Download DNA Microarray Analysis of Altered Gene Expression in Cadmium

J Occup Health 2003; 45: 331–334
Journal of
Occupational Health
Review
DNA Microarray Analysis of Altered Gene Expression in Cadmiumexposed Human Cells
Shinji KOIZUMI1 and Hirotomo YAMADA2
1
Department of Hazard Assessment and 2Department of Health Effects Research, National Institute of Industrial
Health, Japan
Abstract: DNA Microarray Analysis of Altered Gene
Expression in Cadmium-exposed Human Cells:
Shinji K O I Z U M I , et al. Department of Hazard
Assessment, National Institute of Industrial
Health—Cadmium (Cd) is a heavy metal known to be
toxic and carcinogenic, but its mechanism of action
remains to be elucidated. Development of the DNA
microarray technology has recently made the
comprehensive analysis of gene expression possible,
and it could be a powerful tool also in toxicological
studies. With microarray slides containing 7,000–9,000
genes, we have been studying the gene expression
profiles of a human cell line exposed to Cd. By
exposure to a non-lethal concentration of Cd, 46
upregulated and 10 downregulated genes whose
expression levels changed twofold or greater were
observed. The expression of genes related to cellular
protection and damage control mechanisms such as
those encoding metallothioneins, anti-oxidant proteins
and heat shock proteins was simultaneously induced.
In addition, altered expression of many genes involved
in signaling, metabolism and so on was newly
observed. As a whole, a number of genes appear to
be coordinately regulated toward survival from Cd
toxicity. When cells were exposed to a higher
concentration of Cd, more remarkable effects were
observed both in the number of affected genes and in
the extent of altered expression. These findings will
contribute to the understanding of the complicated
biological effects of Cd.
(J Occup Health 2003; 45: 331–334)
Key words: DNA microarray, Cadmium, Gene
expression profile, Stress response
Known biological effects of Cd
Exposures to cadmium (Cd) have occurred both
occupationally and environmentally1). This metal is used
in such occupations as electroplating and the manufactures
of batteries, plastics, paints, alloys and fertilizers. Cd is
also generated as a by-product of the mining of lead, copper
and zinc. As a most striking example of accidental human
exposure, a Japanese population was exposed to Cd through
rice contaminated by a runoff from a mine. Chronic
exposure to Cd causes disorders in the kidney, calcium
metabolism and respiratory system1, 2). Cd has also been
reported to be carcinogenic2, 3).
It is also known that Cd affects the transcription of a
number of genes. Cd induces the human genes with
protective functions 4), including those coding for
metallothioneins (MTs)5, 6) that chelate Cd ions to make
it biologically inert, heat shock proteins (HSPs)7, 8) that
play roles in renaturing damaged proteins and hemeoxygenase 19) that plays an anti-oxidative role. Cd also
induces protooncogenes10, 11) that might be related to the
mechanistic background of carcinogenesis. Its effects
on the sex hormone receptor genes12) suggest endocrinedisrupting activity. However, these effects of Cd on gene
expression probably represent only a fraction, and there
might be a number of unidentified effects on genes
relevant to the expression of toxicity and protection
against it. By a comprehensive screening of altered gene
expression with the new and powerful tool of DNA
microarray technology, we have been trying to obtain a
general picture of the health effects caused by Cd.
DNA Microarray analysis of genes affected by Cd
Received Sep 2, 2003; Accepted Sep 24, 2003
Correspondence to: S. Koizumi, Department of Hazard Assessment
National Institute of Industrial Health, 21–1, Nagao 6-chome,
Tama-ku, Kawasaki 214-8585, Japan
First, we examined the genes whose expression is
changed after exposure to a low concentration of Cd13).
A cultured cell line HeLa S3, that is derived from human
cervical carcinoma, was exposed to 5 µM CdSO4 for 6 h.
