Taylor & Francis Taylor & Francis Group
Toxic/Hazardous Substances Environmental Engincering
Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro
Jana Bilcikova, Veronika Fialkova, Hana Duranova, Eva Kovacikova, Zsolt Forgacs, Agnieszka Gren, Peter Massanyi, Norbert Lukac, Shubhadeep Roychoudhury & Zuzana Knazicka
To cite this article: Jana Bilcikova, Veronika Fialkova, Hana Duranova, Eva Kovacikova, Zsolt Forgacs, Agnieszka Gren, Peter Massanyi, Norbert Lukac, Shubhadeep Roychoudhury & Zuzana Knazicka (2020): Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro, Journal of Environmental Science and Health, Part A, DOI: 10.1080/10934529.2020.1769400
To link to this article: https://doi.org/10.1080/10934529.2020.1769400
Published online: 21 May 2020.
Submit your article to this journal ☒
Article views: 3
Q
View related articles
☒
View Crossmark data ☒ CrossMark
Taylor & Francis Taylor & Francis Group
Check for updates
Copper affects steroidogenesis and viability of human adrenocortical carcinoma (NCI-H295R) cell line in vitro
Jana Bilcikovaª ID, Veronika Fialkovaa (D, Hana Duranovaª, Eva Kovacikovaª, Zsolt Forgacsb, Agnieszka Gren“, Peter Massanyi”, Norbert Lukacª D, Shubhadeep Roychoudhurye, and Zuzana Knazicka”
ªAgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic; bIndependent Researcher, Budapest, Hungary; ‘Department of Animal Physiology and Toxicology, Pedagogical University of Cracow, Cracow, Poland; “Faculty of Biotechnology and Food Sciences, Department of Animal Physiology, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic; eDepartment of Life Science and Bioinformatics, Assam University, Silchar, India; “Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Nitra, Slovak Republic
ABSTRACT
Copper is an environmental risk factor, which has various effects on reproductive endocrinology. In this study human adrenocortical carcinoma (NCI-H295R) cell line was used as an in vitro bio- logical model to study the effect of copper sulfate (CuSO4.5H2O) on steroidogenesis and cytotox- icity. The cell cultures were exposed to different concentrations (3.90, 62.50, 250, 500, 1000 µM) of CuSO4.5H2O and compared to control group (medium without CuSO4.5H2O). Cell viability was measured by the metabolic activity assay. Quantification of sexual steroid production directly from the medium was performed by ELISA assay. Following 48h culture of NCI-H295R cell line in the presence of CuSO4.5H2O a dose-dependent depletion of progesterone release was observed even at the lower concentrations of CuSO4.5H2O. The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 uM) of CuSO4.5H2O, which elicited significant cytotoxic action. Testosterone production decreased significantly, and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O. The cytotoxic effect of CuSO4.5H2O was evident at all concentrations used in the study. The presented data suggest that copper has detrimental effects on sexual steroid hormones and consecutively on reproductive physiology.
ARTICLE HISTORY Received 4 March 2020 Accepted 9 May 2020
KEYWORDS
Copper sulfate; endocrine disruption; NCI-H295R cell line; sexual steroid hormones; cell viability
Introduction
Damage to hormonal systems caused by various endocrine active chemicals (EACs) and endocrine disruptors has been gaining attention, [1] mainly because of human exposure either occupationally or through dietary and environmental routes (water, soil, air etc.). Endocrine dis- ruptors may alter the normal functions of the endocrine system of both wildlife and humans. [2] These can directly affect hormone production by interacting with the enzymes, to interfere with their transport to target organs, to alter natural hormone metabolism, or to inactivate the function of steroidogenesis regulatory proteins (e.g., Steroidogenic Acute Regulatory - StAR). [3] In exposed organisms, environmental contaminants such as pesticides, pollutants, heavy or transition metals, [1,4-8] and other industrial chemicals such as, alkylphenols, [9-12] poly- chlorinated biphenyls [13] and bisphenol A [14-15] may jeopardize proper endocrine functions, [1] including adverse effects on their reproductive system.
