In Vitro Assessment of the Impact of Nickel on the Viability and Steroidogenesis in the Human Adrenocortical Carcinoma (NCI-H295R) Cell Line
Norbert LUKAC1, Zsolt FORGACS2, Hana DURANOVA3, Tomas JAMBOR1, Jirina ZEMANOVA1, Peter MASSANYI1, Barbara TOMBARKIEWICZ4, Shubhadeep ROYCHOUDHURY5, Zuzana KNAZICKA6
1Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic, 2Independent Researcher, Budapest, Hungary, 3AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Slovak Republic, 4Department of Zoology and Animal Welfare, Faculty of Animal Sciences, University of Agriculture in Krakow, Krakow, Poland, 5Department of Life Science and Bioinformatics, Assam University, Silchar, India, ‘Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic
Received February 14, 2020 Accepted July 8, 2020 Epub Ahead of Print September 9, 2020
Summary
Nickel is a ubiquitous environmental pollutant, which has various effects on reproductive endocrinology. In this study, human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro biological model to study the effect of nickel chloride (NiCl2) on the viability and steroidogenesis. The cells were exposed to different concentrations (3.90; 7.80; 15.60; 31.20; 62.50; 125; 250 and 500 µM) of NiCl2 and compared with control group (culture medium without NiCl2). The cell viability was measured by the metabolic activity assay. Production of sexual steroid hormones was quantified by enzyme linked immunosorbent assay. Following 48 h culture of the cells in the presence of NiCl2 a dose-dependent depletion of progesterone release was observed even at the lower concentrations. In fact, lower levels of progesterone were detected in groups with higher doses (≥125 µM) of NiCl2 (P<0.01), which also elicited cytotoxic action. A more prominent decrease in testosterone production (P<0.01) was also noted in comparison to that of progesterone. On the other hand, the release of 170-estradiol was substantially increased at low concentrations (3.90 to 62.50 [M) of NiCl2. The cell viability remained relatively unaltered up to 125 µM (P>0.05) and slightly decreased from 250 µM of NiCl2 (P<0.05). Our results indicate endocrine disruptive effect of NiCl2 on the release of progesterone and testosterone in the NCI-H295R cell line.
Although no detrimental effect of NiCl2 (≤62.50 uM) could be found on 17B-estradiol production, its toxicity may reflect at other points of the steroidogenic pathway.
Key words
Nickel chloride . Sexual steroid hormones . Cell viability . NCI-H295R cell line . Endocrine disruption
Corresponding author
H. Duranova, AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. Andreja Hlinku 2, 949 76 Nitra, Slovak Republic. E-mail: hana.duranova@uniag.sk
Introduction
Several environmental contaminants are recognized as endocrine disruptors (EDs), which may adversely affect the reproductive functions of humans, as well as wildlife species (Kabir et al. 2015, Vitku et al. 2015, Yang et al. 2015, Roychoudhury et al. 2016, Kolatorova et al. 2017, Jambor et al. 2018, Jambor et al. 2019). This heterogeneous group of exogenous substances has the ability to alter functions of the endocrine system with a subsequent negative impact on
the cellular behaviour and health in an intact organism. Endocrine disruptors may be found in a variety of products, such as pesticides, household items, cosmetics or plastic packaging. It is likely that some EDs are structural analogues of steroids, having similar effects as true hormones, high levels of which may have disproportionate consequences (Sanderson 2006, Svechnikov et al. 2010). They can strongly affect reproductive and endocrine functions in several ways (Andersen et al. 2002), either by directly affecting the hormone production through interaction with the appropriate enzymes, or through interfering with their transport to target organs to alter natural hormone metabolism or even to inactivate the function of steroidogenesis regulatory proteins (e.g. Steroidogenic Acute Regulatory - StAR) (Sanderson and van den Berg 2003).
