Journal of Inorganic Biochemistry 117 (2012) 140–146

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Bis(L-cysteinato)zincate(lI) as a coordination compound that induces

metallothionein gene transcription without inducing cell-stress-related
gene transcription
Tomoki Kimura a,⁎, Kengo Yoshida b, c, Chika Yamamoto d, Minako Suzuki a, Tomoko Uno a, Masakazu Isobe a,
Hiroshi Naka e, Shuji Yasuike f, Masahiko Satoh g, Toshiyuki Kaji h, Masanobu Uchiyama b, c
a
Department of Toxicology, Faculty of Pharmaceutical Sciences, Setsunan University, 45–1, Nagaotoge-cho, Hirakata, Osaka 573–0101, Japan
b
Advanced Elements Chemistry Laboratory, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan
c
Advanced Elements Chemistry Laboratory, the Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351–0198, Japan
d
Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa, Ishikawa 920–1181, Japan
e
Research Center for Materials Science, Nagoya University, Chikusa, Nagoya 464–8602, Japan
f
Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa, Ishikawa 920–1181, Japan
g
Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, 1–100 Kusumotocho, Chikusaku, Nagoya 464–8650, Japan
h
Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278–8510, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Zinc is an essential micronutrient, deficiency of which results in growth retardation, immunodeficiency, and
Received 27 February 2012 neurological diseases such as dysgeusia. Several zinc coordination compounds are used for zinc supplemen-
Received in revised form 25 July 2012 tation; however, supplemented zinc ions have no specificity and interact with various groups of molecules.
Accepted 31 July 2012
Here, we found that, from a library of 30 zinc coordination compounds, bis(L-cysteinato)zincate(II), designat-
Available online 29 August 2012
ed Z01, functioned as a metallothionein (MT) inducer. Z01 induced MT expression mediated by the transcrip-
Keywords:
tion factor MTF-1, without inducing cell-stress-related heme oxygenase-1 gene expression at specific
Zn coordination compound concentration. The zinc ion was necessary for the MT induction. 65Zn incorporation following treatment
Metallothionein with 65Zn-labeled Z01 suggested that Z01 did not act as zinc ionophore despite its hydrophilicity. Electropho-
MTF-1 retic mobility shift assays revealed that Z01 facilitates MTF-1–MRE complex formation, and, by inference,
Cell-stress transfer of zinc from Z01 to MTF-1. Phosphorylated ERK levels were increased by ZnSO4 treatment but not
Transcription by Z01. Although our data do not definitely prove that Z01 is an MTF-1-specific activator, our observations
suggest that zinc coordination compounds can regulate zinc distribution and act as zinc donors for specific
molecules.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction metal response element (MRE)-binding transcription factor-1 (MTF-1)

Zinc is an essential micronutrient and plays a diverse role in many
biological processes by serving as a structural component of proteins,
an essential cofactor in enzymes, and a modulator of signal transduc-
tion cascades within the cell [1–3]. The intracellular zinc concentra-
tion is tightly regulated by various sets of proteins, including zinc
efflux and influx transporter protein families, such as the ZnT proteins
(SLC30A) and ZIP proteins (SLC39A), as well as zinc-binding proteins
and metallothioneins (MTs) [4]. Furthermore, at least three thousand
proteins have one or more zinc-binding sites [5]. The functions of
some proteins are directly modulated by zinc ions; for example,
⁎ Corresponding author at: Department of Toxicology, Faculty of Pharmaceutical
Sciences, Setsunan University, Japan. Tel./fax: +81 72 866 3107.
E-mail address: tomoki@pharm.setsunan.ac.jp (T. Kimura).
has six C2H2-type zinc fingers [6]. Zinc binding to all six fingers serves
DNA-binding ability [7].
Zinc has a variety of physiological activities. It suppresses tran-
scription factor STAT3 activation in Th17 cells [8], by blocking the
association of STAT3 with JAK kinase and gp 130. The hydrolase
(phosphatase) activity of protein tyrosine phosphatase 1B (PTP1B)
is also inhibited by zinc [9]. Zinc increases phosphorylation of recep-
tor tyrosine kinases and triggers growth factor signals. Zinc also
inhibits adenylate cyclase activity and thereby inhibits cAMP signal-
ing in N18TG2 neuroblastoma cells [10,11]. These facts suggest that
the function of zinc proteins may be modulated by intracellular zinc
trafficking in many cases. We hypothesize that zinc compounds are
useful for zinc delivery to modulate zinc protein function(s).
Specific changes in metal distribution by chemicals (coordination
compounds and metal chelators) control cellular processes. Mutation
of the ATP7B gene, which encodes a copper transporter that is
expressed in hepatocytes and effluxes excess copper into the bile,

