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Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line
Zuzana Knazicka ª , Norbert Lukac ª , Zsolt Forgacs b , Eva Tvrda ª , Jana Lukacova ª , Jana Slivkova ª , Łukasz Binkowski “ & Peter Massanyi ª
b Department of Animal Physiology , Slovak University of Agriculture , Nitra , Slovak Republic National Institute of Chemical Safety , Budapest , Hungary
” Institute of Biology, Pedagogical University of Cracow , Cracow , Poland Published online: 17 Dec 2012.
To cite this article: Zuzana Knazicka , Norbert Lukac , Zsolt Forgacs , Eva Tvrda , Jana Lukacova , Jana Slivkova , Łukasz Binkowski & Peter Massanyi (2013) Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental Engineering, 48:3, 348-353, DOI: 10.1080/10934529.2013.726908
To link to this article: http://dx.doi.org/ 10.1080/10934529.2013.726908
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Effects of mercury on the steroidogenesis of human adrenocarcinoma (NCI-H295R) cell line
ZUZANA KNAZICKA1, NORBERT LUKAC1, ZSOLT FORGACS2, EVA TVRDA1, JANA LUKACOVA1, JANA SLIVKOVA1, ŁUKASZ BINKOWSKI3 and PETER MASSANYI1
1 Department of Animal Physiology, Slovak University of Agriculture, Nitra, Slovak Republic
2 National Institute of Chemical Safety, Budapest, Hungary
3Institute of Biology, Pedagogical University of Cracow, Cracow, Poland
In this study the NCI-H295R human adrenocortical carcinoma cell line was used as an in vitro biological model to study the effect of mercury (HgCl2) on the steroidogenesis. The cells were cultured for 48 h with addition of 1.0; 5.0; 25; 50 or 100 M of HgCl2 and compared to control. Cell viability was measured by the MTT (metabolic activity) assay estimation for the mitochondria structural integrity. Quantification of testosterone and progesterone directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Concentration-dependent depression in testosterone production was detected particularly for higher concentration of Hg2+. Progesterone production was also decreased, but at the lower concentrations (1.0 and 5.0 (M) of Hg2+ this decline was lower compared to depression of testosterone. The cell viability significantly decreased at 25 µM and higher concentration of Hg2+. However, at 25 pM Hg2+ exposure the cell viability remained relatively high (> 80%). Results of the study indicate dose- dependent decreases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl2 exposure. The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology.
Keywords: Mercury chloride, steroid hormones, cell viability, cell line H295R.
Introduction
Currently, there is increasing evidence that various chem- icals introduced in the environment have the potential to disrupt the endocrine system, which may result in adverse effects on sexual differentiation, growth, and development. It is possible for certain environmental contaminants (i.e., metals) to cause or contribute to a hormonal disruption and to interfere with the function of key enzymes involved in steroid synthesis.[1] Changes in the endocrine system are useful indicator of metal exposure and potential toxicity.[2]
Mercury (Hg) is one of the oldest toxicants known[3] and is considered to be a risk factor of the environment and food chain.[4,5] Mercury occurs as elemental Hg as well as in inorganic and organic compounds, although all having different toxicological properties. This heavy metal is circulated naturally in the biosphere. In addition Hg is released into the environment each year by human activi- ties, such as combustion of fossil fuels and other industrial
releases.[6] Nevertheless, recent studies indicate that anthro- pogenic sources have the greatest contribution in the envi- ronment.[7] The usual way of human exposure to Hg is via food, dermal absorption or by inhalation of vapor.[3]
Mercuric chloride (HgCl2) is a highly reactive com- pound, which can harm cells by a variety of mechanisms including direct interactions with sulfhydryl (SH-) groups of proteins and enzymes.[8] Mercuric ion (Hg2+) is able to form many stable complexes with biologically important substances, such as SH- groups. The affinity of mercury for SH- groups is a major factor underlying the biochemi- cal properties of Hg and mercury compounds. [6] Mercury binding to these groups can produce changes in protein structure and alter binding conditions in prosthetic enzyme groups[9] and block receptor bindings[10] as well as K+ or Ca2+ ion flows in the cell membranes.[11,12]
This can affect the cell membrane potential, intracel- lular and intercellular signaling. Mercury is a potential neurotoxic agent[13], which accumulates in female follicu- lar fluid [14], liver, kidney[15,16] and muscle.[17] Its presence in agricultural systems is of concern due to its potential tox- icity.[5] Exposure to a high concentration of Hg causes an increase in reproductive problems[18], which can be reflected in the process of steroidogenesis. The endocrine disruptive effects of Hg have recently become one of the major public
Address correspondence to Norbert Lukac, Department of An- imal Physiology, Faculty of Biotechnology and Food Sciences Slovak University of Agriculture in Nitra, Tr. A., Hlinku 2 Nitra, SK-94976, Slovak Republic; E-mail: norolukac@gmail.com Received February 22, 2012.