Nickel (Ni) is a widely distributed metal that is industrially applied in various mineral forms (Lu et al. 2005). Dusts from volcanic emissions, the weathering of rocks and soils, biological cycles and solubilisation of Ni compounds from soils (Sunderman 2004) represent the main natural sources of atmospheric (7.0-12.0 ng of Ni/m3 of air; 150 ng of Ni/m3 of air near point sources) and aqueous (3-10 µg of Ni/l of water in surface water and groundwater) Ni (ATSDR 2005). Anthropogenic sources of Ni pollution include mining, smelting and refining activities, burning of fossil fuels, sewage incineration and plastic production (Yu 2005). The major source of exposure to Ni for the general population is the food chain (Pandey and Srivastava 2000, Llamas and Sanz 2008). It usually enters the body via food and water consumption, although inhalation exposure in occupational settings is the primary route for Ni-induced toxicity (Ankel-Fuchs and Thauer 1988). Based on the average levels of Ni consumption through water (4.0-8.6 µg/day) and food (69.0-162.0 µg/day), the daily per oral intake of the metal was estimated to be 0.001-0.0024 mg/kg/day for an average adult human being weighing 70 kg (ATSDR 2005). Another source of non-occupational exposure to Ni is tobacco smoking, and each cigarette is estimated to contain 1.1-3.1 µg of Ni (Cempel and Nikel 2006).
Although Ni is considered to be an essential micronutrient (Eisler 1998), it has a number of effects in the cell (Das 2009). It plays an important role in DNA, RNA and protein structure and/or function (Pandey and Srivastava 2000). Nickel also serves as a cofactor or a structural component of several metalloenzymes
(Przybyla et al. 1992). Deficiency is rare due to a low level of requirement, and relatively high availability in the diet, but experiments have shown that at cellular levels Ni deprivation may result in changes in the membrane properties and other structures (Das and Dasgupta 1997, Das 2009). On the contrary, high quantity of Ni is injurious for animal and human health (Pandey et al. 1999, Pandey and Srivastava 2000). More recently, several reports have showed that Ni is able to induce toxicological, physiological and histopathological alterations in a number of animal species (Pane et al. 2003, Bersenyi et al. 2004, Brix et al. 2004, Gupta et al. 2006, Krockova et al. 2011, Lukac et al. 2011). Soluble Ni compounds are likely to be human carcinogens (Costa 1991, Costa et al. 2005), and toxic and/or carcinogenic effects of such Ni compounds may be associated with Ni-mediated oxidative damage to DNA, proteins and inhibition of cellular antioxidant defences (Rodriguez et al. 1996). There is sufficient evidence that Ni ions (Ni2+) have potential toxic effects on the reproductive system (Das and Dasgupta 2000). Animal studies referred to the negative effects of Ni2+ on the structure and function of testis, seminal vesicles, prostate gland (Pandey et al. 1999, Forgacs et al. 2001, Massanyi et al. 2003, Massanyi et al. 2007, Zemanova et al. 2007), and spermatozoa concentration as well as motility (Das and Dasgupta 2000, Lukac et al. 2011). Nickel salts are also capable of inducing morphological changes such as, degeneration of testicular germinal epithelium (Benson et al. 1988, Pandey et al. 1999), testicular sarcomas as well as functional disorders including inhibition of spermatogenesis (Mathur et al. 1977, Yokio et al. 2003) and steroidogenesis (Das and Dasgupta 2002, Krockova et al. 2011). Such negative effects may ultimately lead to sterility (Massanyi et al. 2007).
Steroidogenesis can be tested using a number of cell lines or primary culture with gonadal tissue, but the most widely used assay utilizes a human adrenocortical carcinoma (NCI-H295R) cell line. Such in vitro steroidogenesis screening assays are used to examine the impact of endocrine active chemicals/substances (EACs) capable of altering steroid biosynthesis (Ding et al. 2007, Fialkova et al. 2018). Progesterone, testosterone and estradiol are the main steroid hormones that play essential roles during the regulation of reproduction in vertebrates and are also involved in numerous other processes related to development and growth (Hecker and Giesy 2008). The present study investigated the effects of nickel chloride (NiCl2) on the viability and steroidogenesis of
the NCI-H295R cell line. Specifically, we examined the dose-dependent changes of NiCl2 as a potential endocrine disruptor in relation to the release of progesterone, testosterone and 17ß-estradiol by NCI-H295R cell line in vitro. The NCI-H295R cell line was derived from H295 cells, which were established from a primary hormonally active adrenocortical carcinoma (Gazdar et al. 1990, Rainey et al. 2004). This cell line has physiological characteristics of zonally undifferentiated human fetal adrenal cells (Staels et al. 1993, Harvey and Everett 2003), and represent an unique in vitro model system having the ability to produce all of the steroid hormones found in the adult adrenal cortex and the gonads, allowing testing the effects on both corticosteroid synthesis together with the production of sexual steroid hormones (Gazdar et al. 1990). Another advantage of the H295R cell bioassay is that it can be used to evaluate the enzymatic activities of steroidogenic genes (Hilscherova et al. 2004). In fact, the NCI-H295R Steroidogenesis Assay has been included in the Tier1 Screening Battery of the United States Environmental Protection Agency’s (EPA) Endocrine Disruptor Screening Program (EDSP). The test guideline of the H295R Steroidogenesis Assay (TG 456) has been further validated by the Organization for Economic Cooperation and Development (OECD 2011).