This concentration is higher than the known blood
concentrations in occupationally exposed workers2, 14), but
did not affect cell viability within the period of
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J Occup Health, Vol. 45, 2003
Figure. Genes upregulated and downregulated by Cd
Genes the expression of which was altered by exposure to 5 µM CdSO4 for 6 h
are summarized. Upregulation and downregulation are indicated by upward
and downward arrows, respectively.
experimental exposure. RNA was prepared from
unexposed and Cd-exposed cells by the acid guanidiniumphenol-chloroform (AGPC) method15), and was inspected
to be free of degradation by agarose gel electorphoresis
followed by ethidium bromide staining, as well as
northern blotting with probes specific to a few mRNA
species. With these samples, DNA microarray analysis
was carried out by Incyte Genomics, Inc. (St. Louis,
MO, USA). Messenger RNAs from unexposed and Cdexposed cells were reverse-transcribed into complementary DNAs (cDNAs), that were simultaneously
labeled with fluorescent dyes Cy3 and Cy5, respectively.
The DNA microarray slide used for the target gene search
contained 7,075 spots of human DNA. The mixture of
the labeled cDNAs from unexposed and Cd-exposed cells
was hybridized with the DNA probes on the array. The
fluorescence intensity of the spots was measured for Cy3
and Cy5, and the induction ratio of each gene was
calculated based on these signals. For the exposure at a
higher concentration of Cd (50 µM), analysis was carried
out in the same way with a microarray slide with 9.182
genes.
Gene expression altered by a low concentration
of Cd
The numbers of genes induced and repressed by 5 µM
Cd twofold or greater were 46 and 10, respectively, out of
the 7,075 genes on the array13). The genes with relatively
high fluorescence signals (and therefore considered to be
more reliable) are shown in the figure. Some of these
genes were functionally related to protection and damage
control. Cd also affected genes involved in signaling,
which may be relevant to the control of the above and/or
other genes. The induction ratio is indicated in parenthesis
for each gene mentioned below.
(a) Genes relevant to protective functions
As expected from previous findings4), several stress
response genes were induced13). With regard to MTs,
isoform genes including the MT-IL (58.8 x), MT-IE (6.5
x), MT-IB (5.3 x) and MT-III (3.8 x) genes were induced.
Since the MT-IL gene is considered to be nonfunctional16), and the expression of the MT-III gene is
almost restricted to brain and unaffected by Cd17, 18), the
apparent expression of these genes is possibly due to
cross-hybridization between isoform sequences closely
related to each other. Similar artifacts can often occur in
microarray experiments, which must always be carefully
inspected. It should also be noted that not all the MT
isoform genes were present on the microarray slide used,
that is, the absence of certain isoforms in the figure does
not always mean that they are not expressed in HeLa cells.
It has been suggested that Cd exposure generates
reactive oxygen species (ROS) through indirect
Shinji KOIZUMI, et al.: Gene Expression Affected by Cadmium
mechanisms such as depletion of antioxidant molecules19).
Consistent with this, several genes that have anti-oxidative
functions20) were upregulated, including those coding for
the heavy and light chains of ferritin (protein limiting the
availability of Fe for oxygen radical formation; 3.4x and
2.1x, respectively), Mn SOD (Mn superoxide dismutase:
enzyme reducing superoxide generated in mitochondria;
2.5x) and γGCS (γ-glutamylcysteine synthetase: key
enzyme of glutathione biosynthesis; 2.0x).
(b) Genes involved in damage control
A series of HSP genes, which code for chaperones and
co-chaperones required to renature damaged proteins21–23),
were upregulated. These include the genes encoding
HSP70–1 (11.6x), HSP70–6 (7.7x), HSP70–5 (7.1x),
HSP110α (4.4x), HSP40 (2.8x), Hop (HSP70/HSP90
organizing protein; 2.4x), annexin I (2.1x), HSP90β
(2.0x) and HSP60–1 (2.0x).
It was found that several genes involved in the ubiquitin
system24) were induced: the genes coding for ubiquitin B
(signal protein for proteolysis; 2.2x), Nedd4 (neuronal
precursor cell-expressed developmentally down-regulated
4: ubiquitin-protein ligase; 2.1x), Mif1 (involved in
ubiquitin-dependent proteolysis; 2.2x), and Cacy BP
(calcyclin binding protein: involved in ubiquitindependent proteolysis; 2.1x). These genes are thought
to play a role in the degradation of proteins unable to be
renatured. Interestingly, it has been reported that the
ubiquitin system is important for resistance to Cd in
yeast25). Expression of the ataxin-2 (2.9x), reported to be
involved in apotosis26), was also enhanced.
