Short Communication
Human Adrenocortical Carcinoma (NCI-H295R) Cell Line as an In Vitro Cell Culture Model for Assessing the Impact of Iron on Steroidogenesis
(NCI-H295R cell line / ferrous sulphate heptahydrate / endocrine disruption / sexual steroid hormones / cell viability)
Z. KŇAŽICKÁ1, V. FIALKOVÁ2*, H. ĎÚRANOVÁ2, J. BILČÍKOVÁ2,3,
E. KOVÁČIKOVÁ2, M. MIŠKEJE2, V. VALKOVÁ2, Z. FORGÁCS4,
S. ROYCHOUDHURY5, P. MASSÁNYI6, N. LUKÁČ6
1Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic
2 AgroBioTech Research Centre, Slovak University of Agriculture, Nitra, Slovak Republic
3Department of Genetics and Plant Breeding, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, Nitra, Slovak Republic
4Independent Researcher, Budapest, Hungary
5Department of Life Science and Bioinformatics, Assam University, Silchar, India
‘Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovak Republic
Abstract. The aim of this in vitro study was to exam- ine the dose-dependent effects of iron as a potential endocrine disruptor in relation to the release of sex- ual steroid hormones by a human adrenocortical carcinoma (NCI-H295R) cell line. The cells were ex- posed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of FeSO4.7H,O and compared with the control group (culture medium without FeSO4.7H2O). Cell viability was measured by the metabolic activity
The study was financially supported by the Scientific Agency of the Slovak Republic VEGA 1/0163/18, APVV-16-0289, APVV- 15-0543 and SUA grants 33/2019 and 35/2019.
Veronika Fialková, AgroBioTech Research Centre, Slovak Uni- versity of Agriculture in Nitra, Tr. Andreja Hlinku 2, 949 76 Nitra, Slovak Republic. Phone: (+421) 376 414 926; e-mail: veronika. fialkova@uniag.sk
Abbreviations: ATCC - American Type Culture Collection, ATP - adenosine triphosphate, Ctrl - control group, CV - coefficient of variation, DMEM/F12 - Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture, ED - endocrine disruptor, ELISA - enzyme-linked immunosorbent assay, Fe - iron, FeSO4.7H2O - ferrous sulphate heptahydrate, IRPs - iron regula- tory proteins, MTT - [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide], MTP - microtitre plates, NCI-H295 - pluripotent adrenocortical carcinoma cell, NCI-H295R - human adrenocortical carcinoma cell line, SD - standard deviation, TfR - transferrin membrane receptor, TPP - techno plastic products, x - arithmetic mean.
assay. Quantification of sexual steroid production was performed by enzyme-linked immunosorbent assay. Following 48 h culture of the cells in the pres- ence of FeSO4.7H2O, significantly (P < 0.001) in- creased production of progesterone was observed at the lowest concentration (3.90 uM) of FeSO1.7H,O, whereas the lowest release of progesterone by NCI- H295R cells was noted after addition of 1000 µM of FeSO4.7H2O, which did not elicit cytotoxic action (P> 0.05). Testosterone production was substantially increased at the concentrations ≤ 62.50 uM of FeSO4.7H,O. Lower levels of testosterone were re- corded in the groups with higher concentrations (≥ 250 µM) of FeSO4.7H2O (P> 0.05). The presented data suggest that iron has no endocrine disruptive effect on the release of sexual steroid hormones, but its toxicity may be reflected at other points of the steroidogenesis pathway.
Introduction
Endocrine disruptors (EDs) can strongly affect repro- ductive and endocrine functions in several ways, either by directly affecting hormone production through the interaction with appropriate enzymes, or through inter- fering with their transport to target organs to alter the natural hormone metabolism or even inactivating the function of steroidogenesis regulatory proteins (San- derson and van den Berg, 2003).