Materials and Methods
Cell culture
The NCI-H295R cell line 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, Budapest, Hungary; OGYI/31762- 9/2010) according to previously established and
specifically validated protocols (Hilscherova et al. 2004, Zhang et al. 2005, Hecker et al. 2006, Hecker and Giesy 2008, OECD 2011).
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 additional passages using new NCI-H295R batches from frozen 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 °℃ and 5.0% 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 1.2 g/l NaHCO3 (Sigma- Aldrich, St. Louis, MO, USA), 5.0 ml/l of ITS+Premix (BD Bioscience, San Jose, CA, USA) and 12.5 ml/l of BD Nu-Serum (BD Bioscience, 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). After trypsinization, cells were plated at the appropriate density to obtain 90-100 % confluency. Cell density was determined using a hemocytometer (Fig. 1) and adjusted with culture medium to a final concentration of 300000 cells/ml. The cell suspensions were plated (with final volume of 1.0 ml/well) into sterile plastic 24-well plates (TPP, Grainer, Germany) for estimation of sexual steroid hormones (50-60 % confluency of cells). For cytotoxicity evaluation, the cells (100 ul/well) were seeded into 96-well plates (MTP, Grainer, Germany). The seeded plates were incubated at 37 ℃ and 5.0 % CO2 atmosphere for 24 h to allow the cells to attach to the wells (Knazicka et al. 2013).
A
B
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 3.90; 7.80; 15.60; 31.20; 62.50; 125; 250 and 500 µM nickel chloride (NiCl2; ≥98 %; Sigma-Aldrich, St. Louis, MO, USA), respectively. Cell cultures were set in 24 and 96-well plates (MTP, Grainer, Germany). Following treatment, the cells were maintained for 48 h. The experimental groups A-H (exposed to different concentrations of NiCl2) with control group (Ctrl) (culture medium without NiCl2) were compared.
Cell viability
The viability of the cells exposed to NiCl2 was evaluated by the metabolic activity (MTT) assay (Mosmann 1983). This colorimetric assay measures the conversion of a yellow tetrazolium salt [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide i.e. MTT), to blue formazan particles by mitochondrial succinate dehydrogenase enzyme of intact mitochondria of living cells. Formazan was measured spectrophotometrically. Following the termination of NiCl2 exposure, the cells were stained with MTT (Sigma- Aldrich, St. Louis, MO, USA) at a final concentration of 0.2 mg/ml. After 2 h incubation (37 ℃, and 5.0% CO2 atmosphere), the cells and the formazan crystals were dissolved in 150 ul of acidified (0.08 M HCI) isopropanol (CentralChem, Bratislava, Slovak Republic). The absorbance was determined at a measuring 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 group (i.e. absorbance of formazan from cells not exposed to NiCl2).
