal
metaplasia, dysplasia, and gastric cancer. Cancer Res. 60, 1995–2001.
Garrick, M.D., Singleton, S.T., Vargas, F., Kuo, H.C., Zhao, L., Knöpfel, M., Davidson, T.,
Costa, M., Paradkar, P., Roth, J.A., Garrick, L.M., 2006. DMT1: which metals does it
transport? Biol. Res. 39, 79–85.
Giacconi, R., Cipriano, C., Muti, E., Costarelli, L., Maurizio, C., Saba, V., Gasparini, N.,
Malavolta, M., Mocchegiani, E., 2005. Novel -209A/G MT2A polymorphism in old
patients with type 2 diabetes and atherosclerosis: relationship with inflammation
(IL-6) and zinc. Biogerontology 6, 407–413.
Goering, P.L., Klaassen, C.D., 1984. Tolerance to cadmium-induced toxicity depends on
presynthesized metallothionein in liver. J. Toxicol. Environ. Health 14, 803–812.
Gundacker, C., Wittmann, K.J., Kukuckova, M., Komarnicki, G., Hikkel, I., Gencik, M.,
2009. Genetic background of lead and mercury metabolism in a group of medical
students in Austria. Environ. Res. 109, 786–796.
Hayashi, Y., Hashizume, T., Wakida, K., Satoh, M., Uchida, Y., Watabe, K., Matsuyama, Z.,
Kimura, A., Inuzuka, T., Hozumi, I., 2006. Association between metallothionein
genes polymorphisms and sporadic amyotrophic lateral sclerosis in a Japanese
population. Amyotroph. Lateral Scler. 7, 22–26.
Hu, H., 1998. Bone lead as a new biologic marker of lead dose: recent findings and
implications for public health. Environ. Health Perspect. 106, 961–967.
Ikebuchi, H., Teshima, R., Suzuki, K., Terao, T., Yamane, Y., 1986. Stimultaneous
induction of Pb-metallothionein-like protein and Zn-thionein in the liver of rats
given lead acetate. Biochem. J. 223, 541–546.
Jacob, C., Maret, W., Vallee, B.L., 1998. Control of zinc transfer between thionein,
metallothionein, and zinc proteins. Proc. Natl. Acad. Sci. U. S. A. 95, 3489–3494.
Järup, L., 2003. Hazards of heavy metal contamination. Br. Med. Bull. 68, 167–182.
Järup, L., Akesson, A., 2009. Current status of cadmium as an environmental health
problem. Toxicol. Appl. Pharmacol. 238, 201–208.
Kayaalti, Z., Söylemezoğlu, T., 2010. The polymorphism of core promoter region on
metallothionein 2A-metal binding protein in Turkish population. Mol. Biol. Rep. 37,
185–190.
Kayaalti, Z., Mergen, G., Söylemezoğlu, T., 2010. Effect of metallothionein core promoter
region polymorphism on cadmium, zinc and copper levels in autopsy kidney
tissues from a Turkish population. Toxicol. Appl. Pharmacol. 245, 252–255.
Kayaaltı, Z., Sahiner, L., Durakoğlugil, M.E., Söylemezoğlu, T., 2011. Distributions of
interleukin-6 (IL-6) promoter and metallothionein 2A (MT2A) core promoter region
gene polymorphisms and their associations with aging in Turkish population. Arch.
Gerontol. Geriatr 53, 354–358.
Kelly, E.J., Quaife, C.J., Froelick, G.J., Palmiter, R.D., 1996. Metallothionein I and II protect
against zinc deficiency and zinc toxicity in mice. J. Nutr. 126, 1782–1790.
Kita, K., Miura, N., Yoshida, M., Yamazaki, K., Ohkubo, T., Imai, Y., Naganuma, A., 2006.
Potential effect on cellular response to cadmium of a single-nucleotide A – N G
polymorphism in the promoter of the human gene for metallothionein IIA. Hum.
Genet. 120, 553–560.
Koizumi, S., Suzuki, K., Ogra, Y., Yamada, H., Otsuka, F., 1999. Transcriptional activity
and regulatory protein binding of metal-responsive elements of the human
metallothionein-IIA gene. Eur. J. Biochem. 259, 635–642.
Landrigan, P.J., Boffetta, P., Apostoli, P., 2000. The reproductive toxicity and
carcinogenicity of lead: a critical review. Am. J. Ind. Med. 38, 231–243.
Liu, Y., Liu, J., Habeebu, S.M., Waalkes, M.P., Klaassen, C.D., 2000. Metallothionein-I/II
null mice are sensitive to chronic oral cadmium-induced nephrotoxicity. Toxicol.
Sci. 57, 167–176.