0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.07.021
 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146
leads to Wilson's disease, which is characterized by hyper-accumulation
of copper in the liver [12]. The copper chelator D-penicillamine, which
sequesters excess hepatic copper, thereby lowering the concentration,
is an effective treatment for Wilson's disease. Water-solubilized zinc
ionophores are reported to have potential as anti-cancer agents
[13,14]. Yoshikawa et al. found that zinc compounds could function as
a new type of orally active anti-diabetic medicine [15]. Polaprezinc
(catena-(S)-[μ-[Nα-(3-aminopropionyl) histidinato(2-)-N1, N2, O: Nτ]-
zinc]), a coordination compound consisting of zinc and L-carnosine,
improves hypogeusia, a symptom of zinc deficiency, without serious
side effects [16,17]. Polaprezinc also has an anti-ulcer effect and is used
clinically to treat gastric ulcers in Japan. These examples suggest that
regulation of the intracellular distribution of metals can effectively modu-
late cellular processes. However, the targets of these chemicals, for exam-
ple the copper donor of D-penicillamine and the protein(s) that receive
zinc from these zinc coordination compounds, have not been clarified yet.
Here, to examine whether zinc coordination compounds can serve
as specific zinc protein functional modulators, we constructed a library
of zinc complexes. The transcription factor MTF-1 acts as cytosolic zinc
sensor to activate the expression of specific genes, including MT
and SLC30A1 [18]. MTF-1 has 6 Cys2His2-type zinc fingers. Canonical
Cys2His2-type zinc fingers typically bind one zinc ion with high affinity
(10−9 − 10 −12 M). Each zinc finger in MTF-1 also bind one zinc ion, but
have zinc binding affinities in the nanomolar to sub-micromoler range.
Zinc fingers in MTF-1 would be saturated with zinc under conditions of
zinc excess. Zinc-sensing transcription by MTF-1 involves zinc binding
to its unique zinc finger domain. So, MT-I mRNA levels were determined
to assess MTF-1 activation, and cell stress-mediated gene transcription
was monitored by measuring the induction of heme oxygenase 1
(HO-1) mRNA, whose transcription is mediated by the cell-stress-
related transcription factor nuclear factor-erythroid 2-related factor 2
(Nrf2) [19,20]. From the library, we found a zinc complex that activates
MT-I gene expression without inducing HO-1 gene expression. We also
determined the mechanism of this MTF-1 activation.
2. Materials and methods
2.1. Materials
Pyrithione (Py; 1-hydroxypyridine-2-thione) and zinc pyrithione
(ZnPy; 1-hydroxypyridine-2-thione zinc salt) were purchased from
Sigma (St. Louis, MO, USA). The zinc coordination compounds were
synthesized using previously described protocols referenced in Table 1
(Z01, Z02 and Z05) and Supplemental Table S1 (Z06–Z42) [21–40].
Zinc acetylacetonate (Z27) was purchased from Sigma-Aldrich.
(Toluene-3,4-dithiolato)zinc (Z28) and bis(8-quinolinolato)zinc
(Z32) were purchased from Tokyo Chemical Industry (Tokyo, Japan).
Table 1
Zinc coordination compounds.
No Name Structure
Z01 Na2[Zn(cys)2]
Z02 Zn(his)2
Z05 Zn(mal)2
141
2.2. Cell culture
Mouse embryo fibroblasts (MEFs) prepared from wild-type (WT)
mice and MTF-1 knockout (MTF-KO) mice were transformed and se-
lected as described previously [41]. MEFs were cultured in Dulbecco's
modified Eagle's medium (DMEM) — 4.5 g/L glucose supplemented
with 10% fetal bovine serum (FBS). BALB/3 T3 A31-1-1 cells, a
mouse embryonic fibroblast cell line, were provided by the JCRB,
Japan, JCRB1356. Hepa 1–6 cells (mouse hepatoma cells), HepG2
cells (human hepatoma cells), and HEK293 cells (human embryonic
kidney cells) were provided by the RIKEN BRC (Tsukuba, Japan)
through the National Bio-Resource Project of the MEXT, Japan.
These cells were cultured in DMEM — 1.0 g/L glucose supplemented
with 10% FBS at 37 °C in 5% CO2/95% air. During experiment, cells
were cultured in these medium containing FBS.
2.3. Isolation of RNA and quantification of specific mRNAs
Cells were cultured in 12-well culture plates and treated with
ZnSO4, CdCl2, or the indicated coordination compounds for 3 h before
RNA extraction. Total RNAs were isolated with Isogen (Nippon Gene,