Hormonal analysis
At the end of 48 h NiCl2 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 sexual steroid hormones measurements. Enzyme linked immunosorbent assay (ELISA) was used for the quantification of progesterone, testosterone and 17ß-estradiol (Dialab GmbH, Wiener Neudorf, Austria) directly from the aliquots of the medium. According to the manufacturer’s data, the sensitivity of testosterone assay was 0.075 ng/ml, and the intra- and inter-assay coefficients of variation were 4.6 % and 7.5 %, respectively. Cross- reactivity with 5a-dihydrotestosterone was 16.0 %. The
sensitivity of progesterone assay was 0.05 ng/ml, and the intra- and inter-assay coefficients of variation were ≤4.0 % and ≤9.3 %, respectively. The intra- and inter- assay coefficients of variation for the 170-estradiol assay were ≤9.0 % and ≤10.0%, and the sensitivity was 8.68 pg/ml. The absorbance was determined at a wavelength 450 nm using a microplate reader (Anthos MultiRead 400, Austria) and the data were evaluated by WinRead 2.30 computer software. Values were expressed in percentage of the untreated control (control groups served as 100 %). Forscolin, prochloraz and aminoglutethimide (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1 % DMSO were used as positive controls.
Statistical analysis
Obtained data were statistically analyzed using the PC program GraphPad Prism 3.02 (GraphPad Software Incorporated, San Diego, California, USA). Descriptive statistical characteristics (arithmetic mean, minimum, maximum, standard deviation and coefficient of variation) were evaluated. Homogeneity of variance was assessed by Bartlett’s test. One-way analysis of variance (ANOVA) and the Dunnett’s multiple comparison tests were used for statistical evaluations. The level of significance was set at *** (P<0.001); ** (P<0.01) and * (P<0.05). Three inde- pendent experiments were performed.
Results
Cell viability
The cell viability remained relatively unaltered up to 125 µM (P>0.05) and slightly decreased from 250 µM of NiCl2 (P<0.05). The cytotoxic effect of NiCl2 (<50 %) was very distinct (P<0.01) in the group with the highest concentration (500 µM) of NiCl2 (Fig. 2).
Release of progesterone by human adrenocortical carcinoma (NCI-H295R) cell line
Following 48 h culture of NCI-H295R cell line in the presence of NiCl2, a dose-dependent depletion (P<0.01) of progesterone release was observed in all the experimental groups, even at the lowest concentration (3.90 µM) of NiCl2 used in the study (19.56+4.00 ng/ml). Lower levels of progesterone were detected in groups with higher doses (≥125 µM) of NiCl2 (P<0.01) as shown in Table 1. In the control group, progesterone production (100 %) was 21.05+4.40 ng/ml. The percentage changes of progesterone release after NiCl2 exposure are presented in Fig. 3.
150
absorbance % of control group
100
*
**
50
0
Ctrl
3.90
7.80
15.60
31.20
62.50
125
250
500
NiCI2 (LM)
| Groups | Control | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 | 125 | 250 | 500 |
|---|---|---|---|---|---|---|---|---|---|
| Ctrl | H | G | F | E | D | C | B | A | |
| NiCl2 (IM) | |||||||||
| X | 21.05 | 19.56 ** | 11.07 ** | 10.59 ** | 11.70 ** | 9.93 ** | 7.33 ** | 6.18 ** | 5.79 ** |
| minimum | 15.24 | 15.64 | 6.24 | 6.84 | 8.27 | 6.88 | 5.27 | 4.25 | 3.55 |
| maximum | 28.25 | 24.12 | 14.25 | 15.26 | 14.85 | 14.49 | 10.58 | 8.18 | 7.58 |
| ± S.D. | 4.40 | 4.00 | 3.26 | 3.06 | 2.65 | 3.00 | 1.99 | 1.62 | 1.35 |
| CV (%) | 20.91 | 20.46 | 29.48 | 28.96 | 22.96 | 30.24 | 27.15 | 26.13 | 23.41 |
| % | 100.00 | 92.92 | 52.59 | 47.51 | 55.58 | 47.19 | 34.83 | 29.37 | 27.48 |
X - arithmetic mean, ± S.D. - standard deviation, CV (%) - coefficient of variation. The level of significance was set at *** (P<0.001), ** (P<0.01) and * (P<0.05). Ctrl - control group.
progesterone release % of control group
150
**
100
**
**
**
**
50
**
**
**
0
Ctrl
3.90
7.80
15.60
31.20
62.50
125
250
500
NiCl2 (uM)
Release of testosterone by human adrenocortical carcinoma (NCI-H295R) cell line
Testosterone production decreased significantly (P<0.01) at all the concentrations of NiCl2 used in the study (Table 2). Furthermore, this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was (P<0.01) noted at 125 µM of NiCl2 (1.22+0.74 ng/ml) in comparison with control group (10.75±3.45 ng/ml). The percentage changes of testosterone release after NiCl2 exposure are presented in Fig. 4.