Iron (Fe) has a widespread use and key roles in bio- logical processes, including a bilateral role in the organ-
ism and its endocrine disrupting potential, which may lead to reproductive disorders (Lucesoli et al., 1999; Escobar-Morreale, 2012; Rossi et al., 2016; Ng et al., 2019). It participates in various physiological, regulato- ry and biochemical processes (Carter, 1995; Lieu et al., 2001; Gozzelino and Arosio, 2016). This trace element has a crucial role in the human body as part of metallo- proteins, as well as enzymes that are associated with en- ergetic reactions (Dev and Babitt, 2017). Iron metabo- lism is one of the most complex processes involving many organs and tissues, the interaction of which is critical for Fe homeostasis (Yiannikourides and Latunde- Dada, 2019). Transferrin is the primary transport protein for Fe and represents an essential Fe pool. When need- ed, Fe enters the cell via transferrin membrane receptor (TfR)-mediated endocytosis. The micronutrient subse- quently dissociates throughout the cytosol and is taken up by ferritin, the most effective Fe storage protein. Both transferrin and ferritin are regulated by Fe regula- tory proteins (IRP1/IRP2) found in the cytoplasm (Mackenzie et al., 2008). If due to genetic, lifestyle, and environmental factors transferrin is unable to effectively regulate the amount of Fe in the body, this will accumu- late to toxic levels (Wang and Pantopoulos, 2011). Cellular Fe homeostasis is maintained by the IRP sys- tem. When cellular Fe levels are low, IRPs regulate ex- pression of numerous Fe homeostasis proteins to inhibit translation of Fe transporter or storage proteins (such as ferritin and ferroportin), which leads to an increase in Fe uptake, decrease in Fe storage, and export (Muckenthaler et al., 2008). Among the numerous proteins involved in the Fe metabolism, hepcidin is the key regulator of sys- temic Fe levels (Yiannikourides and Latunde-Dada, 2019). At physiological levels, Fe and its compounds have not been reported to be toxic for animals and hu- man organisms (Marzec-Wróblewska et al., 2012). Nevertheless, disturbances in the regulative absorption mechanism can appear due to pathological conditions or prolonged intake of high Fe doses. In these cases, since the capacity for storage of Fe in ferritin is exceeded, the metal is complexed to a phosphate or a hydroxide to form hemosiderin (or it is bound to proteins), and in this form it is present in the liver (Kabata-Pendias and Mukherjee, 2007). Indeed, Fe excess in the organism is associated with the metal deposition in organs through- out the body (mainly the liver, heart and endocrine glands) and relates to their specific damage. On the oth- er hand, the most severe consequence of Fe depletion is Fe deficiency, anaemia, which is considered the most common nutrition deficiency worldwide (Clark, 2008). Moreover, it was recently suggested that Fe acts as a “double-edged sword” based on the ability to either main- tain cellular homeostasis as a micronutrient or to over- turn this balance as a catalyst responsible for serious structural and functional alterations (Tvrda et al., 2015).
Reports concerning the effects of Fe in the area of steroid hormone biosynthesis pathway are relatively scarce, and therefore, this in vitro study examined the
effects of Fe (in the form of ferrous sulphate heptahy- drate - FeSO4.7H,O) as a potential ED in relation to the release of sexual steroid hormones by the NCI-H295R cell line. This cell line was derived from pluripotent adrenocortical carcinoma (NCI-H295) cells. The NCI- H295R cell line has physiological characteristics of zonally undifferentiated human foetal adrenal cells, and represents a unique in vitro model system having the ability to produce all the steroid hormones found in the adult adrenal cortex and gonads, thus allowing testing the effects of diverse EDs on both corticosteroid synthe- sis together with production of sexual steroid hormones (Gazdar et al., 1990).
