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Endocrine disruptive effects of cadmium on steroidogenesis: Human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing
Zuzana Knazickaª, Zsolt Forgacs”, Jana Lukacovaª, Shubhadeep Roychoudhury“, Peter Massanyiª & Norbert Lukaca
Department of Animal Physiology, Slovak University of Agriculture, Nitra, Slovak Republic
b Department of Molecular and Cell Biology, National Institute of Chemical Safety, Budapest, Hungary
Department of Life Science and Bioinformatics, Assam University, Silchar, India Published online: 27 Feb 2015.
To cite this article: Zuzana Knazicka, Zsolt Forgacs, Jana Lukacova, Shubhadeep Roychoudhury, Peter Massanyi & Norbert Lukac (2015) Endocrine disruptive effects of cadmium on steroidogenesis: Human adrenocortical carcinoma cell line NCI- H295R as a cellular model for reproductive toxicity testing, Journal of Environmental Science and Health, Part A: Toxic/ Hazardous Substances and Environmental Engineering, 50:4, 348-356, DOI: 10.1080/10934529.2015.987520
To link to this article: http:// dx.doi.org/10.1080/10934529.2015. 987520
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Endocrine disruptive effects of cadmium on steroidogenesis: Human adrenocortical carcinoma cell line NCI-H295R as a cellular model for reproductive toxicity testing
ZUZANA KNAZICKA1, ZSOLT FORGACS2, JANA LUKACOVA1, SHUBHADEEP ROYCHOUDHURY3, PETER MASSANYI1 and NORBERT LUKAC1
1 Department of Animal Physiology, Slovak University of Agriculture, Nitra, Slovak Republic
2 Department of Molecular and Cell Biology, National Institute of Chemical Safety, Budapest, Hungary
3 Department of Life Science and Bioinformatics, Assam University, Silchar, India
Cadmium (Cd) is a known endocrine disruptor with the ability to affect the production of hormones involved in the regulation of reproductive processes. In this study human adrenocortical carcinoma cell line NCI-H295R was used as an in vitro biological model to study the effect of cadmium (CdCl2) on steroidogenesis. The cell cultures were exposed to different concentrations of CdCl2 (1.90, 3.90, 7.80, 15.60, 31.20 and 62.50 p.M) and compared to control (medium without CdCl2). Cell viability was measured by the metabolic activity (MTT) assay for estimation of mitochondria structural integrity. Quantification of sexual steroid production directly from aliquots of the medium was performed by enzyme linked immunosorbent assay (ELISA). Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion in progesterone production was observed at the lower concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥ 31.20 µM) of CdCl2, which elicited significant (P < 0.01) cytotoxic action, too. Cadmium decreased testosterone release in the whole applied range even at the lower concentration of CdCl2. The release of 17B-estradiol decreased as well, but the decline was less pronounced compared to decrease of progesterone and testosterone. The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 (1.90-62.50 }M) used in the study. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and significantly (P <0.01) decreased at 15.60 p.M and higher concentrations of CdCl2. These results suggest that cadmium has endocrine disruptive effects on sexual steroid synthesis even at very low concentrations.
Keywords: Cadmium chloride, cell viability, endocrine disruption, NCI-H295R cell line steroid hormones.