Release of 17ß-estradiol by human adrenocortical carcinoma (NCI-H295R) cell line
The 17-estradiol production was substantially increased at low concentrations (3.90 to 62.50 µM) of NiCl2. However, the increment was not statistically
significant (P>0.05) in comparison with control group (Fig. 5). The lowest release of 17ß-estradiol by NCI-H295R cell line was recorded in groups with high concentrations (≥125 µM) of NiCl2, which released similar levels of 17ß-estradiol (Table 3).
Discussion
Hormonal effects are believed to play an important role in the reproductive toxicology of Ni at both the neuroendocrine and gonadal levels in the hypothalamic-pituitary-gonadal (HPG) axis (Forgacs et al. 2012). The effects of Ni on steroido- genesis have been described recently; however, the results vary depending on the experimental model, duration of exposure as well as the doses used. The present study on the impact of NiCl2 on the NCI-H295R
| Groups | Control | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 | 125 | 250 | 500 |
|---|---|---|---|---|---|---|---|---|---|
| Ctrl | H | G | F | E | D | C | B | A | |
| NiCl2 (uM) | |||||||||
| X | 10.75 | 4.42 | 3.18 | 1.98 | 4.96 | 1.46 | 1.22 | 2.18 | 1.86 |
| minimum | 6.54 | 2.12 | 1.84 | 0.48 | 3.02 | 0.27 | 0.25 | 0.88 | 0.57 |
| maximum | 16.44 | 7.28 | 5.87 | 3.54 | 7.12 | 2.71 | 2.33 | 3.19 | 3.21 |
| ± S.D. | 3.45 | 2.02 | 1.47 | 1.18 | 1.71 | 0.90 | 0.74 | 0.89 | 1.07 |
| CV (%) | 32.12 | 45.66 | 46.33 | 59.52 | 34.45 | 61.85 | 60.43 | 41.05 | 57.51 |
| % | 100.00 | 41.10 | 29.55 | 18.41 | 46.14 | 13.53 | 11.30 | 20.23 | 17.26 |
X - arithmetic mean, ± S.D. - standard deviation, CV (%) - coefficient of variation. The level of significance was set at *** (P<0.001), ** (P<0.01) and * (P<0.05). Ctrl - control group.
testosterone release % of control group
150
100
**
**
50
**
**
**
**
**
**
0
Ctrl
3.90
7.80
15.60
31.20
62.50
125
250
500
NiCl2 (LM)
17ß-estradiol release % of control group
250
200
150
100
50
0
Ctrl
3.90
7.80
15.60
31.20
62.50
125
250
500
NiCl2 (μM)
| Groups | Control | 3.90 | 7.80 | 15.60 | 31.20 | 62.50 | 125 | 250 | 500 |
|---|---|---|---|---|---|---|---|---|---|
| Ctrl | H | G | F | E | D | C | B | A | |
| NiCl2 (IM) | |||||||||
| X | 1.10 | 1.69 | 2.08 | 1.66 | 1.42 | 1.34 | 0.89 | 0.86 | 0.91 |
| Minimum | 0.74 | 0.94 | 1.02 | 0.80 | 0.84 | 0.94 | 0.64 | 0.76 | 0.51 |
| Maximum | 1.68 | 2.88 | 2.57 | 2.39 | 2.78 | 2.51 | 1.30 | 1.05 | 1.06 |
| ±S.D. | 0.29 | 0.67 | 0.57 | 0.68 | 0.71 | 0.60 | 0.20 | 0.09 | 0.18 |
| CV (%) | 26.32 | 39.86 | 27.57 | 41.23 | 49.90 | 45.05 | 21.87 | 10.49 | 20.35 |
| % | 100.00 | 154.70 | 167.00 | 151.70 | 129.60 | 122.40 | 81.73 | 78.37 | 83.21 |
X - arithmetic mean, ± S.D. - standard deviation, CV (%) - coefficient of variation. The level of significance was set at *** (P<0.001), ** (P<0.01) and * (P<0.05). Ctrl - control group.