Copper (Cu) is an essential trace element [16-17] and plays important roles in various physiological, enzymatic and regulatory processes. [18-19] Moreover, it is a component of
numerous of metalloenzymes and metalloproteins, [20-21] which are involved in cellular energy and antioxidant metabolism. [22-23] Apparently, its deficiency can affect cata- lytic activity of the Cu-dependent enzymes (24) and may also limit the function of Cu-binding proteins. [25] It can lead to several physiological changes in the body, such as ataxia, anemia or duodenal hypoxia. [26-27] Copper is an important biological trace element required for normal metabolism, [28] however in recent years, concentrations of Cu in the envir- onment have increased due to anthropogenic activities and mining operations, 29] exposing humans to potentially harmful Cu levels. The estimated intakes of Cu in the gen- eral population are 0.15 mg/day from drinking water, and approximately 2.0 mg/day from food. [30] Environmental sources also include inhalation of Cu in airborne particles (0.1-4.0 µg Cu/day). [31] Probably the most important dan- ger is the occupational exposure to extreme Cu levels, which may result in abnormal rise in plasma Cu concentration and bring about adverse effects. Normal levels of Cu in an adult human’s blood ranges from 70 to 140 µg/dL. [33] Significantly increased serum levels of Cu (108.57-113.74 µg/ dL) have been reported in automobile workers compared to
unexposed controls (85.42 µg/dL). [32] Similar serum levels of Cu (101.74 µg/dL) have also been found in workers from Cu handling industry. [34] Higher than normal levels of Cu in human blood have also been associated with nausea, diar- rhea, vomiting, digestive disorders, (35) gastrointestinal disor- ders, acute intravascular hemolysis and neurological diseases which may be of grave concern. [36-38]
Over the last four decades, research on the effects of Cu on the reproductive functions have increased 2-3 folds. [39] Reproductive and developmental effects of Cu have been well-documented both in vivo and in vitro experiments. [40-43] Excessive Cu intake has been implicated with negative effects on the reproductive system, [44-45] including toxicity in the epididymis, [46) testis and scrotum of mammals. [47-48] Several experimental studies have demonstrated Cu- induced suppression of spermatogenesis and spermatozoa motility. [8,49-51) The negative effects of Cu on the male as well as the female reproductive organs may ultimately lead to a reduced fertility. (52) Interestingly, both the deficiency as well as the excess of Cu is capable of affecting the process of steroidogenesis. [53-57]
Sexual steroid hormones are considered to be the key fac- tors in the regulation of reproduction in vertebrates and are also involved in numerous other processes related to devel- opment and growth. Hence, chemical substances that can disrupt the production of steroid hormones are believed to have direct links with adverse outcomes. [58] Cell lines are considered ideal for direct study of biological effects as well as toxicity of chemical substances, including the process of steroidogenesis. The present study investigated the effects of copper sulfate (CuSO4.5H2O) on cytotoxicity as well as steroidogenesis using human adrenocortical carcinoma (NCI-H295R) cell line. Specifically, we examined the dose- dependent changes of CuSO4.5H2O as an endocrine dis- ruptor in relation to release of sexual steroid hormones (progesterone and testosterone) by adrenocortical carcinoma 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, Budapest; OGYI/ 31762-9/2010) according to previously established and vali- dated specific protocols. [58-6 [58-63]
After initiation of the NCI-H295R culture from the ori- ginal ATCC batch cells were cultured for five passages and the 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 expos- ure studies. The cells were grown in 75 cm2 plastic cell cul- ture flasks (TPP Techno Plastic Products AG, Switzerland) in an incubator under standard conditions (37℃ and 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 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 ster- ile 0.25% trypsin-EDTA (Sigma-Aldrich, St. Louis, MO, USA). After trypsinization, cells were plated at the appropri- ate density to obtain 90-100% confluency. Cell density was determined using a hemocytometer and adjusted with cul- ture medium to a final concentration of 300 000 cells/mL. The cell suspensions were plated (with final volume of 1.00 mL/well) into sterile plastic 24-well plates (TPP, Grainer, Germany) for estimation of sex steroid hormones (50-60% confluency of cells). For cytotoxicity evaluation the cells (100 µL/well) were seeded into 96-well plates (MTP, Grainer, Germany). The seeded plates were incubated at 37°℃ and 5% CO2 atmosphere for 24h to allow the cells to attach to the wells. [6 [6]
In vitro exposure
After 24h attachment period the cell culture medium was removed from the plates and replaced with a new medium supplemented with 3.90, 62.50, 250, 500 and 1000 µM cop- per sulfate (CuSO4.5H2O; ≥ 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 E1 - E5 (exposed to different concentrations of CuSO4.5H2O) were compared to the control group (Ctrl) (medium without CuSO4.5H2O).
Cytotoxicity evaluation
The viability of the cells exposed to CuSO4.5H2O was eval- uated by the metabolic activity (MTT) assay. [64] This colori- metric assay measures the conversion of a yellow tetrazolium salt [3-(4,5-dimetylthiazol-2-yl)-2,5-diphenylte- trazolium bromide] (MTT), to blue formazan particles by mitochondrial succinate dehydrogenase of intact mitochon- dria of living cells. Formazan was measured spectrophoto- metrically. Following the termination of CuSO4.5H2O exposure, the cells were stained with MTT (Sigma-Aldrich, St. Louis, MO, USA) at a final concentration of 0.20 mg/mL. After incubation (37℃, and 5% CO2 atmosphere) for 2h, the cells and the formazan crystals were dissolved in 150 µL of acidified (0.08 M HCl) isopropanol (CentralChem, Bratislava, Slovak Republic). The absorbance was determined at a measuring wavelength of 570 nm against 620 nm as ref- erence by a microplate reader (Anthos MultiRead 400, Austria). The data were expressed in percentage of the con- trol group (i.e., absorbance of formazan from cells not exposed to CuSO4.5H2O).