Introduction
Cadmium (Cd; atomic number 48; relative atomic mass 112.40) is a toxic metal that belongs to group IIB in the periodic table. It occurs in nature at low concentrations, mainly in association with the sulfide ores of zinc (Zn), lead (Pb), and copper (Cu). However, due to the wide- spread nature of its occurrence, it is presented in measur- able amounts in almost everything that we eat, drink, and breathe.[1] This transition metal has been reviewed by the International Register of Potentially Toxic Chemicals of the United Nations Environment Program, and included
on the list of chemical substances considered to be poten- tially dangerous at the global level.[2]
Exposure to Cd occurs as a result of atmospheric emis- sion during Cd production and processing, from combus- tion of fossil energy sources, waste and sludge, phosphate fertilizers, and deposition of waste and slag at disposal sites.[3] Cigarette smoking is also a high source of Cd expo- sure. 4) Meat, fish, and fruits generally contain up to 50 µg Cd kg-1 on fresh weight basis, whereas vegetables, pota- toes, and grain products may contain up to 150 µg Cd/kg fresh weight.[3] Cadmium is an industrial and environmen- tal contaminant unique among metals because of its non- biodegradable nature, long environmental persistence, extremely protracted biological half-life, low rate of excre- tion from the body and predominant storage in soft tissue (primarily liver and kidney).[5, 6] Cadmium has been identi- fied as a human carcinogen by the International Agency for Research on Cancer17 and the National Toxicology Program.[8, 9] However, the precise mechanism of carcino- genesis caused by Cd remains unknown.[10]
Although Cd is not essential for growth and develop- ment in mammals, it generally follows the metabolic pathways of essential elements as Zn and Cu.[11, 12] Cad- mium has a strong preferential affinity for the liver and the kidney over a wide range of exposure levels. In gen- eral, about 50% of the total body burden is found in these two organs.[13] Cadmium causes tissue damage in humans, animals and many toxicological studies have found the functional and structural changes in the kid- neys, liver, lungs, bones, ovaries and fetal effects.[14-17] Reproductive organs, such as the testis and placenta, are sensitive to the toxic effects of Cd.[10] Its action may be either direct, affecting the gonads and accessory organs, or indirect via interference with the hypothalamus-pitui- tary-gonadal axis.[18]
Increasing evidence demonstrated that environmental exposure to Cd is associated with male infertility and the poor semen quality.[19, 20] Several studies showed that Cd induces apoptosis in testicular germ cells.[21, 22] As a well- known endocrine disrupting chemical, Cd not only regu- lates hypothalamus and pituitary hormone secretion, [23, 24] but also disrupts steroidogenesis including the syntheses of androgen, progesterone and estrogen, leading to suppres- sion of reproductive functions.[10, 25] This metal inhibits the expression of testicular steroidogenic acute regulatory (StAR) protein, which is responsible for the rate-limiting step in steroidogenesis.[25, 26] In addition, Cd exposure down-regulates the level of cytochrome P450 cholesterol side-chain cleavage (P450scc) enzyme, cytochrome P450 17-hydroxysteroid dehydrogenase (P450178), 17B-hydrox- ysteroid dehydrogenase (17-HSD) and other steroido- genic enzymes.[25, 27]
Sexual steroid hormones are key factors involved in the regulation of reproduction in vertebrates and are also involved in numerous other processes that are related to development and growth. Thus, chemicals that can disrupt the production of steroid hormones may be directly linked to adverse outcomes for these processes.[28] The cell lines are an ideal biological object to study the direct effects of different chemical and physical factors on steroidogenesis. Therefore, in the present study the human adrenocortical carcinoma cell line NCI-H295R was used as a model system for detection of the effect of CdCl2 on the production of sexual steroid hormones in vitro. This cell line was derived from H295 cells, which were established from a primary hormonally active adrenocortical carci- noma.[29, 30]
The NCI-H295R 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 pro- duction of sexual steroid hormones.[29] Another advantage of the H295R cell bioassay is that it can be used to evalu- ate the enzymatic activities of steroidogenic genes.[31, 32] The H295R Steroidogenesis Assay has been included in
the Tier1 Screening Battery of United Staes Environmen- tal Protection Agency (EPA) Endocrine Disruptor Screen- ing Program (EDSP). The test guideline of the H295R Steroidogenesis Assay (TG 456) was validated in Organi- zation for Economic Cooperation and Development (OECD).[33]
The objective of our study was to determine the effects of cadmium (CdCl2) on the steroidogenesis of human adre- nocortical carcinoma cell line (NCI-H295R). Specifically, we examined the dose-dependent changes of CdCl2 as endocrine disruptor in relation to release of progesterone, testosterone and 17ß-estradiol by adrenocortical carci- noma cells in vitro.
Materials and methods
Cell culture
The human adrenocortical carcinoma cell line (NCI- H295R) 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 Chemical Safety, Buda- pest; OGYI/31762-9/2010) according to previously estab- lished and validated protocols.[31-34] After initiation of the NCI-H295R culture from the original ATCC batch cells were cultured for five passages and these cells were split and frozen down in liquid nitrogen (-196℃). The cells for the experiments were cultured for a minimum of five addi- tional passages using new NCI-H295R batches from fro- zen stocks prior to initiation of the exposure studies.