Material and Methods
Cell culture
The NCI-H295R cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cells were cultured in a Good Laboratory Practice certified laboratory (National Institute of Che- mical Safety, Budapest, Hungary; OGYI/31762-9/2010) according to previously established and specifically validated protocols (Zhang et al., 2005; Hecker et al., 2006; OECD, 2011). After initiation of the NCI-H295R culture from the original ATCC batch, cells were cul- tured 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 cul- ture flasks (TPP - Techno Plastic Products AG, Trasa- dingen, Switzerland) in an incubator under standard conditions (37 ℃ and 5.0 % CO2). 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) supple- mented with 1.2 g/l sodium bicarbonate (Sigma- Aldrich), 5.0 ml/l of ITS+Premix (BD Bioscience, San Jose, CA) and 12.5 ml/l of BD Nu-Serum (BD Bio- science). The medium was changed 2-3 times per week and cells were detached from the flasks for sub-cultur- ing using sterile 0.25 % trypsin-EDTA (Sigma-Aldrich). After trypsinization, cells were plated at the appropriate density to obtain 90-100 % confluence. The cell number was determined using a haemocytometer and adjusted with culture medium to a final concentration of 300,000 cells/ml. The cell suspensions were plated (with a final volume of 1.0 ml/well) into sterile plastic 24-well plates (TPP, Grainer, Frickenhausen, Germany) for estimation of sexual steroid hormones (with 50-60 % confluence of cells). For cytotoxicity evaluation, the cells (100 ul/well) were seeded into 96-well microtitre plates (MTP, Grainer). The seeded plates were incubated at 37 ℃ and 5.0 % CO, for 24 h to allow the cells to attach to the wells (Knazicka et al., 2013).
In vitro exposure
After 24 h attachment period, the cell culture medium was removed from the plates and replaced with new me- dium supplemented with 3.90, 62.50, 250, 500, and 1000 µM FeSO4.7H2O (≥ 99 %; Sigma-Aldrich), re- spectively. Cell cultures were set in 24 and 96-well plates (MTP, Grainer). Following treatment, the cells were maintained for 48 h. The experimental groups (ex- posed to different concentrations of FeSO4.7H2O) with the control group (Ctrl) (culture medium without FeSO4.7H2O) were compared.
Cell viability
The viability of the cells exposed to FeSO4. 7H2O was evaluated by the metabolic activity (MTT) assay (Mosmann, 1983). This colorimetric assay measures the conversion of a yellow tetrazolium salt (3-(4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan particles by the mitochondrial succi- nate dehydrogenase enzyme of intact mitochondria of living cells. Formazan was measured spectrophotomet- rically. Following termination of FeSO4.7H,O exposure, the cells were stained with MTT (Sigma-Aldrich) at a fi- nal concentration of 0.2 mg/ml. After 2 h incubation (37 °℃ and 5.0% CO2), the cells and the formazan crys- tals were dissolved in 150 ul of acidified (0.08 M hydro- chlorid acid) isopropanol (CentralChem, Bratislava, Slovak Republic). The absorbance was determined at a measuring wavelength of 570 nm against 620 nm as a reference by a microplate reader (Anthos MultiRead 400, Eugendorf, Austria). The data were expressed in percentage of the control group (i.e., absorbance of formazan from cells not exposed to FeSO4.7H2O).
Hormone measurement
At the end of 48 h FeSO4.7H2O exposure, 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 hormone measurements. Enzyme-linked immunosorb- ent assay (ELISA) was used for quantification of pro- gesterone and testosterone (Dialab GmbH, Wiener Neudorf, Austria) directly from the aliquots of the me- dium. According to the manufacturer’s data, the sensitiv- ity 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 sensitivity of testosterone assay was 0.075 ng/ml, and the intra- and inter-assay coefficients of variation were 4.6 % and 7.5 %, respec- tively. Cross-reactivity with 5a-dihydrotestosterone was 16.0 %. The absorbance was determined at a wavelength of 450 nm using a microplate reader and the data were evaluated by WinRead 2.30 computer software. In order to verify the performance of the NCI-H295R cells, a quality control plate was run in parallel to each NCI- H295R assay in accordance with the OECD guideline (2011). Forscolin, prochloraz and aminoglutethimide
(Sigma-Aldrich) dissolved in 0.1% dimethyl sulphoxide were used as positive controls.
Statistical analysis
The data were statistically analysed using the GraphPad Prism 3.02 program (GraphPad Software Incorporated, San Diego, CA). Descriptive statistical characteristics - arithmetic mean (x), minimum, maximum, standard de- viation (± SD), and coefficient of variation (CV) were evaluated. Homogeneity of variance was assessed by Bartlett’s test. One-way analysis of variance and Dun- nett’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 independent experiments were performed.