140
Absorbance (%) of control group
120
100
80
60
40
20
0
0
ctrl
3.90
62.50
250
500
1000
CuSO4.5H2O (μM)
Legend: The cytotoxicity was assessed using the MTT assay following CuSO4.5H2O exposure. Each point represents the arithmetic mean (± S.D.) absorbance in % of (untreated) controls determined in three independent experiments (n = 3). The number of replicate wells was 7-10 at each point. A decline in absorbance reflects a decline in cell viability. The statistical difference between the values of control and treated cells was indicated by asterisks *** P < 0.001; ** P < 0.01 and * P < 0.05 (One-way ANOVA with Dunnett’s mul- tiple comparison test).
Hormonal analysis
At the end of 48h CuSO4.5H2O exposure, the aliquots of the culture medium were removed from the 24-well cell cul- ture plates and after centrifugation supernatant was collected and frozen at -80℃ until steroid hormones measurements. Enzyme linked immunosorbent assay (ELISA) was used for the quantification of testosterone and progesterone 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, and the intra- and inter-assay coefficients of variation were 4.6% and 7.5%, respectively. Cross-reactivity with 5x-dihydroxytestosterone was 16%. The sensitivity of progesterone 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 (Anthos MultiRead 400, Austria) microplate reader. Values were expressed in percentage of the untreated control (control groups served as 100%). Forscolin, pro- chloraz and aminoglutethimide (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.1% DMSO were used as posi- tive controls.
Statistical analysis
Obtained data were statistically analyzed by GraphPad Prism 3.02 (GraphPad Software Incorporated, San Diego, California, USA). Descriptive statistical characteristics (arith- metic 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 test were used for statistical evaluations. The level of
| Group | Control | 3.90 | 62.50 | 250 | 500 | 1000 |
|---|---|---|---|---|---|---|
| Ctrl | E1 | E2 | E3 | E4 | E5 | |
| CuSO4.5H2O (LM) | ||||||
| Progesterone | ||||||
| x (ng/ml) | 15.47 | 10.71 *** | 8.64 *** | 7.54 *** | 4.13 *** | 1.71 *** |
| Minimum | 8.42 | 6.32 | 5.66 | 5.33 | 2.07 | 1.23 |
| Maximum | 23.23 | 15.47 | 15.59 | 12.30 | 6.37 | 2.96 |
| ± S.D. | 5.34 | 3.56 | 3.52 | 2.54 | 1.37 | 0.54 |
| CV (%) | 34.50 | 33.24 | 40.67 | 33.62 | 33.05 | 31.69 |
| % | 100.00 | 59.17 | 47.02 | 42.85 | 26.05 | 9.22 |
| Testosterone | ||||||
| x (ng/ml) | 8.28 | 4.47 *** | 2.63* | 2.54 *** | 1.08 *** | 0.41 *** |
| minimum | 4.84 | 2.16 | 1.21 | 1.58 | 0.51 | 0.19 |
| maximum | 12.76 | 6.73 | 5.46 | 3.91 | 2.05 | 0.87 |
| ± S.D. | 2.69 | 1.62 | 1.49 | 0.75 | 0.51 | 0.24 |
| CV (%) | 32.53 | 36.27 | 56.59 | 29.41 | 46.79 | 57.84 |
| % | 100.00 | 48.49 | 31.58 | 30.74 | 13.91 | 4.96 |
Legend: 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.
significance was set at *** P<0.001; ** P <0.01 and * P < 0.05. To eliminate heterogeneity of variance among the data of experiments, cell viability and steroid hormone levels were expressed as % of control groups. Three independent experiments (n =3) were performed.
Results
Cell viability
The cytotoxic effect of CuSO4.5H2O was significantly (P<0.001) detected in all experimental groups (3.90- 1000 µM of CuSO4.5H2O). The cell viability remained slightly high (80%) at 3.90 µM of CuSO4.5H2O and signifi- cantly (P <0.001) decreased (≤ 35%) at 250 uM or higher concentrations of CuSO4.5H2O (Figure 1).
Release of progesterone by human adrenocortical carcinoma (NCI-H295R) cell line
Following 48 h culture of human adrenocortical carcinoma (NCI-H295R) cell line in the presence of CuSO4.5H2O a dose-dependent depletion (P <0.001) of progesterone release was observed in all experimental groups, even at the lowest concentration (3.90 µM) of CuSO4.5H2O used in the study (10.71 ± 3.56 ng/ml). The lowest levels of progesterone were detected in groups with the higher doses (≥ 250 [M) of CuSO4.5H2O (P<0.001) (Table 1). The control mean pro- gesterone production (100%) was 15.47 ±5.34 ng/ml. The percentage changes of progesterone release after CuSO4.5H2O exposure are presented in Figure 2.