The cells were grown in 75 cm2 plastic cell culture flasks (TPP Techno Plastic Products AG, Switzerland) in an incubator under standard conditions (37℃ with a 5% CO2 atmosphere). Subsequently, the cells were grown in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient mixture (DMEM/F12; Sigma- Aldrich, St. Louis, MO, USA) supplemented with 1.20 g/ L NaHCO3 (Sigma-Aldrich, St. Louis, MO, USA), 5.00 mL/L of ITS+Premix (BD Bioscience, San Jose, CA, USA) and 12.50 mL L-1 of BD Nu-Serum (BD Biosci- ence, San Jose, CA, USA).
The medium was changed 2-3 times per week and cells were detached from flasks for sub-culturing using sterile 0.25% trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA). Cell density was determined using a hemocytometer and adjusted with culture medium to a final concentration of 300 000 cells mL-1. The cell suspensions were plated (with final volume of 1.00 mL well-1) into sterile plastic 24-well plates (TPP, Grainer, Germany) for estimation of steroid hormones. For cytotoxicity evaluation the cells (100 µL well-1) were seeded into 96-well plates (MTP, Grainer, Germany). The seeded plates were incubated at 37℃ with a 5% CO2 atmosphere for 24 h to allow the cells to attach to the wells.[35]
In vitro exposure
After 24 h attachment period the cell culture medium was removed from the plates and replaced with a new medium supplemented with 1.90; 3.90; 7.80; 15.60; 31.20 and 62.50 MM cadmium chloride (CdCl2; Sigma-Aldrich, St. Louis, MO, USA). Cell cultures were set in 96-well plates (MTP, Grainer, Germany) after 48 h of CdCl2 exposure. The experimental groups (exposed to different concentra- tions of CdCl2) with control (Ctrl) (medium without CdCl2) were compared.
Cytotoxicity evaluation
The viability of the cells exposed to CdCl2 was evaluated by the metabolic activity (MTT) assay.[36] This colorimet- ric assay measures the conversion of a yellow tetrazolium salt [3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), to blue formazan particles by mitochon- drial succinate dehydrogenase of intact mitochondria of living cells. Formazan was measured spectrophotometri- cally. Following the termination of CdCl2 exposure, the cells were stained with MTT (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.20 mg mL-1. After 2 h incubation (37℃, with a 5% CO2 atmosphere), the cells and the formazan crystals were dissolved in 150 ML of acidified (0.08 M HCI) isopropanol (Central- Chem, Bratislava, Slovak Republic). The absorbance was determined at a measuring wavelength of 570 nm against 620 nm as reference by a microplate reader (Anthos Multi- Read 400, Austria). The data were expressed in percentage of the control (i.e., absorbance of formazan from cells not exposed to CdCl2).
Release of hormones
At the end of 48 h of CdCl2 exposure, the aliquots of the culture medium were removed from the 24-well cell culture plates and after centrifugation the supernatant was collected and frozen at -80℃ until steroid hor- mones measurements. Enzyme linked immunosorbent assay (ELISA) was used for the quantification of testos- terone, progesterone and 17B-estradiol directly from the aliquots of the medium. The ELISA kits were pur- chased from Dialab GmbH (Wiener Neudorf, Austria). According to the manufacturer’s data the sensitivity of testosterone assay was 0.075 ng mL-1, and the intra- and inter-assay coefficients of variation were 4.60% and 7.50%, respectively.
Cross-reactivity with 5a-dihydroxytestosterone was 16.00%. The sensitivity of progesterone assay was 0.05 ng mL-1, and the intra- and inter-assay coefficients of varia- tion were ≤ 4.00% and ≤ 9.30%, respectively. The intra- and inter-assay coefficients of variation for the 17B-estra- diol assay were ≤ 9.00% and ≤ 10.00%, and the sensitivity was 8.68 pg/mL. The absorbance was determined at
a wavelength 450 nm using an Anthos MultiRead 400 (Anthos MultiRead 400, Austria) microplate reader and the data were evaluated by WinRead 2.30 computer soft- ware. Values were expressed in percentage of the untreated controls (control groups solved as 100%).