(c) Genes involved in signaling
We also found that Cd affects a group of genes that
code for proteins related to signal transduction13). This
includes the genes coding for RXRγ (retinoid X receptor
γ : nuclear receptor; 2.4x), IL-1α (interleukin-1 α:
cytokine; 2.1x), C-193 (cytokine inducible nuclear protein;
2.1x), importin β2 (nuclear transport receptor; 2.0x), vabl2 (v-abelson murine leukemia oncogene homolog 2
product: homolog of tyrosine kinase; 2.0x) , RAI3 (retinoic
acid induced 3: G-protein; 2.0x) and rit42 (reduced in
tumor 42kd: stress-inducible protein causing growth arrest;
2.0x) that were upregulated, and NKG2D (neutral killer
group 2D: surface receptor of natural killer cells; 0.34x)
that was downregulated. These genes may be relevant to
the regulation of the Cd-inducible genes identified in this
study and/or other Cd-regulated genes (Figure). Although
at present it is unclear with what regulatory pathways the
signaling proteins are concerned, these findings will be
useful for the elucidation of Cd signaling.
(d) Other genes
In addition, the altered expression of genes that code
for several metabolic enzymes and other proteins was
333
detected 13) . These genes were those coding for
spermidine/spermine acetyltransferase (key enzyme of
polyamine catabolism; 4.4x), keratin 17 (epithelial
structural protein; 2.5x), ATA1 (amino acid transporter
system A1: transporter of amino acids; 2.3x), 1,4α-glucan
branching enzyme 1 (glycogen branching enzyme; 2.3x),
bactercidal/permeability-increasing protein (antibacterial,
endotoxin-neutralising protein; 2.3x), cytosolic malic
enzyme (enzyme producing NADH; 2.2x) and lysine
hydroxylase 2 (enzyme catalyzing hydroxylation of Lys
in collagen; 2.1x) that were upregulated, and MATIIα
(methionine adenosyltransferase IIα: enzyme synthesizing
S-adenosylmethionine; 0.37x) that was downregulated.
These changes may reflect the detrimental effects of Cd or
recovery processes from them.
Gene expression profile after exposure to a high
concentration of Cd
We are also trying to list up genes affected by a higher
concentration of Cd. RNA extracted from HeLa cells
exposed to 50 µM CdSO4 for 6 h was subjected to DNA
microarray analysis as well. Although the outcome is
now under inspection and analysis (Yamada and Koizumi,
to be published), its outline is as follows. Out of 9,182
genes on a human DNA array, 82 genes were upregulated
and 75 genes were downregulated twofold or greater.
(i) As compared with the low dose exposure, further
activation of genes involved in protection and damage
control was observed. The number of induced genes was
increased, and many of the genes activated by 5 µM Cd
showed higher induction ratios.
(ii) Gene expression appeared to be directed toward
growth inhibition, protein degradation and apoptosis.
(iii) Expression of a lot of genes involved in signaling
and transcriptional activation was altered.
Seven genes whose expression had been observed to
increase in this study were further inspected for their
induction by northern blotting. All of these genes were
confirmed to be Cd-inducible, demonstrating high
reliability of the microarray analysis and data handling.
Usually higher induction ratios were obtained in the
northern blot analysis.
Summary
Our studies successfully detected known Cd-inducible
genes such as the MT and HSP genes, demonstrating the
DNA microarray screening functioned well. In addition,
the expression of many genes relevant to recovery from
damage and signaling was newly found to be modulated
by Cd. It appeared that a number of cellular functions
were synchronously directed to survival, to resist the
detrimental effects caused by Cd. For many of these
changes in gene expression, their biological significance
remains ambiguous. However, they are expected to serve
as important clues to depict a general picture of Cd
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J Occup Health, Vol. 45, 2003
response. Recently, microarray screenings of genes
affected by Cd have been reported also by other
laboratories, although these studies were less
comprehensive in the numbers of genes examined. They
used different biological sources such as a human T cell
line27) and mouse liver28). Several of the genes detected
in these studies were observed in common with our study,
but there were many genes unique to each screening.
Although careful inspection is required before concluding
the validity of altered expression, their data as well as
ours might provide information about the tissue-specific
effects of Cd.
Acknowledgments: The authors thank Mrs. K. Suzuki
for assistance in preparing the manuscript.
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