Release of testosterone by human adrenocortical carcinoma (NCI-H295R) cell line
Testosterone production decreased significantly (P <0.001) and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at 1000 µM of CuSO4.5H2O (0.41 ± 0.24 ng/ml) in comparison to the control group (8.28 ±2.69 ng/ml) (Table 1). The
140
Progesterone release (%)
120
of control group
100-
80.
60
40.
20.
0
0
ctrl
3.90
62.50
250
500
1000
CuSO4.5H2O (LM)
140
Testosterone release (%)
120
of control group
100
80
60
40
I
20
0
0
ctrl
3.90
62.50
250
500
1000
CuSO4.5H2O (IM)
percentage changes of testosterone release after CuSO4.5H2O exposure are presented in Figure 3.
Discussion
The H295 cell line has been established from a primary hor- monally active adrenocortical carcinoma. [65-66] H295R is a sub-population of H295 and they represent a unique model system providing the possibility to measure not only the changes in gene expression, but also detection of alterations in steroid hormone production. [59,67] The NCI-H295R cell line has been shown to express most of the key steroido- genic enzymes coded by a number of genes. [60,68,69]
In vitro Steroidogenesis Screening Assay is used to assess the impact of EACs capable of altering steroid biosynthesis. [59,68]
Although characterized by a lower sensitivity to cytotoxicity in comparison to other cell lines, NCI-H295R is an effective screening toll for identification of chemical substances affect- ing biosynthesis of steroid hormones. [3,65,70] In fact, the NCI-H295R Steroidogenesis Assays 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). [61]
Copper can affect multiple points of steroidogenesis path- way, inhibiting enzymes important for hormone synthesis. Recently, the effects of Cu on steroidogenesis have been described, by a number of researchers, but results vary depending on the various experimental models, time-dur- ation of exposure, as well as the doses used. [53,55,71,72] Yang et al. [55] demonstrated that rats (Sprageu Dawley) treated with 12.50 mg/kg/day of copper nanoparticles (Cu NPs) had significantly decreased serum concentrations of progesterone without a change of estradiol levels. Furthermore, the authors showed that Cu NPs downregulated several steroi- dogenesis-related genes including HSD3B1, HSD3B6, HSD3B and upregulated HSD17B gene. Chattopadhyay et al. [71] investigated the dose-dependent effect of copper chloride (CuCl2) on male reproductive functions in immature rats. Their results indicated that CuCl2 at the doses of 2000 or 3000 µg/kg/day caused a significant decrease in accessory reproductive organ (seminal vesicle, ventral prostate) weight, inhibition of testicular 17ß-HSD activity together with the degeneration of testicular spermatogenic cells and reduction in serum testosterone, FSH and LH levels were observed. On the other hand, the dose of 1000 µg/kg/day of CuCl2 sig- nificantly increased testicular activity of steroidogenic enzymes and stimulated testicular spermatogenesis with ele- vated levels of testosterone and LH, but serum FSH levels did not change significantly. In another previous study, the same authors found that the dose 2000 µg/kg/day of CuCl2 resulted in significant rise of adrenal weight, 45-3ß-HSD activity and serum corticosterone level in both adult and immature male rats. The lower dose (1000 µg/kg/day) of CuCl2 did not affect these parameters, but caused a signifi- cant decrease in adrenal 45-3ß-HSD activity and serum cor- ticosterone level in immature male rats.
High levels of Cu were detected in the follicular fluid and granulosa cells from small, medium and large antral follicles and also in atretic follicles of goat ovaries. [74] In vitro dis- ruptive effect of Cu on hormone release was demonstrated in animal cells, such as bovine theca cells, [75] bovine ovar- ian follicle granulosa cells, [76) porcine ovarian granulosa cells [43] and recently also in human granulosa cells. [77] Roychoudhury et al. [43] demonstrated the effect of Cu on insulin-like growth factor (IGF-I) release by porcine ovarian granulosa cells. Results indicated that the release of IGF-I is stimulated by 2.0 µg/mL CuSO4.5H2O dose used, but lower doses (0.33 - 1.0 µg/mL CuSO4.5H2O) did not have any influence on IGF-I release. [42-43] It was observed that Cu administration in granulosa cells released IGF-I,
progesterone (P4) and induced expression of peptides related to proliferation and apoptosis. In another work a Cu dose of 51.60 µg/mL was reported to reduce androstendione production without affecting the cell number of bovine theca cells. [75] However, there is lack of data describing the effect of Cu on the human cell lines. Therefore, this study was aimed to determine the effect of CuSO4.5H2O on steroidogenesis and cytotoxicity of human adrenocorti- cal carcinoma (NCI-H295R) cell line. Our results indicate that CuSO4.5H2O is able to disturb the sexual steroid pro- duction. The cytotoxic effect of CuSO4.5H2O was evident (P <0.001) at all concentrations used in the study (3.90 - 1000 µM) in comparison to the control group. Copper significantly (P <0.001) decreased the release of steroid hormones (progesterone and testosterone) at the entire range of concentrations used. Similar effects of heavy metals (cadmium, mercury) were also reported in our pre- vious studies in the human adrenocortical carcinoma (NCI-H295R) cell line. [6,7]
In the present study, the lowest levels of progesterone were detected at higher doses (≥ 250 µM) of CuSO4.5H2O (P <0.001). Production of steroid hormone testosterone also decreased significantly (P <0.001), and this decline was more prominent in comparison to that of progesterone. The lowest release of testosterone was recorded at the highest experimental dose (1000 µM) of CuSO4.5H2O.