Statistical analysis
Obtained data were statistically analyzed by the PC program GraphPad Prism 6.00 (GraphPad Software Incorporated, San Diego, CA, USA). Descriptive statis- tical characteristics (arithmetic mean, minimum, maxi- mum, standard deviation and coefficient of variation) were evaluated. One-way analysis of variance (ANOVA) and the Dunnett’s multiple comparison test were used for statistical evaluations. The level of significance was set at *** P < 0.001; ** P < 0.01 and P < 0.05. *
Results
Cell viability
The cytotoxic effect of CdCl2 was significant (P < 0.01) for all concentrations. However, the cell viability remained relatively high (>75%) up to 7.80 µM of CdCl2 and signif- icantly (P < 0.01) decreased from 15.60 µM and higher concentration of CdCl2 (Fig. 1).
150
A
absorbance % of control
100
**
**
B
**
-
50
**
**
**
0
Ctrl
1.90
3.90
7.80
15.60
31.20
62.50
CdCl2 (PM)
Release of progesterone by adrenocortical carcinoma (NCI-H295R) cells
Following 48 h culture of the cells in the presence of CdCl2 a concentration-dependent depletion of progesterone release was observed even at low concentrations of CdCl2. The lowest amount of progesterone was significantly detected in groups with the higher doses (≥31.20 M) of CdCl2 (Table 1). The control mean release of progesterone (100%) was 26.72± 9.68 ng mL-1. The percentage changes of progesterone release after CdCl2 exposure are described in Figure 2.
Release of testosterone by adrenocortical carcinoma (NCI-H295R) cells
The testosterone production was decreased as well, but this decline was more evident at 7.80 p.M (8.82 ± 2.26 ng mL-1) of CdCl2 in comparison to the decrease in proges- terone release. However, only a low testosterone release was detected at 3.90 p.M of CdCl2 (13.31± 4.65 ng mL-1), which was not significant (P > 0.05) when compared to the control (14.02± 3.15 ng mL-1) (Table 2). The percent- age changes of testosterone release after CdCl2 exposure are described in Figure 3.
Release of 17B-estradiol by adrenocortical carcinoma (NCI-H295R) cells
The 178-estradiol production also decreased (Fig. 4), but this decline was less pronounced comparing the decrease in release of progesterone and testosterone. The highest release of 17B-estradiol by adrenocortical carcinoma (NCI-H295R) cells was recorded in control (1.09± 0.31 pg mL-1). The results are shown in Table 3.
| CdCl2 (µM) | |||||||
|---|---|---|---|---|---|---|---|
| Groups | Control | 1.90 | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 |
| Ctrl | F | E | D | C | B | A | |
| x (ng/ml) | 26.72 | 19.03 | 20.97 | 22.93 | 9.85 | 5.34* | 2.49 ** |
| Minimum | 15.24 | 11.77 | 10.89 | 8.15 | 4.88 | 2.44 | 1.25 |
| Maximum | 42.88 | 34.24 | 35.07 | 32.53 | 12.25 | 6.88 | 3.88 |
| S.D. | 9.68 | 13.84 | 9.17 | 10.60 | 2.70 | 1.58 | 0.90 |
| CV (%) | 36.24 | 43.05 | 43.71 | 46.25 | 27.37 | 29.55 | 36.15 |
| % | 100.00 | 71.20 | 78.47 | 85.80 | 36.86 | 19.99 | 9.31 |
x - arithmetic mean, S.D. - standard deviation, CV (%) - coefficient of variation.
P < 0.05; ** P < 0.01; *** P < 0.001.
150
progesterone release % of control
100
50
*
**
0
Ctrl
1.90
3.90
7.80
15.60
31.20
62.50
CdCl2 (M)
Discussion
Currently, there is increased evidence that various chemi- cals introduced to the environment have the potential to disrupt the endocrine system,[37] which may result in adverse effects on differentiation, growth and develop- ment. It is possible for certain environmental contami- nants (including metals) to cause or contribute to a hormonal disruption and interfere with functions of the key enzymes involved in steroidogenesis.[38] According to several research studies, Cd can affect multiple points of the steroidogenesis pathway, inhibiting enzymes important for hormone synthesis.[10, 39, 40] Recently, the effects of Cd on steroidogenesis have been described, but results vary depending on the experimental model, time-duration of exposure and the dose used. Therefore, the general objec- tive of this study was to provide information of the impact of Cd on steroidogenesis.