concerns. There is sufficient evidence from animal studies supporting the disruptive effects of Hg on the functions of the thyroid, adrenal gland, ovary and testis, although sev- eral factors make it difficult to extrapolate the animal data to humans.[3]
In the present study the human adrenocortical carci- noma cell line H295R was used as a model system for de- tection of the toxic effect of HgCl2 on the production of sex steroid hormones in vitro. This cell line was derived from NCl-H295 cells, which were established from a primary hormonally active adrenocortical carcinoma. The H295R cell line expresses genes (CYP11A, CYP11B1, CYP11B2, CYP17, CYP19, CYP21) that encode all the key enzymes (3B-HSD1,38-HSD2, 178-HSD1, 178-HSD2) of steroido- genesis.[19,20]
This is a unique property because in vivo expression of these genes is tissue and developmental stage-specific. These cells represent unique in vitro model system having the ability to produce all steroid hormones found in the adult adrenal cortex and the gonads, allowing testing the effects of corticosteroid synthesis and the production of sex steroid hormones.[19] Another advantage of the H295R cell bioassay is that it can be used to evaluate the enzy- matic activities of steroidogenic genes.[21,22] The objective of our study was to determine the effects of various con- centrations of Hg2+ on the steroidogenesis in the H295R adrenocarcinoma cell line.
Materials and methods
Cell culture
The H295R human adrenocortical carcinoma cell line (ATCC # CRL-2128) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in a Good Laboratory Practice (GLP) certified laboratory (National Institute of Chemi- cal Safety, Budapest; OGYI/31762-9/2010) according to previously established and validated protocols.[21-24]
After initiation of the H295R culture from the original ATCC batch cells were cultured for five passages and these cells were splitted and frozen down in liquid nitrogen. Cells for the experiments were cultured for a minimum of five additional passages using new H295R batches from frozen stocks prior to initiation of the exposure studies.
The H295R cells were grown in 75 cm2 plastic cell cul- ture flasks (TPP Techno Plastic Products AG, Switzerland) in an incubator at 37℃ with a 5% CO2 atmosphere. The cells were grown in a 1:1 mixture of Dulbecco’s Modi- fied Eagle’s Medium and Ham’s F-12 Nutrient mixture (DMEM/F12; Cat. # D-2906; Sigma, St. Louis, MO, USA) supplemented with 1.2 g/L NaHCO3, 5 mL/L of ITS+ Premix (Cat. # 354352; BD Bioscience, San Jose, CA, USA) and 12.5 mL/L of BD Nu-Serum (Cat. # 355100; BD Bio- science). The medium was changed 2-3 times per week and
cells were detached from flasks for sub-culturing using ster- ile 0.25% trypsin-EDTA (Cat. # D-8537; Sigma).
Cell density was determined using a hemocytometer and adjusted with culture medium to a final concentration of 300,000 cells/mL. The cell suspensions were plated (with final volume of 1.0 mL/well) into sterile plastic 24-well plates (TPP) for steroid measurements. For cytotoxicity evaluation the cells (100 [L /well) were seeded into 96-well plates (TPP). The seeded plates were incubated at 37℃, with a 5% CO2 atmosphere for 24 h to allow the cells to attach to the wells.
In vitro exposure
After a 24-h attachment period the cell culture medium was removed from the plates and replaced with new medium supplemented with 1; 5; 25; 50 or 100 M mercury chloride (HgCl2; Sigma). Cell cultures were maintained for 48 h.
Cytotoxicity evaluation
The viability of the cells exposed to HgCl2 was evaluated by the metabolic activity (MTT) assay.[25] This colorimetric as- say measures the conversion of a yellow tetrazolium salt (3- (4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), to blue formazan particles by mitochondrial suc- cinate dehydrogenase of intact mitochondria of living cells. Formazan was measured spectrophotometerically.
Following the termination of HgCl2 exposure, the cells were stained with MTT (Cat. # M-2128; Sigma) at con- centration 0.2 mg/mL. After 2 h incubation (37℃, with a 5% CO2 atmosphere), the cells and the formazan crys- tals were dissolved in 150 pL of acidified (0.08 M HCI) isopropanol. The optical density was determined at a mea- suring wavelength of 570 nm against 620 nm as reference by a microplate reader (Anthos MultiRead 400, Austria). The data were expressed in percentage of the control (i.e., opti- cal density of formazan from cells not exposed to HgCl2).