Micronutrients are closely related to the synthesis of total testosterone. [78] In animal studies, Cu has been shown to be toxic for testosterone synthesis, with a correlation with the degree and the amount of accumulation. [71] Our pre- sented data showed that testosterone seemed to be more vulnerable than progesterone to CuSO4.5H2O exposure sug- gesting multiple sites of action of this trace metal in steroi- dogenesis. Disorders of the testosterone synthesis could result in a reducted activity of the key enzymes involved in the biosynthesis of testosterone. Hormone-specific action of Cu was reported in another recent study investigating its effects (0.10, 0.50 and 1.06 µg/mL) on the human ganulosa cells. Results of the study confirmed the suggested vulner- ability of testosterone secretion compared to progesterone. The authors showed that the treatment with 1.06 µg/mL Cu significantly increased estradiol secretion while significantly decreasing testosterone levels, but did not affect progester- one secretion. 771 The different sites of action of metal cati- ons in steroid hormone biosynthesis were also observed in Leydig cells. [79] Authors examined the effect of Cu2+ and other metal cations (Ca2+, Cd2+, Fe3+, Mg2+, Ni2+, Hg2+, Pb2+, 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 were noted with Cd2+, Co2+, Cu2+, Hg2+, Ni2+ and Zn2+ treatment. Cu2+ caused a decrease in testosterone production only at the highest dose used in that study (5000 µM). Surprisingly, Cd2+, Co2+, Ni2+ and Zn2+, which caused depletion in hCG and db-cAMP stimulated testosterone production, also caused significant increases in 20x-hydroxycholesterol and pregnenolone stimulated
testosterone production over untreated and similarly stimu- lated cultures. Ng and Liu [80] found similar effects on ster- oidogenesis caused by Cd2+, Co2+, Cu2+, Hg2+, but at a lower dose (100 µM).
Investigation of cell viability and in vitro production of sexual steroid hormones proved to be sensitive for assess- ment of a direct action of EACs. Some metals have adverse effects in experimental animals, but not in the cell culture models. [81] The results of our in vitro study revealed the cytotoxic effect of CuSO4.5H2O was significant (P <0.001) at all experimental doses (3.90 - 1000 µM of CuSO4.5H2O) used in the study. The viability of NCI-H295R cells in cul- ture after 48 h remained slightly high (80%) at 3.90 µM of CuSO4.5H2O and significantly (P<0.001) decreased (≤ 35%) at 250 µM or higher concentrations of CuSO4.5H2O. Ng and Liu [80] found that heavy metals (Cd2+, Co2+, Cu2+, Hg2) at a concentration of 100 uM exerted an adverse effect on the viability of adrenal glands (zona glomerulosa, fascicu- lata and reticularis) of rats, and Leydig cells of the testis, with mercury being the most potent. Due to the decreased cell viability, corticotropin-stimulated corticosterone produc- tion by the adrenal decapsular cells and luteinizing hor- mone-stimulated testosterone production by Leydig cells were also observed. Their results indicated a direct toxic action of these heavy metals on the steroid-producing cells in the adrenal gland and testis.
Conclusion
Even at very low concentrations, Cu is believed to play a negative role on steroid hormone synthesis which is involved in the regulation of reproductive processes. Testosterone release seemed more vulnerable than proges- terone to Cu exposure suggesting multiple sites of action of this metal in the steroidogenic pathway. It may be assumed that the effect of enzymatic action of 17B-hydroxysteroid dehydrogenase is more sensitive, which further results in the decreased release of testosterone in comparison to progesterone and thereby the effect of enzymatic action of 3ß-hydroxysteriod dehydrogenase converting pregnenolone to progesterone apart from other possible points of the pathway. However, further confirmatory studies may clarify the precise molecular mechanism of action of Cu on the sexual steroid production and their metabolites whose pro- duction is conditioned by the steroidogenic enzymes.