Results and Discussion
In the present in vitro study, the viability of cells (Fig. 1) increased substantially (P<0.001) at the lowest concentration (3.90 uM) of FeSO4.7H2O in comparison with the control group. It can be postulated that this con- centration could stimulate cell proliferation through ox- idation-reduction reactions. Generally, cancer cells ex- hibit an increased demand for intracellular Fe, because the metal has a pivotal role in cellular homeostasis as a substrate or cofactor of enzymes that participate in cell proliferation (Steegmann-Olmedillas, 2011). Further- more, Fe is needed for formation of Fe-sulphur clusters in mitochondrial succinate dehydrogenase (Eid et al., 2017). Probably, at the lowest concentration, Fe pro- vides proper activity of this enzyme via the nicotina- mide adenine dinucleotide-cytochrome oxidase systems and protects cells. The biological systems are equipped with a myriad assortment of mechanisms that regulate Fe homeostasis and serve to prevent insufficiency or toxicity of the metal (Eid et al., 2017). Our results of the MTT assay did not confirm the cytotoxic effect of Fe. The lowest viability of cells was found after addition of the highest concentration (1000 µM) of FeSO4.7H2O (P> 0.05). This observation points to another possible mechanism of its toxicity that could be reflected at other points of the steroidogenesis pathway. Equally, the ex- perimental study by Ng and Liu (1990) confirmed that Fe (tested up to a concentration of 100 uM FeCl2) had no deleterious effect on the cell viability and hormone- induced steroidogenesis of Leydig cells and cells in the adrenal gland. The toxic effects of Fe on cells were de- scribed in another study by Bauckman et al. (2015). Ovarian carcinoma cell lines treated with 250 umol/l of non-transferrin-bound Fe during 24 h induced mito- chondrial damage, reduced expression of outer mito- chondrial membrane proteins, increased reactive oxy- gen species levels, and reduced cell viability.
The results of this study extend our knowledge about effects of several heavy metals on steroidogenesis path- ways in the NCI-H295R cell line. Hence, they contrib- ute to the establishment of this cellular model system for
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absorbance (%) of control group
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Ctrl
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FeSO4.7H2O (u.M)
the study of cell behaviour after exposure to various metals. Our previous results revealed significantly de- creased viability of the NCI-H295R cell line after expo- sure to mercury (≥ 25 uM of HgCl2; P< 0.05) (Knazicka et al., 2013), cadmium (1.90-62.50 µM of CdCl,; P < 0.01) (Knazicka et al., 2015), nickel (≥ 125 µM of NiCl2; P>0.05) (Lukac et al., 2020) and copper (3.90-1000 µM of CuSO2.5H,O; P < 0.001) (Bilcikova et al., 2020), which elicited cytotoxic action. It is reasonable to be- lieve that even exposure to low concentrations of some metals may be sufficient to affect the steroidogenic pathway. However, our results here demonstrate that FeSO4.7H,O has a beneficial action at the lowest con- centration tested (3.90 uM). This may be explained by the unique position of Fe among other biometals, and the transferrin cycle and regulation of Fe homeostasis acts to keep the amount of free ferrous iron (Fe2+) at the lowest possible level. In cancer cells, upregulation of Fe regulatory proteins including transferrin, TfR-1 and fer- ritin has been found to further induce the level of acces- sible Fe ion pool (called labile Fe pool), which acts as a crossroads of metabolic pathways of Fe-associated physiological or pathological processes (Jamnogkan et al., 2017; Wang et al., 2019). Recently, Weber et al. (2020) suggested Fe homeostasis as a key function of cancer cell viability and proliferation independent of metabolic and adenosine triphosphate (ATP)-associated processes. Moreover, their findings demonstrated that Fe supplementation protects cells from cytotoxic death upon release of degradative enzymes or heavy metals and restores mitochondrial function even in the condi- tion of lysosomal dysfunction. This indicates the special role of Fe homeostasis, uptake and regulation in cancer cells.