Tokyo, Japan) according to the manufacturer's protocol. Reverse tran-
scription was carried out with random hexamers and a PrimeScript
RT reagent kit (TaKaRa Bio, Otsu, Japan). The cDNAs were subjected
to real-time PCR analysis using mouse MT-I-, human MT-IIA, and
human/mouse GAPDH-specific TaqMan® primers (Life Technologies,
Carlsbad, CA, USA), as well as human HO-1 specific primers (forward
primer: 5′-agtcaggcagagggtgatagaag-3′, reverse primer: 5′-cataaag
ccctacagcaactgtc-3 ′) and mouse HO-1 specific primers (forward
primer: 5′-acagccccaccaagttcaaacagct-3′, reverse primer: 5′-ctgtcag
catcacctgcagctcctc-3′). The amount of MT or HO-1 mRNA was normal-
ized against the amount of GAPDH mRNA.
2.4. Transfection and luciferase reporter assay
The reporter plasmid pGL4.12-MT − 264/+42 containing the
mouse MT-I promoter, which spans bases − 264 to + 42 (relative to
the transcription start site), was used for the MT-I promoter reporter
assay. The promoter constructs were cloned into a Firefly reporter
plasmid, pGL4.12[luc2CP], as previously described [41]. For transfec-
tion, FuGENE 6 (Roche, Basel, Switzerland) was used for MEFs. MEFs
were cultured in 12-well culture plates and transfected at 70%–80%
confluence using FuGENE 6 according to the manufacturer's proto-
cols. Briefly, 3 μL of FuGENE 6 was diluted with 100 μL of serum-
and antibiotic-free Opti-MEM medium (Life Technologies). Then
1.0 μg of Firefly reporter plasmid pGL4.12-MT–264/+42 and
0.25 ng of the control Renilla reporter plasmid pRL-SV40 were
Formula, MW Reference
C6H22Na2O10N2S2Zn, 457.74 [21]
C12H18N6O6Zn, 407.7 [21]
C12H15O8.5Zn, 360.64 [22]
142 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146
added; the mixture was incubated for 15 min at room temperature.
The FuGENE 6:DNA complex remained on the cells for 24 h. For the
luciferase assay, the cells were lysed using 1 × Passive Lysis Buffer
(Promega, Madison, WI, USA), and luciferase activity was measured
using the Dual-Luciferase Reporter Assay System (Promega) and a
luminometer (GloMax 20/20n Luminometer; Promega). Firefly lucif-
erase activity was normalized to Renilla luciferase activity for normal-
ization of the transfection efficiency.
2.5. Quantification of intracellular zinc
Cells were cultured in 10-cm dishes and treated with ZnSO4, Z01,
or ZnPy for 1 h before cell collection. The cell layer was washed twice
with Ca 2+, Mg 2+-free phosphate-buffered saline (PBS) and solubi-
lized in 0.5 mL SDS buffer solution (50 mM Tris, 2% SDS, 10% glycerol,
pH 6.8). The cell lysate was incubated for 10 min at 95 °C; the portion
of the lysate was degraded with nitric acid–H2O2 in an aluminum
dry-block bath at 130 °C for 2 days and then diluted with 0.1 M nitric
acid and used for determination of the zinc concentration by means of
inductively coupled plasma mass spectrometry (ICP-MS, ELAN DRC II,
PerkinElmer, MA, USA).
65
2.6. Zn incorporation assay
Radioactive 65Zn (270 MBq/nmol) was purchased from RIKEN (Wako,
Japan) as a 0.1 N HCl solution. For 65ZnSO4 treatment, the 65ZnCl2 solution
was diluted with 100 vol of 100 mM ZnSO4 (5.4 MBq/mmol). For synthe-
sis of 65Zn-labeled Z01 (65Zn\Z01), 10 pmol of 65Zn and 0.5 mmol of
ZnCl2 were used as zinc sources. MEFs were cultured in 12-well culture
plates and treated with 100 μM 65Zn-labeled Z01 (about 50 cpm/nmol)
or 65ZnSO4 (about 50 cpm/nmol) for 1 h at 4 °C, 1 h at 37 °C, 3 h at
4 °C, and 3 h at 37 °C. After the indicated times, the cells were washed
three times with ice-cold PBS. Cells were collected, and radioactivity
was measured by using a γ-counter (Auto Well Gamma System
ARC-380CL; Aloka, Tokyo, Japan).
2.7. Calculation of logP values
The logP values of Z01 and ZnPy were calculated by using the
ALOGPS 2.1 program (http://www.vcclab.org/lab/alogps/) [42].
2.8. In vitro transcription/translation
Recombinant mouse MTF-1 proteins were synthesized in vitro
with the TnT T7 quick coupled transcription/translation system
(Promega) using 1.0 μg of plasmid (pcDNA3.1/hygromycin (+) plas-
mid expressing flag-tagged mouse MTF-1) per reaction as previously
described [43].
2.9. Electrophoretic mobility shift assay
Binding reactions in the electrophoretic mobility shift assay was
performed using binding buffer containing 12 mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) (pH 7.9), 5 mM MgCl2,
