Oleh:

MERKURI PUSPASARI PROGRAM MAGISTER ILMU BIOMEDIK

FAKULTAS KEDOKTERAN UNIVERSITAS BRAWIJAYA
SUKATMAN MALANG
2019
NIM: 176070100011004
 As a result of natural and human activities, the occurrence of metals is becoming
ubiquitous and highly mobile in the environmental components. For instance,
cadmium (Cd), Cobalt (Co), manganese (Mn), lead (Pb) were extensively
detected in surface water and groundwater
Among various toxicological mechanisms of action, metals have been considered
as endocrine-disrupting chemicals (EDCs) through interference with steroid
receptors, such as estrogen receptor (ER) and androgen receptor (AR).

Our recent studies demonstrated that glucocorticoid receptor(GR) and

Introduction mineralocorticoid receptor (MR), members of steroid receptor subfamily, were
potential targets for EDCs.
GR mediates the effects of cortisol and other glucocorticoids on immune,
metabolic, endocrine and nervous systems.
MR binds to a class of mineralocorticoids, such as aldosterone, and plays
instrumental roles in Naþ/Kþ homeostasis, cell proliferation, inflammation and
fibrosis. Bellows College
Materials and methods

1. Chemicals
Cortisol (>98% pure), analytical standards of BaCl2, CdCl2, Cr(NO3)3,
CoCl2, CuCl2, Pb(NO3)2, LiCl, MnCl2, K2Cr2O7, Na2MoO4, Na2WO4,
NiCl2, SrCl2, SnCl2, Ti(SO4)2, and ZnCl2.

2. Plasmid Constructs
The expression plasmid pF25GFP-hGRa and the reporter plasmid
pMMTV-luc containing mineralocorticoid response element (MRE)
and glucocorticoid response element (GRE).

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3. Cell culture and cell proliferation assay
Chinese hamster ovary K1 (CHO-K1) cells, mouse macrophage cells
J774A.1 and human hepatocellular carcinoma cell line (SMMC-7721).
The cells were cultured in phenol red-free Dulbecco's modified eagle
medium (DMEM) supplemented with charcoal/dextran-treated FBS (pH
6-8).
The cell proliferation assays for CHOK1 and J774A.1 were conducted
after 24 h exposure.

4. Dual-luciferase reporter assays for hGR and hMR

5. Real-time quantitative PCR
6. Statistical analysis
The statistical analysis was performed using SPSS version 16.0 and
Origin 8.0.

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 (A) The highest non-cytotoxic concentrations
Results observed for CHO-K1 were 105M for BaCl2,
Cr(NO3)3, CoCl2, CuCl2, Pb(NO3)2, LiCl,
Na2WO4, SnCl2 and Ti(SO4)2, 106M for MnCl2,
NiCl2 and ZnCl2, 107M for K2Cr2O7, Na2MoO4
and SrCl2, and 108M for CdCl2.
Fig. 1. Agonistic and
(B) Agonistic effects of metals in GR reporter gene
antagonistic effects of assays. CHO-K1 cells were transiently
metals in GR transfected with pMMTV-luc, pRL-TK and
transactivation assays. pF25GFP-hGRa and then incubated with DMEM
without chemicals (negative control), 107M
cortisol (positive control), or metals at non-
cytotoxic concentrations.

(C) Antagonistic effects of metals in GR reporter

gene assays. Transiently transfected CHO-K1
cells were exposed to metals at non-cytotoxic
concentrations in presence of 107M cortisol.

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 Fig. 2. The concentration-response antagonistic effects of metals
in GR transactivation assays.

A) The concentration-response antagonistic effects of 109 to 105M BaCl2, CoCl2, CuCl2,

Pb(NO3)2.
B) The concentration-response antagonistic effects of 109 to 105M LiCl, SnCl2, and 1010
to 106M ZnCl2.

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 Fig. 3. Inhibitory effects of metals on cortisol-induced expression of
glucocorticoid-responsive genes in J774A.1 cells.

(A) The highest non-cytotoxic concentrations observed for J774A.1 cells

were 105M for BaCl2, Pb(NO3)2, LiCl, SnCl2, ZnCl2, and 106M for
CoCl2 and CuCl2.
(B) (B) The relative expression levels of GILZ in J774A.1 cells after exposure
to 107M cortisol (positive control), or metals at non-cytotoxic
concentrations in the presence of 107M cortisol for 24 h.

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 Fig. 4. Agonistic and antagonistic effects in MR transactivation assays.

(A) Agonistic effects of metals in MR reporter gene

assays. Transiently transfected CHO-K1 cells were
incubated with DMEM without chemicals (negative
control), 109.5M aldosterone (positive control), or
metals.

(B) Antagonistic effects of metals in MR reporter gene

assays. Transiently transfected CHO-K1 cells were
exposed to metals at non-cytotoxic concentrations
in presence of 109.5M aldosterone.

(C) The concentration-response antagonistic effects of

109 to 105MPb(NO3)2, LiCl, SnCl2, 1010 to 106M
MnCl2, and 1012 to 108M CdCl2.

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 Fig. 5. Effects of metals on the aldosterone-induced
SMMC-7721 cell growth suppression.

A. The highest non-cytotoxic concentrations observed for SMMC-7721 were 108M for CdCl2, 106M for Pb(NO3)2, LiCl
and SnCl2, or 109M for MnCl2.
B. SMMC-7721 cells were incubated with DMEM without chemicals (negative control), 106M aldosterone (positive
control), or the combination of 106 M aldosterone and metals at non-cytotoxic concentrations.

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THANK YOU…..