Our results suggest a direct toxic action of Cd on the ste- roid-producing cells and subsequent changes in hormonal release. Cadmium decreased the release of progesterone, testosterone and 17B-estradiol in the whole applied range even at a very low concentration (1.90 M) of CdCl2, while the cell viability remained relatively high (> 75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased from 15.60 p.M and higher concentrations of CdCl2. These results clearly confirm previous reports by Forgacs et al.[41] and Ocztos et al.,[42] who observed the effect of Ni2+, Hg2+ and Cd2+ on the progesterone and testosterone production of H295R cells. Similar results were also obtained in our previous study with mercury.[35]
Cadmium disrupts steroid biosynthesis in a variety of cells.[18] The recent studies conducted using cultured
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| CdCl2 (M) | |||||||
|---|---|---|---|---|---|---|---|
| Groups | Control | 1.90 | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 |
| Ctrl | F | E | D | C | B | A | |
| x (ng/ml) | 14.02 | 9.56 | 13.31 | 8.82* | 4.10 ** | 2.65 ** | 2.21 ** |
| Minimum | 9.22 | 4.03 | 8.28 | 4.97 | 2.13 | 1.08 | 1.02 |
| Maximum | 18.50 | 14.66 | 22.24 | 12.28 | 7.12 | 4.88 | 3.88 |
| S.D. | 3.15 | 4.26 | 4.65 | 2.26 | 1.78 | 1.37 | 0.87 |
| CV (%) | 22.49 | 44.51 | 34.96 | 25.66 | 43.37 | 51.77 | 39.36 |
| % | 100.00 | 68.13 | 94.83 | 46.57 | 19.84 | 18.88 | 15.74 |
Legend: x - arithmetic mean, S.D. - standard deviation, CV (%) - coeffi- cient of variation. *P < 0.05; ** P < 0.01; *** P < 0.001.
human placental trophoblastic cells suggest that Cd reduces progesterone synthesis by inhibiting the gene expression of the low-density lipoprotein (LDL) receptor, which controls the internalization of cholesterol into ste- roidogenic cells,[43] cytochrome P450scc, which coverts pregnenolone to progesterone.[44] On the other hand, Cd administered to female rats during estrus and diestrus resulted in increased serum progesterone level[39, 45, 46] and stimulated progesterone synthesis in both cultured porcine granulosa cells[47] and JAR choriocarcinoma cells, a malignant trophoblast cell line.[48]
The results of our present study indicate dose-dependent decrease in progesterone release by NCI-H295R cell line in culture following a 48-h in vitro CdCl2 exposure. The lowest amount of progesterone was detected in groups
150
testosterone release % of control
100
*
50
**
**
**
0
Ctrl
1.90
3.90
7.80
15.60
31.20
62.50
CdCl2 (AM)
with the higher doses (≥ 31.20 [M) of CdCl2, which eli- cited significant cytotoxic action, too. Massanyi et al.[49] suggested that degeneration and luteinisation of granulosa cells also cause the progesterone production. Basal proges- terone production of cadmium-treated cells remained unchanged (20 ng mL-1) or was enhanced; this supports the theory of Cd - calcium competition.[50]
Henson and Chedrese[51] discussed the dual effects of Cd on progesterone synthesis in their review. They suggested that low concentrations of Cd stimulate P450scc gene transcription resulting in enhancement of the steroidogenic pathway, whereas high concentrations of Cd inhibit P450scc activity resulting in the suppression of progester- one synthesis. Results of the study by Smida et al.[52] sup- port the concept that depending on the concentration, Cd2+ can exert dual effects on steroidogenesis.