Conflict of interest
The authors declare that there are no conflicts of interest.
Funding
This work was financially supported by the Scientific Agency of the Slovak Republic VEGA No. 1/0163/18, No. 1/0038/19, APVV-15-0543 and co-funded by European Community under project No. 26220220180: Building Research Center, AgroBioTech”.
ORCID
Jana Bilcikova ID http://orcid.org/0000-0003-1738-1113 Veronika Fialkova (D http://orcid.org/0000-0002-3361-0724 Norbert Lukac ID http://orcid.org/0000-0002-4565-2083
References
[1] Sanderson, J. T. The Steroid Hormone Biosynthesis Pathway as a Target for Endocrine-Disrupting Chemicals. Toxicol. Sci. 2006, 94, 3-21. DOI: 10.1093/toxsci/kfl051.
[2] Kabir, E. R .; Rahman, M. S .; Rahman, I. A Review on Endocrine Disruptors and Their Possible Impacts on Human Health. Environ. Toxicol. Pharmacol. 2015, 40, 241-258. DOI: 10.1016/j.etap.2015.06.009.
[3] Sanderson, J. T .; Berg, M. Interactions of Xenobiotics with the Steroid Hormone Biosynthesis Pathway. Pure Appl. Chem. 2003, 75, 1957-1971. DOI: 10.1351/pac200375111957.
[4] Arabi, M .; Mohammadpour, A. A. Adverse Effects of Cadmium on Bull Spermatozoa. Vet. Res. Commun. 2006, 30, 943-951. DOI: 10.1007/s11259-006-3384-3.
[5] Roychoudhury, S .; Massanyi, P. In vitro Copper Inhibition of the Rabbit Spermatozoa Motility. J. Environ. Sci. Health Part A 2008, 43, 658-663.
[6] Knazicka, Z .; Lukac, N .; Forgacs, Z .; Tvrda, E .; Lukacova, J .; Slivkova, J .; Binkowski, Ł .; Massanyi, P. Effects of Mercury on the Steroidogenesis of Human Adrenocarcinoma (NCI-H295R) Cell Line. J. Environ. Sci. Health A. 2013, 48, 348-353. DOI: 10. 1080/10934529.2013.726908.
[7] Knazicka, Z .; Forgacs, Z .; Lukacova, J .; Roychoudhury, S .; Massanyi, P .; Lukac, N. Endocrine Disruptive Effects of Cadmium on Steroidogenesis: Human Adrenocortical Carcinoma Cell Line NCI-H295R As a Cellular Model for Reproductive Toxicity Testing. J. Environ. Sci. Health, Part A 2015, 50, 348-356. DOI: 10.1080/10934529.2015.987520.
[8] Knazicka, Z .; Bezakova, J .; Bistakova, J .; Jambor, T .; Massanyi, P .; Bojnanska, T .; Lukac, N. Dávkovo a časovo závislý účinok chloridu meďnatého na samčí reprodukčný system in vitro. Folia Medica Cassoviensia 2016, 70, 9-16.
[9] Lukacova, J .; Knazicka, Z .; Tvrda, E .; Gren, A .; Lukac, N .; Massanyi, P. The Impact of Nonylphenol (NP) on the Spermatozoa Motility in vitro. J. Microbiol. Biotechnol. Food Sci. 2012, 1, 1551-1560.
[10] Lukac, N .; Lukacova, J .; Pinto, B .; Knazicka, Z .; Tvrda, E .; Massanyi, P. The Effect of Nonylphenol on the Motility and Viability of Bovine Spermatozoa in vitro. J. Environ. Sci. Health Part A 2013, 48, 973-979. DOI: 10.1080/10934529.2013.762744.
[11] Jambor, T .; Lukacova, J .; Tvrda, E .; Knazicka, Z .; Forgacs, Z .; Lukac, N. The Impact of 4-Nonylphenol on the Viability and Hormone Production of Mouse Leydig Cells. Folia Biol (Praha) 2016, 62, 34-39.
[12] Bistakova, J .; Forgacs, Z .; Bartos, Z .; Szivosne, M. R .; Jambor, T .; Knazicka, Z .; Tvrda, E .; Libova, L .; Goc, Z .; Massanyi, P .; Lukac, N. Effects of 4-Nonylphenol on the Steroidogenesis of Human Adrenocarcinoma Cell Line (NCI-H295R). J. Environ. Sci. Health A. 2017, 52, 221-227. DOI: 10.1080/10934529.2016. 1246936.