Hormone measurement
At the end of 48 h HgCl2 exposure, the aliquots of the culture medium were removed from the 24-well cell cul- ture plates and after centrifugation the supernatant was collected and frozen at -80℃ until steroid measurements. Quantification of testosterone (T) and progesterone (P) di- rectly from the aliquots was performed using enzyme linked immunosorbent assay (ELISA). The ELISA kits (Cat. # K00234 and K00225) were purchased from Dialab GmbH (Austria). According to the manufacturer’s data the sen- sitivity of T assay is 0.075 ng/ml, and the intra- and inter-assay coefficient of variations are 4.6 and 7.5%, re- spectively. Cross-reactivity with 5a-dihydroxytestosterone is 16%. The sensitivity of the P assay was 0.05 ng/ml, and the intra- and inter-assay coefficients of variation were ≤ 4
and ≤ 9.3%, respectively. The absorbance was determined at a wavelength 450 nm using an Anthos MultiRead 400 (Biochrom Ltd, UK) microplate reader and the data were evaluated by WinRead 2.3 computer software. The data were expressed in percentage of the untreated controls.
Statistical analysis
Obtained data were statistically analyzed by the PC pro- gram GraphPad Prism 3.02 (GraphPad Software Incorpo- rated, San Diego, California, USA). Descriptive statistical characteristics (mean, minimum, maximum, standard devi- ation and coefficient of variation) were evaluated. One-way analysis of variance (ANOVA) and the Dunnett’s multiple comparison test were used for statistical evaluations.
Results
Cell viability
The viability of cells significantly decreased at 25 uM or higher concentration of Hg2+. However, at 25 pM Hg2+ ex- posure the cell viability was relatively high (> 80%) (Fig. 1).
Sexual steroid production
In the 48 h incubated H295R cell cultures significant concentration-dependent depression in T production was detected even at 1.0 pM (4.85 ± 2.45 ng/ml) or higher concentration of Hg2+ (Fig. 2; Table 1). The control mean
optical density % of control
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testosterone % of control
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HgCl2 [uM]
terstosterone production (100%) was 12.21 ± 4.90 ng/ml (Table 1). The progesterone (P) production was also de- creased, but at the lower concentrations (1.0 and 5.0 pM) of Hg2+ this decline (to 93.42-0.05%) was less pronounced comparing to depression of testosterone (Fig. 3). The con- trol mean P production (100%) was 18.53 ± 4.80 ng/ml (Table 2).
Discussion
The results of present study indicate dose-dependent de- creases in both testosterone and progesterone production of H295R cell culture following a 48 h in vitro HgCl2 ex- posure detected at low concentration, which does not elicit
| Control | 1.0 | 5.0 | 25 | 50 | 100 | |
|---|---|---|---|---|---|---|
| Ctrl | E | D | C | B | A | |
| Groups | HgCl2 (µM) | |||||
| x [ng/ml] | 12.21 | 4.85 ** | 5.51 ** | 1.06' | 0.36* | 0.88 ** |
| minimum | 6.69 | 2.59 | 2.84 | 0.89 | 0.10 | 0.05 |
| maximum | 18.5 | 8.29 | 8.24 | 1.38 | 0.78 | 2.05 |
| S.D. | 4.90 | 2.45 | 2.59 | 0.22 | 0.30 | 0.92 |
| CV (%) | 40.16 | 50.51 | 47.04 | 20.27 | 83.40 | 104.53 |
| % | 100 | 39.72 | 45.13 | 8.68 | 3.0 | 7.2 |
Legend: x - mean, S.D. - standard deviation, CV (%) - coefficient of variation.
** (P < 0.01); * (P < 0.05).
progesterone % of control
125
100
*
75
50
25
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-
**
**
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HgCl2 [uM]
significant cytotoxic action. Reports concerning the effects of Hg (in the form of HgCl2) are sufficiently presented at cellular and molecular levels,[7] but research in the area of hormonal system is insufficient. Therefore, the general ob- jective of this study was to provide other information of its impact on the steroidogenesis.
Mercury can affect multiple points of the steroidogen- esis pathway, inhibiting enzymes important for hormone synthesis. [7] According to McVey et al. [25] Hg is capable of directly inhibition of enzymes within the steroidogene- sis pathway, leading to decreased hormone production. Our results also report a direct toxic action of Hg on the steroid- producing cells and subsequent changes in hormone con- centration. Vachhrajani and Chowdhury [26] examined the testicular steroidogenesis after an intraperitoneal adminis-
| Groups | Control | 1.0 | 5.0 | 25 | 50 | 100 |
|---|---|---|---|---|---|---|
| Ctrl | E | D | C | B | A | |
| HgCl2 (p.M) | ||||||
| x [ng/ml] | 18.53 | 11.79* | 17.31 | 1.43 ** | 0.007 ** | 0.01 ** |
| minimum | 12.14 | 7.92 | 11.88 | 1.09 | 0.00 | 0.00 |
| maximum | 23.58 | 15.21 | 22.24 | 2.02 | 0.02 | 0.02 |
| S.D. | 4.80 | 2.99 | 4.37 | 0.41 | 0.009 | 0.01 |
| CV (%) | 25.89 | 25.40 | 25.26 | 28.65 | 127.66 | 115.47 |
| % | 100 | 63.63 | 93.42 | 7.72 | 0.38 | 0.05 |
x - mean, S.D. - standard deviation, CV (%) - coefficient of variation. ** (P< 0.01); * (P < 0.05).