When used in concentrations greater than 0.6-3.0 µM, CdCl2 stimulated transcription of the P450 scc gene and the steroidogenic pathway in stable porcine granulosa cell line JC-410. At high (5.0 (M) concentration CdCl2 inhib- ited P450 scc gene promoter activity and progesterone syn- thesis and it has cytotoxic effects that produce changes in cell morphology and cell death. Dual effects of Cd2+ have been described in other experimental models as well.[53, 54] Generally, there is a lack of data describing the effect of Cd on granulosa cells[51, 52] mostly monitoring the biochemical aspects of toxicity. The effects of CdCl2 observed in the present study ranged at concentrations between 1.90 and 62.50 p.M. Based on our results, it is reasonable to think that even exposure to low concentrations of CdCl2 may be sufficient to affect the steroidogenic pathway.
The production of testosterone may be disrupted by Cd without inducing a loss of testosterone-producing cells by
150
17B-estradiol release % of control
100
50
0
Ctrl
1.90
3.90
7.80
15.60
31.20
62.50
CdCl2 (M)
| CdCl2 (AM) | |||||||
|---|---|---|---|---|---|---|---|
| Groups | Control | 1.90 | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 |
| Ctrl | F | E | D | C | B | A | |
| x (pg/mL) | 1.09 | 0.80 | 0.91 | 0.94 | 0.88 | 0.83 | 0.84 |
| Minimum | 0.53 | 0.57 | 0.88 | 0.73 | 0.70 | 0.69 | 0.58 |
| Maximum | 1.68 | 1.05 | 0.97 | 1.05 | 1.09 | 0.97 | 1.08 |
| S.D. | 0.31 | 0.16 | 0.03 | 0.13 | 0.14 | 0.15 | 0.22 |
| CV (%) | 28.28 | 20.19 | 3.54 | 14.03 | 15.43 | 18.13 | 25.87 |
| % | 100.00 | 73.69 | 84.23 | 86.75 | 80.77 | 76.44 | 77.06 |
Legend: x - arithmetic mean, S.D. - standard deviation, CV (%) - coeffi- cient of variation.
*P < 0.05; ** P < 0.01; *** P < 0.001.
necrosis.[10] Laskey and Phelps[55] examined effect of Cd2+ and other metal cations (Co2+, Cu2+, Hg2+, Ni2+ and Zn2+) on in vitro Leydig cell testosterone production. The results showed no change in Leydig cell viability with any metal cation treatment during the 3 h incubation. Dose- response depletion in both hCG- and db-cAMP-stimulated testosterone production were noted with Cd2+, Co2+, Cu2+, Hg2+, Ni2+ and Zn2+ treatment. Surprisingly, Cd2+, Co2+, Ni2+ and Zn2+, which caused a depletion in hCG- and db-cAMP-stimulated testosterone production, caused significant increases in HCHOL- and PREG-stimu- lated testosterone production over untreated and similarly stimulated cultures.
This indicated that these cations may act at multiple sites within the Leydig cell. Zeng et al.[56] reported that serum testosterone levels were significantly increased by chronic oral Cd exposure (50, 100 and 200 ppm) for 3 months. Although the mechanism underlying the Cd- induced increase in the serum testosterone levels is unclear. The authors consider that chronic oral Cd exposure might have induced endocrine homeostasis disruption through a mechanism different from that associated with other routes of Cd administration, e.g., subcutaneous or intra- peritoneal administration used in most studies.
Our presented data showed that testosterone seem to be more vulnerable than progesterone and 17B-estradiol to cadmium exposure suggesting multiple sites of action of this metal in steroidogenesis. Disorders of the testosterone synthesis could result in a reduction of the activity of the key enzymes implied in the biosynthesis of testosterone.
Reduction in testosterone hormone was manifested by lowered follicle stimulating hormone and luteinizing hor- mone plasma levels,[57-59] apart from Cd-induced decrease in total testosterone hydroxylase activity,[60] which resulted in a dramatic decrease in testosterone hormone levels. In turn, this decline may be responsible for signifi- cant decrease in the hCG-stimulated serum testosterone levels,[61] interference of Cd with cAMP in testis and
depression of protein kinase.[62] Massanyi et al.[49] exam- ined the effects of CdCl2 on ultrastructure and steroido- genesis in cultured porcine ovarian granulosa cells in vitro. Evaluation of steroidogenesis indicated that CdCl2 induces an increase in progesterone production and a decrease in 17B-estradiol production, which are required at further stages of reproduction.