Our presented data suggest a direct impact of Fe on the steroid-producing NCI-H295R cells and subsequent changes in hormonal release. Following 48 h culture of the cells, significantly (P < 0.001) increased progester- one production was observed at the lowest concentra- tion (3.90 uM) of FeSO4.7H2O (35.33 ± 17.28 ng/ml) in comparison with the control group (17.48 ± 6.44 ng/ml). The lowest release of progesterone by the NCI-H295R cell line was noted at the highest concentration (1000 µM) of FeSO4.7H,O (12.61 ± 5.86 ng/ml), which, however, did not elicit cytotoxic action (P > 0.05) (Table 1). Testosterone production was substantially increased at low concentrations (3.90 to 62.50 µM) of FeSO2.7H2O. Lower levels of testosterone were recorded in the groups with higher concentrations (≥ 250 µM) of FeSO4.7H,O (P> 0.05), whereas this decline was more prominent in comparison with that of progesterone (Table 2). The lowest release of testosterone by the NCI-H295R cell line was detected at the highest concentration (1000 µM) of FeSO4.7H,O (1.40 ± 0.40 ng/ml) in comparison with the control group (2.68 ± 1.95 ng/ml). Altogether, the findings of the present in vitro study suggest that Fe has no endocrine disruptive effect on the release of sexual steroid hormones involved in the regulation of repro- ductive processes. However, testosterone release seem- ed to be more vulnerable than progesterone when NCI-H295R cells were exposed to FeSO4.7H2O, sug- gesting multiple sites of action of this metal in the ster- oidogenesis pathway. It may be assumed that the effect of enzymatic action of 17ß-hydroxysteroid dehydroge- nase is more sensitive, which further results in the de- creased release of testosterone in comparison to proges- terone. Additional studies are required to define the precise molecular mechanism of action of Fe on the
| Groups | Control Ctrl | 3.90 E | 62.50 D | 250 | 500 | 1000 A |
|---|---|---|---|---|---|---|
| C | B | |||||
| FeSO4.7H2O (uM) | ||||||
| x (ng/ml) | 17.48 | 35.33 *** | 24.93 | 18.16 | 16.86 | 12.61 |
| minimum | 5.96 | 14.80 | 6.74 | 6.96 | 5.11 | 8.43 |
| maximum | 25.43 | 59.54 | 36.21 | 26.17 | 27.21 | 19.31 |
| ± SD | 6.44 | 17.28 | 11.72 | 7.96 | 8.00 | 5.86 |
| CV (%) | 36.85 | 48.91 | 47.00 | 43.80 | 47.44 | 46.48 |
Ctrl - control group; CV (%) - coefficient of variation; ± SD - standard deviation; x - arithmetic mean. The level of significance was set at *** (P < 0.001).
| Groups | Control Ctrl | 3.90 E | 62.50 D | 250 | 500 | 1000 A |
|---|---|---|---|---|---|---|
| C | B | |||||
| FeSO4.7H2O (u.M) | ||||||
| x (ng/ml) | 2.68 | 3.30 | 3.02 | 2.30 | 2.39 | 1.40 |
| minimum | 1.16 | 1.66 | 0.75 | 0.46 | 1.04 | 0.94 |
| maximum | 7.20 | 5.75 | 5.38 | 4.45 | 3.82 | 1.90 |
| ± SD | 1.95 | 1.52 | 1.82 | 1.43 | 1.00 | 0.40 |
| CV (%) | 72.76 | 45.96 | 60.09 | 62.22 | 41.54 | 28.19 |
Ctrl - control group; CV (%) - coefficient of variation; ± SD - standard deviation; x - arithmetic mean. The statistical difference be- tween the values of Ctrl and treated cells was not recorded (P > 0.05).
sexual steroid production and their metabolites, whose production is dependent on steroidogenic enzymes.
Conflict of interest
The authors report no conflicts of interest. The au- thors alone are responsible for the content and writing of the paper.
Acknowledgements
The authors are thankful to the colleagues and techni- cal staff from the National Institute of Chemical Safety in Budapest, Hungary.
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