[13] Pinto, B .; Garritano, S. L .; Cristofani, R .; Ortaggi, G .; Giuliano, A .; Amodio Cocchieri, R .; Cirillo, T .; De Giusti, M .; Boccia, A .; Reali, D. Monitoring of Polychlorinated Biphenyl Contamination and Estrogenic Activity in Water, Commercial Feed and Farmed Seafood. Environ. Monit. Assess. 2008, 144, 445-453. DOI: 10.1007/s10661-007-0007-6.
[14] Kendig, E. L .; Buesing, D. R .; Christie, S. M .; Cookman, C. J .; Gear, R. B .; Hugo, E. R .; Kasper, S. N .; Kendziorski, J. A .; Ungi, K. R .; Williams, K .; Belcher, S. M. Estrogen-Like Disruptive Effects of Dietary Exposure to Bisphenol A or 17x-Ethinyl Estradiol in CD1 Mice. Int. J. Toxicol. 2012, 31, 537-550. DOI: 10.1177/1091581812463254.
[15] Lukacova, J .; Jambor, T .; Knazicka, Z .; Tvrda, E .; Kolesarova, A .; Lukac, N. Dose- and Time-Dependent Effects of Bisphenol A on Bovine Spermatozoa in vitro. J. Environ. Sci. Health, Part A 2015, 50, 669-676. DOI: 10.1080/10934529.2015.1011963.
[16] Craig, P. M .; Galus, M .; Wood, C. H. M .; McClelland, G. B. Dietary Iron Alters Waterborne Copper-Induced Gene Expression in Soft Water Acclimated Zebrafish (Danio rerio). Am. J. Physiol. Regul. Integr. Comp. Physiol. 2009, 296, 362-373.
[17] Yunus, E. U .; Mustafa, S .; Mustafa, A. T .; Baki, H. Determination of Lead, Copper and Iron in Cosmetics, Water, Soil and Food Using Polyhydroxybutyrate-B-Polydimethyl Siloxane Preconcentration and Flame Atomic Absorption Spectroscopy. Anal. Lett. 2015, 48, 1163-1179.
[18] Dobrzanski, Z .; Kolacz, R .; Bodak, E. Heavy Metals in Animal Environment. Medycyna Weterynaryjna 1996, 52, 570-574.
[19] Tang, S. H. Study of Interaction between Gelatin and Copper (II) Using Synchronours Fluorescence Spectroscopy. J. Photographic Sci. Photochem. 2005, 23, 102-107.
[20] Agarwal, K .; Sharma, A .; Talukder, G. Clastogenic Effects of Copper Sulphate on the Bone Marrow Chromosomes of Mice in vivo. Mutat. Res. 1990, 243, 1-6. DOI: 10.1016/0165- 7992(90)90115-Z.
[21] Kong, Y. Q .; Chen, L. Q .; Li, E. C .; Du, Z. Y .; Ding, Z. L. Growth and Antioxidant Activity of Juvenile Oriental River Prawn Macrobrachium Nipponense, Fed Diets Containing Different Copper Levels Under Nitrite Exposure. Global Adv. Res. J. Agricul. Sci. 2014, 3, 119-122.
[22] Gaetke, L. M .; Chow, C. K. Copper Toxicity, Oxidative Stress and Antioxidant Nutriens. Toxicology 2003, 189, 147-163. DOI: 10.1016/S0300-483X(03)00159-8.
[23] Aydemir, B .; Kiziler, A. R .; Onaran, I .; Alici, B .; Ozkara, H .; Akyolcu, M. C. Impact of Cu and Fe Concentrations on Oxidative Damage in Male Infertility. BTER 2006, 112, 193-203. DOI: 10.1385/BTER:112:3:193.
[24] Prohaska, J. R. Copper. In: Present Knowledge in Nutrition; Erdman, J., Macdonald, I., Zeisel, S., Eds. Wiley, Blackwell, Oxford; 2012; pp 873-896
[25] Prohaska, J. R. Impact of Copper Deficiency in Humans. Ann. N.Y. Acad. Sci. 2014, 1314, 1-5. DOI: 10.1111/nyas.12354.
[26] Kumar, N .; Crum, B .; Petersen, R. C .; Vernino, S. A .; Ahlskog, J. E. Copper Deficiency Myelopathy. Arch. Neurol. 2004, 61, 762-766. DOI: 10.1001/archneur.61.5.762.
[27] Matak, P .; Zumerle, S .; Mastrogiannaki, M .; El Balkhi, S .; Delga, S .; Mathieu, J. R. R .; Canonne-Hergaux, F .; Poupon, J .; Sharp, P. A .; Vaulont, S .; Peyssonnaux, C. Copper Deficiency Leads to Anemia, Duodenal Hypoxia, Upregulation of HIF-2% and Altered Expression of Iron Absorption Genes in Mice. PLoS One 2013, 8, e59538DOI: 10.1371/journal.pone.0059538.