tration of HgCl2 (5.0; 10 µg/kg) and methylmercury (50; 100 µg/kg of MeHg) for 90 days. Both (HgCl2, MeHg) inhibited the activity of 3B-hydroxysteroid dehydrogenase (3B-HSD) in the rat, leading to a significant decrease in serum testosterone levels and an induced cellular disinte- gration of Leydig cells.
Nishiyama et al. [27] reported that the adrenocorti- cotropic hormone (ACTH) production in rat adrenal corti- cal cells was not affected by mercury application at 1.0 p.M. In contrast, when the concentration reached 100 pM, mer- cury exerted adverse effects on the viability of isolated rat adrenal cortical cells. Thus, the concentration of mercury applied to the culture system seems to be critical. There was a reduction in ACTH-stimulated corticosterone pro- duction by adrenal decapsular cells as well as luteiniz- ing hormone-stimulated testosterone production by Leydig cells.[3, 28] Our presented data showed, that all concentra- tions of HgCl2 (1.0-100 [M) inhibited the release of testos- terone by the H295R cell line. Disorders of the testosterone synthesis could result in a reduction of the activity of the key enzymes implied in the biosynthesis of testosterone.[25] Similar results were also found in our previous study with cadmium on the human cell line H295R.[29]
The extent of the adverse effects induced by Hg depends on the chemical form of mercury at the time of exposure, dose, duration, route of administration [6,30] and various an- imal species.[31] Our experiment found that the exposure of human adrenocarcinoma cells to Hg depends on dose. The observed data demonstrated a negative influence of high Hg doses (≥25 [M HgCl2) on the progesterone release. Burton and Meikle[32] found that mitochondrial conversion of cholesterol to pregnenolone was inhibited in testicular tissue following mercury (MeHg) exposure and this could inhibit steroidogenesis. Veltman and Maines[33] studied the synthesis of corticosterone in rats following exposure to HgCl2, and determined that there was an increase in spe- cific cytochrome P450 enzymes (CYPs) in the mitochon- drial fraction of the adrenal glands, which in turn caused an increase in side-chain cleavage of cholesterol and a sev- enfold increase in the rate of production of pregnenolone.
Mercury blocks the conversion of 17a-hydroxy progesterone to 11-deoxycortisol and ultimately corticos- terone. Steroid 21-hydroxylase (CYP21) contains more free cysteine sulfhydryl residues within its active site than any other CYPs in the adrenal cortex, thus Hg could bind to SH- groups in the active site and block 17a- hydroxyprogesterone from binding. As a result, an increase in plasma progesterone could then be observed, since it is not being converted to cortisol by CYP21. Mercury as an environment risk factor has general cytotoxic effects, which may be shown in certain endocrine tissues. [7] Therefore, in this study the effects of various concentrations of Hg on the survival of human adrenocarcinoma cells were tested. The cytotoxic effect was detected at doses ≥25 pM of HgCl2. This result is in agreement with previous studies indicating
the high degree of Hg toxicity to human liver carcinoma cells.[34]
Additionally, Hg induced the cytotoxicity of primary neuronal cells.[35] The measurement of cell viability and in vitro sexual steroid production proved to be sensitive for assessing a direct action of environmental chemical factors. Ng and Liu[28] found that HgCl2 exposure led to a reduced cell viability in the adrenal glands of rats, which was linked to a decreased corticosterone production. The authors ex- plained that HgCl2 had a specific toxic mechanism on the adrenal glands as well as the Leydig cells of the testis.
Conclusion
The results suggest that Hg has detrimental effects on steroid hormone synthesis also at very low concentrations and consecutively on reproductive physiology. Results of this study clearly indicate endocrine disruptive and repro- ductive toxicological effects of Hg2+ far below its cyto- toxic concentration. Testosterone production seemed more vulnerable than progesterone to Hg2+ exposure suggesting multiple sites of action of this metal ion in steroidogenesis. Further studies are needed to clarify the precise mechanism of action of Hg2+ on the sexual steroid production.
Acknowledgments
This work was supported by the Scientific Agency of the Slovak Republic VEGA No. 1/0532/11 and KEGA No. 013SPU-4/2012.
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