60 mM KCl, 0.5 mM dithiothreitol, 2 μg of poly (dI-dC) with 0.5 μL
of recombinant MTF-1 protein in a total volume of 20 μL. The MTF-1
protein was incubated with ZnSO4, or the indicated coordination
compounds in the binding buffer for 15 min at 37 °C. Then 50 fmol
of 32P-labeled double-stranded MRE oligonucleotide (MREs-sense;
5′-GATCCAGGGAGCTCTGCACACGGCCCGAAAAGTA-3′) was added, and
the mixture was placed on ice for 30 min. After this incubation,
protein-DNA complexes were separated electrophoretically at 4 °C
on 8% nondenaturing polyacrylamide gels. The gels were dried, and
the labeled complexes were detected by radioluminography using a
Fuji BAS1500 phosphorimager (Fujifilm, Tokyo, Japan).
2.10. Western blotting analysis
MEFs were pre-cultured for 24 h in DMEM — 1.0 g/L glucose
supplemented with 1% FBS. After the pre-culture, MEFs were washed
three times with ice-cold PBS, scraped into PBS, and collected by
centrifugation. Cell lysate was prepared by sonication in lysis buffer
(20 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Triton
X-100; 2.5 mM sodium pyrophosphate; 1 mM β-glycerol phosphate;
1 mM Na3VO4; 1 mg/mL leupeptin; 1 mM phenylmethylsulfonyl fluo-
ride). Loading buffer was added to the protein extracts, and the mixture
was incubated at 65 °C for 15 min. Extract volumes equivalent to 20 μg
of protein were separated by using 10% SDS-PAGE gels, transferred
to polyvinylidene difluouride membranes, and probed using anti-
phosphorylated ERK1/2 (Cell Signaling Technology, Inc., Beverly, MA,
USA) and ERK1/2 (BD Biosciences, Franklin Lakes, NJ, USA) antibodies
followed by an anti-mouse or rabbit IgG antibody conjugated with
horseradish peroxidase. Complexes were detected with Western Light-
ning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences,
Boston, MA, USA).
2.11. Statistical analyses
Data were analyzed with Tukey's test using PASW Statistics 18
software (IBM, Armonk, NY, USA). Differences between groups were
considered significant if P b 0.05.
3. Results and discussion
3.1. Coordination compounds alter MT-I and HO-1 mRNA concentrations
in MEFs
First, we measured MT-I and HO-1 mRNA concentrations to find
MT-I mRNA-specific inducers. We treated MEFs for 3 h with the coor-
dination compounds (see Table 1 and Supplemental Table 1) at a con-
centration of 100 μM; however, when compounds showed cytotoxicity
at 100 μM, we tested them at 20, 1, or 0.1 μM. We also examined ZnSO4,
the strong MT inducer CdCl2, and the Zn ionophore ZnPy to compare
inducing capability. In our experimental condition, background zinc
concentration in culture medium was several μM. Weak MT-I mRNA
induction by 100 μM ZnSO4 was observed without HO-I mRNA in-
duction. Strong MT-I mRNA induction by 200 μM ZnSO4, 10–20 μM
CdCl2, and 100 nM ZnPy was observed with strong HO-I mRNA in-
duction. Most of the compounds induced both MT-I and HO-1
mRNA, whereas a few induced neither MT-I nor HO-1 mRNA, because
of cytotoxicity (Fig. 1A). Z02 strongly induced MT-I mRNA but with
weak HO-1 mRNA induction; this effect was not specific for MT-I
mRNA. In contrast, Z01 induced MT-I mRNA selectively at the con-
centration of 100 μM. High MT-I mRNA induction by Z01 was ob-
served after treatment at 100 μM and then plateaued (Fig. 1B). In
addition, Z01 at 200 μM and 500 μM showed induction of HO-1
mRNA and MT-I mRNA. Too much intracellular Z01 might cause
cell-stress. We also found some compounds that induced both MT
and HO-1. Z12–Z17, which are phenanthrene derivatives, and are
MT plus HO-1 inducers. Almost all phenanthrene derivatives in our
library had high MT-inducing activity and high HO-1-inducing activ-
ity and/or cytotoxicity. Polycyclic aromatic hydrocarbon metabo-
lites, produced by endogenous aldo-keto reductases (AKR), are
known to generate reactive oxygen species (ROS) and enhance
Nrf2-mediated transcription [44]. Phenanthrene derivatives might
be metabolized by AKR and generate ROS under our experimental
conditions.
3.2. The ligand of Z01 does not induce MT-I gene transcription
To examine whether zinc is essential for induction of MT-I mRNA
by Z01, we treated MEFs for 3 h with the ligand of Z01. Z01 consists
 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146 143