cell line suggests a direct action of NiCl2 on the steroid- producing cells and subsequent changes in hormonal release. Nickel significantly decreased the release of progesterone and testosterone in the entire range of concentrations of NiCl2 used in the study whereas the cell viability remained relatively unaltered up to 125 µM (P>0.05) and slightly decreased from 250 µM of NiCl2 (P<0.05). The cytotoxic effect of NiCl2 (<50%) was evident (P<0.01) in the group with the highest concentration (500 µM) of NiCl2. These results clearly confirm reports of Forgacs et al. (2011) and Ocztos et al. (2011), who observed similar effects of Ni2+, Hg2+ and Cd2+ on the release of progesterone and testosterone by NCI-H295R cell line. Using primary gonadal culture, these authors also confirmed that Ni2+ is able to disturb the sexual steroid production far below its cytotoxic concentration. Similar effects of other metals (cadmium,
mercury, copper) have also been reported by our group from studies in the NCI-H295R cell line (Knazicka et al. 2013, 2015, Bilcikova et al. 2020). Earlier, Krockova and Massanyi (2010) reported a dose-dependent decrease in progesterone production by the Leydig cells at the highest concentration of 1000 umol/l of NiCl2. Revesz et al. (2004) previously exposed human ovarian granulosa cells (obtained from women undergoing in vitro fertilization) to 15.60 to 1000 µM of Ni2+ for 48 h in order to determine the site of action of Ni2+. The granulosa cells were stimulated to produce progesterone by using maximally stimulating amounts of human chorionic gonadotropin (0.10 IU/ml hCG) or dibutyryl cyclic adenosine monophosphate (1.00 mM db-cAMP). Dose- dependent depression in both hCG and db-cAMP stimulated progesterone production was seen at 15.60 µM or higher concentration of Ni2+ which is not cytotoxic to
human ovarian granulosa cells. The viability of cells remained unaffected up to 31.25 µM of Ni2+ and decreased significantly at 62.50 µM of Ni2+. Their data further indicated that the effect of Ni2+ on the progesterone production is not due to cytotoxicity, and the cellular site(s) of inhibitory action appears to be subsequent to the membrane receptor and production of db-cAMP. The inhibition of progesterone secretion by granulosa cells (Roychoudhury et al. 2014a, 2015) or rat ovarian fragments (Roychoudhury et al. 2014b) were also induced by other metals. The effect of Ni2+ may be associated with its interactions with other essential divalent metal cations, blocking functional groups, displacing essential metal ions or modifying active conformation of biomolecules (Coogan et al. 1989). Ni2+ is known to inhibit calcium (Ca2+) channels. On the other hand, Ca2+ plays an important role in the regulation of progesterone production as shown in the rat granulosa cells (Tsang and Carnegie 1983). In addition, Ni has been demonstrated to alter the metabolic activity of microsomal monoxygenases, some of which are essential for steroid metabolism (Mattison et al. 1983). Thus, above mentioned findings could also participate in Ni-triggered alterations of progesterone release by NCI-H295R cell line.