McElroy, J.A., Bryda, E.C., McKay, S.D., Schnabel, R.D., Taylor, J.F., 2010. Genetic variation at a
metallothionein 2A promoter single-nucleotide polymorphism in white and black
females in Midwestern United States. J. Toxicol. Environ. Health A 73, 1283–1287.
Memon, A.R., Kazi, T.G., Afridi, H.I., Jamali, M.K., Arain, M.B., Jalbani, N., Syed, N., 2007.
Evaluation of zinc status in whole blood and scalp hair of female cancer patients.
Clin. Chim. Acta 379, 66–70.
Miles, A.T., Hawksworth, G.M., Beattie, J.H., Rodilla, V., 2000. Induction, regulation,
degradation, and biological significance of mammalian metallothioneins. Crit. Rev.
Biochem. Mol. Biol. 35, 35–70.
Moffatt, P., Séguin, C., 1998. Expression of the gene encoding metallothionein-3 in
organs of the reproductive system. DNA Cell Biol. 17, 501–510.
Nartey, N.O., Banerjee, D., Cherian, M.G., 1987. Immunohistochemical localization of
metallothionein in cell nucleus and cytoplasm of fetal human liver and kidney and
its changes during development. Pathology 19, 233–238.
Nilsson, U., Attewell, R., Christoffersson, J.-O., Schutz, A., Ahlgren, L., Skerfving, S.,
Mattsson, S., 1991. Kinetics of lead in bone and bfood after end of occupational
exposure. Pharmacol. Toxicol. 69, 477–484.
Nordberg, M., 1998. Metallothioneins: historical review and state of knowledge. Talanta
46, 243–254.
Pan, J., Plant, J.A., Voulvoulis, N., Oates, C.J., Ihlenfeld, C., 2010. Cadmium levels in
Europe: implications for human health. Environ. Geochem. Health 32, 1–12.
Qu, W., Diwan, B.A., Liu, J., Goyer, R.A., Dawson, T., Horton, J.L., Cherian, M.G., Waalkes,
M.P., 2002. The metallothionein-null phenotype is associated with heightened
sensitivity to lead toxicity and an inability to form inclusion bodies. Am. J. Pathol.
160, 1047–1056.
Quaife, C.J., Findley, S.D., Erickson, J.C., Froelick, G.J., Kelly, E.J., Zambrowicz, B.P.,
Palmiter, R.D., 1994. Induction of a new metallothionein isoform (MT-IV) occurs
during differentiation of stratified squamous epithelia. Biochemistry 33,
7250–7259.
Rana, S.V., 2008. Metals and apoptosis: recent developments. J. Trace Elem. Med. Biol.
22, 262–284.
Robert, F., Forget, D., Li, J., Greenblatt, J., Coulombe, B., 1996. Localization of subunits of
transcription factors IIE and IIF immediately upstream of the transcriptional
initiation site of the adenovirus major late promoter. J. Biol. Chem. 271, 8517–8520.
Samson, S.L., Gedamu, L., 1998. Molecular analyses of metallothionein gene regulation.
Prog. Nucleic Acid Res. Mol. Biol. 59, 257–288.
Silbergeld, E.K., Weaver, V.M., 2007. Exposure to metals: are we protecting the
workers? Occup. Environ. Med. 64, 141–142.
Suzuki, K., Koizumi, S., 2000. Individual metal responsive elements of the metallothionein-IIA gene independently mediate responses to various heavy metal signals. Ind.
Health 38, 87–90.
Tekin, D., Kayaaltı, Z., Aliyev, V., Söylemezoğlu, T., 2011. The effects of metallothionein
2A polymorphism on placental cadmium accumulation: is metallothionein a
Z. Kayaaltı et al. / Toxicology and Applied Pharmacology 256 (2011) 1–7
modifiying factor in transfer of micronutrients to the fetus? J. Appl. Toxicol.
doi:10.1002/jat.1661 [Epub ahead of print].
Vasak, M., 2005. Advances in metallothionein structure and functions. J. Trace Elem.
Med. Biol. 19, 13–17.
Waalkes, M.P., Harvey, M.J., Klaassen, C.D., 1984. Relative in vitro affinity of hepatic
metallothionein for metals. Toxicol. Lett. 20, 33–39.
7
Waalkes, M.P., Liu, J., Goyer, R.A., Diwan, B.A., 2004. Metallothionein-I/II double
knockout mice are hypersensitive to lead-induced kidney carcinogenesis: role of
inclusion body formation. Cancer Res. 64, 7766–7772.
Yoshida, M., Ohta, H., Yamauchi, Y., Seki, Y., Sagi, M., Yamazaki, K., Sumi, Y., 1998. Agedependent changes in metallothionein levels in liver and kidney of the Japanese.
Biol. Trace Elem. Res. 63, 167–175.