Fig. 1. Effect of zinc coordination compounds on MT-I and HO-1 mRNA levels. (A) MEFs were treated with the indicated concentrations of zinc coordination compounds for 3 h, and
then total RNA was isolated. MT-I, HO-1, and GAPDH mRNAs were reverse transcribed and quantified by real-time PCR. MT-I (closed bars) and HO-1 (open bars) mRNA levels were
normalized to GAPDH mRNA levels and are shown as relative values against the group treated with 20 μM CdCl2. Values are expressed as the mean± S.D. of 3–7 independent
experiments. (B) Dose response studies of MT-I and HO-1 mRNA levels treated with zinc coordination compounds for 3 h. Values are expressed as the mean ± S.D. of 3–5 indepen-
dent experiments. *, significantly different from the untreated control (P b 0.05). n.d., not determined. cytotoxic, cytotoxicity under phase-contrast microscopy was observed.

of one zinc and two L-cysteine molecules. A two-fold greater concen-

tration (200 μM) of L-cysteine relative to the concentration of Z01was
used to determine whether L-cysteine could act as an MT-I mRNA
inducer. After treatment with the ligands of Z01, Z02, and Z05, neither
MT-I nor HO-1 transcription was increased (Fig. 2). The ligand of
ZnPy, Py, markedly induced HO-1 mRNA expression with a slight
increase in MT-I mRNA expression. Zinc ions are present in the culture
medium, so Py might form ZnPy with the zinc in the culture medium.
The HO-1 mRNA level after Py treatment was higher than that after
ZnPy treatment.