Our presented data showed that testosterone seemed to be more vulnerable than progesterone and 17-estradiol to NiCl2 exposure suggesting multiple sites of action of this metal in steroidogenesis. Disorders of the testosterone synthesis could result in a reduced activity of the key enzymes involved in the biosynthesis of testosterone. Das and Dasgupta (2002) reported that nickel sulphate (NiSO4) affects steroidogenic enzymes (3ß-hydroxysteroid dehydrogenase and 17ß-hydroxy- steroid dehydrogenase) causing alterations in the testosterone formation in adult rat testes. In another study, Krockova et al. (2011) investigated the effects of NiCl2 on the testosterone secretion, cell viability and apoptosis in mouse Leydig cells in vitro. They demonstrated that NiCl2 decreased the testosterone production at a low dose (15.67 µmol/l) and subsequently confirmed Ni-induced structural and functional alterations in the Leydig cells. Testosterone production by mouse primary Leydig cells culture following an in vitro Ni2+ exposure (62.50 to 1000 µM) was also evaluated by Forgács et al. (1998). Dose-dependent depression in hCG-stimulated testosterone production was found at ≥125 uM or higher dose of Ni2+, while basal testosterone production remained unaffected. They
further showed the effect to be dose-dependent, and is not due to cytotoxicity. Previously, Laskey and Phelps (1991) examined the effect of Ni2+ and other metal cations (Co2+, Cu2+, Hg2+, Cd2+ 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 depression in both hCG- and db-cAMP-stimulated testosterone production was noted with Cd2+, Co2+, Cu2+, Hg2+, Ni2+ and Zn2+ treatment. Surprisingly, Cd2+, Co2+, Ni2+ and Zn2+ caused a depletion in hCG- and db-cAMP- stimulated testosterone production, also caused significant increases in 20a-hydroxycholesterol- and pregnenolone-stimulated testosterone production over untreated and similarly stimulated cultures. This indicates that these cations may act at multiple sites within the Leydig cells. Sun et al. (2003) studied the mechanisms of changes in the genital system caused by NiSO4 in male rats. They observed that the contents of testicular Ni were increased; however, the blood serum contents of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were reduced. It was assumed that the Ni-induced genital system injury in male rats may be related to the decrease in the content of these hormones.
The present study noted that the 170-estradiol production was increased (although non-significantly) at low concentrations (3.90 to 62.50 µM) of NiCl2 (P>0.05). In agreement with our results, no significant changes were observed in serum estradiol levels in rats intraperitoneally injected with NiCl2 (4 mg/kg body weight) (Hfaiedh et al. 2007). In the treated rats, demonstrably increased activity of testicular aromatase was also reported. Taking into account these considerations we presume that the considerably decreased levels of testosterone together with non- significant alterations in release of 17ß-estradiol in the present study could be associated with higher aromatase activity leading to stable estrogen levels as an adaptive response of NCI-H295R cell line to Ni exposure.
As a metalloestrogen, Ni activates estrogen receptor-a (ERa) (Darbre 2006, Forgacs et al. 2012). Martin et al. (2003) examined the ability of metal ions to activate ERa in the human breast cancer cell line (MCF-7). Similar to estradiol, treatment of cells with Cu, Co, Ni, Pb, Hg, Sn, Cr or V stimulated cell proliferation. The metals also decreased the concentration of ERa protein and mRNA, and induced expression of the estrogen-regulated genes, progesterone receptor and pS2.
The ability of such metals to alter gene expression was blocked by an anti-estrogen, suggesting that their activity is probably mediated by ERa. The estrogenic potency of Ni was comparable to that of estradiol.
Moreover, the cytotoxic effect of NiCl2 (<50 %) was evident (P<0.01) in the group with the highest concentration (500 µM/ml) of NiCl2 used in the study. The cell viability remained relatively unaltered up to 125 µM (P>0.05) and slightly decreased from 250 µM of NiCl2 (P<0.05). Ng and Liu (1990) noted that Ni (1.0; 10.0 and 100 uM of NiCl2.6H2O) and other metals tested (including PbCl2, ZnCl2, AlCl3, CrCl3, FeCl2 and LiCl) had no deleterious effect on viability and hormone- induced steroidogenesis of Leydig cells and the cells in the adrenal gland.
Conclusions
The results of the present study indicate the endocrine disruptive effect of NiCl2 on the release of sexual steroid hormones (progesterone and testosterone)
in the human adrenocortical carcinoma (NCl-H295R) cell line even at low (minimum) concentrations. Testosterone release seemed more vulnerable whereas no detrimental effect of NiCl2 could be seen at concentrations ≤62.50 uM of NiCl2 on 17B-estradiol production thereby suggesting multiple sites of action of this metal in the steroidogenic pathway. Further research may clarify the precise molecular mechanism of action of NiCl2 on the sexual steroid production and their metabolites whose production is conditioned by the steroidogenic enzymes.
Conflict of Interest
There is no conflict of interest.
Acknowledgements
This study was financially supported by the Scientific Agency of the Slovak Republic VEGA No. 1/0163/18, APVV-15-0543, APVV-16-0289 and co-funded by European Community under project No. 26220220180: Building Research Centre, “AgroBioTech”.
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