Subsequently, structural and functional alterations in the ovarian granulosa cells after CdCl2 administration were confirmed. A decrease in 17B-estradiol concentrations was observed; in this respect our results are supported by those obtained by Paksy et al.[39] in human granulosa cells. Han et al.[63] showed that 10.0 mg kg-1 CdCl2 on weight gain decreases the secretion of estradiol and progesterone in serum of the pigs during long-term exposure. They con- cluded that Cd might have a direct effect on the function of granulosa cells and lutein cells because of its accumulation in the ovary, and it is also possible that cadmium produces the change of estradiol and progesterone levels through combining with their receptors. Similar results were also reported from female rats. [64, 65]
In addition, Cd acts as metallohormone, which mimics the biosynthesis of estrogen.[66, 67] This metal induces pro- liferation,[68-70] increases the transcription and expression of estrogen regulated genes such as the progesterone recep- tor, [68] and activates estrogen receptor a (ERa). [69] Accord- ing to Martin et al.[71] and Garcia-Morales et al.[68] Cd exhibited estrogen activity through an ER« in MCF-7 human breast cancer cells. Their results suggest that the effects of Cd are mediated directly by ERa and are inde- pendent of estradiol. However, additional studies are required to define the mechanism by which Cd activates steroid receptors.
The measurement of cell viability and in vitro sexual ste- roid production proved to be sensitive for assessing a direct action of environmental chemical factors. The rela- tive cytotoxicity of a variety of Cd has been evaluated in vitro using various cell types, such as alveolar macro- phages,[72] lymphocytes,[73] fibroblasts,[74] red blood cells,[75] ovarian cells,[76] and adrenocortical carcinoma cells.[77, 78] Some metals have adverse effects in experimen- tal animals but not in the cell culture model. Few studies have been conducted on the effect of divalent metals on steroidogenesis of adrenocortical carcinoma cells.[78] Therefore, this study was conducted to ascertain whether Cd has a direct toxic effect on steroidogenesis of cells iso- lated from human adrenocortical carcinoma cell line.
The cytotoxic effect was significantly (P < 0.01) detected at all concentrations of CdCl2 used in the study (1.90- 62.50 p.M). However, the cell viability remained relatively high (> 75%) up to 7.80 µM of CdCl2 and significantly (P < 0.01) decreased from 15.60 MM and higher concen- trations of CdCl2. These results are in agreement with a previous report by Tchounwou et al.[79] indicating the high degree of CdCl2 toxicity to human liver carcinoma cells (HepG2). Ng and Liu[80] noted that Cd (100 µM)
exerted an adverse effect on the viability of isolated rat adrenal capsular (zona glomerulosa), adrenal decapsular (fasciculata et reticularis) and Leydig cells of the testis, which was linked to a decreased corticosterone production and luteinizing hormone-stimulated testosterone produc- tion. The authors explained that CdCl2 had a specific toxic mechanism on the adrenal glands as well as the Leydig cells of the testis.
Conclusions
The results of the present study contribute to the knowl- edge of the effect of cadmium on steroidogenesis, and should also serve to increase the level of awareness of its effect on reproductive physiology. Data obtained from this in vitro study indicate that the release of sexual steroid hormones by adrenocortical carcinoma cells can be associ- ated with the doses of cadmium administration. Testoster- one release seemed more vulnerable than progesterone and 17-estradiol to cadmium exposure. Probably the effect of enzymatic action of 17ß-hydroxysteroid dehydro- genase is more sensitive, which results in decreased release of testosterone in comparison with progesterone/17ß- estradiol and thereby the effect of enzymatic action of 3B- hydroxysteriod dehydrogenase/aromatase.
In conclusion, the present study suggests the endocrine disruptive and reproductive toxicological effects of this metal. Cadmium toxicity may also reflect at other points of the steroidogenesis pathway. Therefore, further studies are needed to clarify the precise mechanism of action of cadmium on the sexual steroid production and their metabolites, whose production is conditioned by steroido- genic enzymes.
Funding
This work was supported by the Scientific Agency of the Slovak Republic VEGA No. 1/0857/14, by European Community under project no 26220220180: Building Research Centre, AgroBioTech and the Tatra Bank Foun- dation 2012/2013.
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