3.3. Z01 increases MT-I promoter–driven luciferase reporter gene

expression and the gene expression is mediated the transcription MTF-1

We examined the effects of Z01 on MT-I promoter activation

(Fig. 3A). Treatment with 100 μM ZnSO4 increased MT-I promoter‐
driven luciferase reporter gene expression. Treatment with 100 μM
Z01 and 0.2 μM ZnPy also increased MT-I promoter‐driven luciferase
reporter gene expression. These result showed that Z01 induced
MT-I gene transcription.
Heavy metal-induced MT-I gene transcription is mediated by
MTF-1. To confirm that the Z01-induced MT-I transcription was also
mediated by MTF-1, we examined the effect of Z01 on MT-I gene tran-
scription in MEFs from MTF-1 knockout mice (MTF-1 KO MEFs). MT-I
induction was not observed in MTF-1 KO MEFs (Fig. 3B). In contrast, Fig. 2. Effect of ligands on MT-I and HO-1 mRNA levels. MEFs were treated with the
indicated concentrations of ligands for 3 h. After treatment, MT-I (closed bars) and
Z01 induced MT-I gene transcription was observed in other HO-1 (open bars) mRNA levels were measured as described for Fig. 1. Values are
MTF-1-expressing cells (BALB/3 T3 A31-1-1 cells and Hepa 1–6 expressed as the mean ± S.D. of 3–5 independent experiments. *, significantly different
cells; Fig. 3C). In human cells, MT-IIA gene transcription was well from the untreated control (P b 0.05).
144 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146

A) Luciferase assay B

Fig. 3. Effect of Z01 on MT-I promoter-driven gene transcription and MTF-1-dependent gene transcription. (A) MEFs were transfected with the Firefly luciferase reporter plasmid
pGL4.12-MT–264/+ 42 and the Renilla reporter plasmid pRL-SV40. Transfected cells were cultured for 24 h and then incubated with the indicated concentrations of Z01. Twelve
hours after treatment, reporter gene expression was measured as luciferase activity. Values are expressed as the mean ± S.D. of 3 independent experiments. *, significantly different
from the untreated control (P b 0.05). (B) MEFs and MTF-1 KO MEFs were treated with the indicated compounds for 3 h. After treatment, MT-I (closed bars) and HO-1 (open bars)
mRNA levels were measured as described for Fig. 1. Values are expressed as the mean ± S.D. of 3–6 independent experiments. *, significantly different from the untreated control
(P b 0.05). (C) BALB/3 T3, Hepa, HepG2, and HEK293 cells were treated with the indicated compounds for 3 h. After treatment, mouse MT-I mRNA (BALB/3 T3 and Hepa cells) and
human MT-IIA mRNA (HepG2 and HEK293 cells) levels were measured as described for Fig. 1. Values are expressed as the mean ± S.D. of 3–5 independent experiments.
*, significantly different from the untreated control (P b 0.05). cytotoxic, cytotoxicity under phase-contrast microscopy was observed.

studied, and the transcription also mediated by MTF-1. Z01-induced
MT-IIA gene transcription was observed in HepG2 and HEK293 cells
(Fig. 3C).
By contrast, HO-1 induction caused by Z01 treatment was ob-
served in MTF-1 KO MEFs, and the induction was much higher than
that in WT MEFs. The induction of HO-1 mRNA in MTF-1 KO MEFs
was also observed after CdCl2, ZnPy, and Z02 treatment. MTF-1 KO
MEFs express extremely low levels of MT [45]; therefore, increasing
the intracellular zinc concentration following treatment with zinc
coordination compounds might be toxic to MT-deficient cells.
3.4. Z01 does not increase intracellular zinc concentration
One hundred micromolar Z01 increased MT-I mRNA to a level that
was much higher than that induced by 100 μM ZnSO4. To examine the
possibility that the high inducibility of Z01 depends on a high intra-
cellular zinc concentration, we measured the total zinc concentration
after ZnSO4 and Z01 treatment by using ICP-MS. However, we found
that the total zinc concentration was the same following both
treatments (Fig. 4A). The intracellular total zinc concentration is
100–500 μM [46]. Zinc ions, supplied as ZnSO4, are incorporated via
zinc importers (ZIPs) [4]. Long-term (24 h) zinc treatment increases
the intracellular zinc concentration [47]; however, a 3-h treatment
with ZnSO4 may be too short to increase the intracellular total zinc
concentration. The intracellular free zinc concentration is estimated
to be ~ 0.4 nM [46], which means that only a small portion of zinc
ions exists as free zinc ions. An increase in the intracellular free zinc
concentration could occur without an increase in the total intracellular
zinc concentration. We measured the intracellular 65Zn concentration
after 1-h and 3-h 65ZnSO4 and 65Zn\Z01 treatments to evaluate the
rate of incorporation. 65Zn was incorporated 3 h after 65ZnSO4 treat-
ment at 37 °C (Fig. 4B gray bar). This increase was not observed at
4 °C (Fig. 4B open bar). A portion of the incorporated zinc ions may
bind to MTF-1 and increase MTF-1-dependent MT gene transcription. In
contrast, no significant increase in intracellular 65Zn concentration was
observed after 65Zn\Z01 treatment at both 4 °C and 37 °C (Fig. 4B
hatched and closed bar). This difference suggests that 65Zn\Z01 is in-
corporated through ZIPs-independent routes. Kim et al. reported that
 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146 145

A) Total zinc concentration B) 65Zn incorporation

Fig. 4. Zinc incorporation after ZnSO4 and Z01 treatment. (A) MEFs were treated with the indicated compounds for 3 h at 37 °C. Then, cell lysates were prepared after the cells were
washed with ice-cold PBS; the total zinc concentration of the lysates was measured by using ICP/MS. (B) MEFs were treated with 65Zn-labeled compounds for 1 h and 3 h at 4 °C
and 37 °C. Then the 65Zn concentration in MEFs was measured as radioactivity. Values are expressed as the mean ± S.D. *, significantly different from the 4 °C group (P b 0.05).

the zinc ionophore ZnPy induces an increase in the intracellular zinc The amount of zinc incorporated after 100 μM Z01 treatment might
concentration within a few minutes and that the maximum zinc con- be similar to that incorporated after 0.4 μM ZnPy treatment.
centration is achieved within 5 minutes [48]. Moreover, when logP
values were estimated by using the ALOGPS 2.1 program, we found 3.6. Possibility of Z01 on selective zinc transfer to MTF
that ZnPy gave a high value (0.58 ±2.32). In contrast, Z01 gave a low
value (−3.79 ± 2.32), suggesting that Z01 is too hydrophilic to be As mentioned previously, zinc ions interact with various sets of
absorbed via the cell surface membrane. The high inducibility of Z01 molecules and modify their functions [5,8–11,18]. Z01 might, there-
was not due to a higher intracellular zinc concentration as a result of a fore, modify the functions of not only MTF-1 but also other molecules.
higher rate of incorporation. Some researchers have studied the To assess whether Z01 selectively transfers zinc to MTF-1, we mea-
application of zinc-amino acid complexes for medical use [15] and ana- sured the phosphorylation of ERK after Z01 treatment. Zinc inhibits
lyzed zinc bioavailability after oral administration [17]. Several the phosphatase activity of PTP1B and increases the phosphorylation
zinc-amino acid complexes, including the zinc-L-cysteine complex
(Z01), showed similar bioavailability. These zinc-amino acid complexes
might be incorporated into cells via the L-type amino acid transporter
LAT [49] or the organic anion transporter OAT [50]. Further investiga-
tion is needed to clarify the role of Z01 in incorporation.

3.5. Z01 facilitates MTF-1–MRE complex formation in vitro

To address the question why Z01 showed high MT-I inducibility

without a higher intracellular zinc concentration, we compared the
abilities of the coordination compounds to promote MTF-1–MRE
complex formation. MTF-1 has 6 Cys2His2-type zinc fingers; the coor-
dination type of these zinc fingers is Zn(S2N2). Zn(S2N2) coordination
compounds could, therefore, transfer zinc to the zinc fingers of
MTF-1. To examine this possibility, we compared the abilities of
ZnSO4, Z01, and ZnPy to promote the formation of MTF-1–MRE com-
plexes. Two micromolar Z01 was needed for the formation of the
MTF-1–MRE complex (Fig. 5). This concentration was similar to the
concentration of ZnPy needed for MTF-1–MRE complex formation.
However, for ZnSO4, 10 μM was needed for the formation of the
MTF-1–MRE complex. The concentration of Z01 was thus lower
than that of ZnSO4. ZnPy has Zn(S2O2) coordination, not Zn(S2N2)
coordination. Zinc coordination compounds, regardless of coordina-
tion type, might transfer zinc ions to MTF-1 more efficiently than Fig. 5. Electrophoretic mobility shift assays of ZnSO4−, Z01-, and ZnPy-dependent
ZnSO4. In the ZnPy treatment experiment, zinc ions may have been MRE-binding activity of MTF-1. MTF-1 was synthesized in vitro using a transcription/
translation system. Synthesized MTF-1 was adjusted to the indicated concentrations
incorporated as the coordination compound, ZnPy, at low concentra-
of ZnSO4, Z01, and ZnPy, and MRE-binding activity was measured as detailed in the
tions (0.1–0.4 μM), and the concentration of incorporated ZnPy and/or Materials and methods section. The arrow demarcates the specific MTF-1–MRE
zinc ions was sufficient to activate MTF-1–MRE binding (Fig. 1A, B). complex, and the line indicates the free MRE.
146 T. Kimura et al. / Journal of Inorganic Biochemistry 117 (2012) 140–146
Fig. 6. Time course of ZnSO4- and Z01-induced ERK1/2 phosphorylation in MEFs. MEFs
were treated with ZnSO4 and Z01 for 5 min and 10 min. Three independent MEF
lysates were subjected to SDS-PAGE and Western blotting with anti-phospho-ERK1/2
and ERK1/2 antibodies. Values shown under the top panel represent the intensity of
the phosphor-ERK1/2 band normalized to that of the ERK band for each sample;
these values are expressed as the mean ± S.D. for three independent experiments.
*, significantly different from the untreated control (P b 0.05).
of ERK [9]. We did not observe a significant increase in pERK after
treatment of cells with 100 μM Z01 (Fig. 6). This result shows that
the zinc of Z01 was selectively utilized for MTF-1 rather than for the
ERK cascade. We measured only one zinc-regulated signaling path-
way of the ERK cascade. To clarify the selectivity of Z01, further inves-
tigation is needed. However, if Z01 can modulate other zinc-regulated
signaling pathways, we can modify its structure to remove such an
effect.
An MTF-1-specific activator might be of use for cancer prevention,
because mouse strains that show higher metal- and γ-ray radiation-
responsive transcription of MTF-1, caused by single nucleotide poly-
morphisms, are resistant to γ-ray-induced lymphoma development
[51]. MT is a key factor for the detoxification of heavy metals by
chelation [52]. Moreover, overexpression of MT protects the myocar-
dium from ischemia/reperfusion injury [53]. Metallothionein is also
an anti-inflammatory mediator [54]. Furthermore, our library of zinc
coordination compounds had only 30 coordination compounds, but
it had structural variety. By using other screening methods, we
might find that one of these zinc coordination compounds regulates
zinc distribution and acts as a zinc donor for specific molecules. We
believe that our results show the potential of coordination com-
pounds as protein-specific activators/inhibitors.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2012.07.021.
Acknowledgements
We are indebted to Dr. Kazuyuki Sato (Laboratory of Pharmaceuti-
cal Chemistry, Faculty of Pharmaceutical Sciences, Setsunan Universi-
ty) for excellent technical assistance.
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