[28] Georgopoulos, A. R .; Yonone-Lioy, M. J .; Opiekun, R. E .; Lioy, P. J. Environmental Copper: Its Dynamics and Human Exposure Issues. J. Toxicol. Environ. Health Part B Crit. Rev. 2001, 4, 341-394.
[29] Romic, M .; Romic, D. Heavy Metals Distribution in Agricultural Topsoils in Urban Area. Environ. Geol. 2003, 43, 795-805. DOI: 10.1007/s00254-002-0694-9.
[30] WHO. 1996. Trace elements in human nutrition and health. Copper. Geneva, Switzerland: World Health Organization, 123-143.
[31] Agency for Toxic Substances and Disease Registry (ATSDR). 2004. Toxicological profile for Copper. U.S. Department of Health and Human Services, Public Health Service: Atlanta, GA.
[32] Ishola, A. B .; Okechukwu, I. M .; Ashimedua, U. G .; Uchechukwu, D .; Michael, E. A .; Moses, O. O .; Okwudili, I. H .; Vaima, H. M .; Itakure, A. U .; Ifeanyichukwu, O. K. Serum Level of Lead, Zinc, Cadmium, Copper and Chromium among Occupationally Exposed Automotive Workers in Benin City. Int. J. Environ. Pollut. Res. 2017, 5, 70-79.
[33] Burtis, C. A .; Ashwood, E. R .; Bruns, D. E. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics (e-book). 5th ed .; Saunders Elsevier: St. Louis, 2012, 2238.
[34] Saha, A .; Karnik, A .; Sathawara, N .; Kulkarni, P .; Singh, V. Ceruloplasmin as a Marker of Occupational Copper Exposure. J. Expo. Sci. Environ. Epidemiol. 2008, 18, 332-337. DOI: 10. 1038/jes.2008.2.
[35] Sabir, S. M .; Khan, S. W .; Hayat, I. Effect of Environmental Pollution on Quality of Meat in District Bagh, Azad Kashmir. Pakistan J. Nutr. 2003, 2, 98-101. DOI: 10.3923/pjn.2003.98. 101.
[36] Ahasan, H. A .; Rafiqueuddin, A. K .; Chowdhury, M. A .; Azhar, M. A .; Kabir, F. Neuromyelitis Optica (Devic’s Disease) Following Chicken Pox. Trop Doct. 1994, 24, 75-76. DOI: 10. 1177/004947559402400211.
[37] Olivares, M .; Araya, M .; Pizarro, F .; Uauy, R. Uauy, R. Nausea Threshold in Apparently Healthy Individuals Who Drink Fluids Containing Graded Concentrations of Copper. Regul. Toxicol. Pharmacol. 2001, 33, 271-275. DOI: 10.1006/rtph.2000.1440.
[38] Bandmann, O .; Weiss, K. H .; Kaler, S. G. Wilson’s Disease and Other Neurological Copper Disorders. Lancet Neurol. 2015, 14, 103-113. DOI: 10.1016/S1474-4422(14)70190-5.
[39] Roychoudhury, S .; Nath, S .; Massanyi, P .; Stawarz, R .; Kacaniova, M .; Kolesarova, A. Copper-Induced Changes in Reproductive Functions: In Vivo and In Vitro Effects (Review). Physiol Res. 2016, 65, 11-22. DOI: 10.33549/physiolres.933063.
[40] Roychoudhury, S .; Massanyi, P .; Bulla, J .; Choudhury, M. D .; Straka, L .; Lukac, N .; Formicki, G .; Dankova, M .; Bardos, L. In vitro Copper Toxicity on Rabbit Spermatozoa Motility, Morphology and Cell Membrane Integrity. J. Environ. Sci. Health. Part A 2010, 45, 1482-1491. DOI: 10.1080/10934529. 2010.506092.
[41] Chattopadhyay, A .; Biswas, N. Testosterone Supplemented Protection on Inhibition of Testicular Function Induced by Copper Chloride. DHR Int. J. Biomed. Life Sci. 2013, 4, 212-223.
[42] Kolesarova, A .; Capcarova, M .; Roychoudhury, S. Metal Induced Ovarian Signaling. 1st ed .; Slovak University of Agriculture in Nitra, Nitra, 2010.
[43] Roychoudhury, S .; Bulla, J .; Sirotkin, A. V .; Kolesarova, A. In vitro Changes in Porcine Ovarian Granulosa Cells Induced by Copper. J. Environ. Sci. Health A 2014, 49, 625-633. DOI: 10. 1080/10934529.2014.865404.
[44] Jockenhovel, F .; Bals-Pratsch, M .; Bertram, H. P .; Nieschlag, E. Seminal Lead and Copper in Fertile and Infertile Men. Andrologia 1990, 22, 503-511. DOI: 10.1111/j.1439-0272.1990. tb02041.x.
[45] Katayose, H .; Shinohara, A .; Chiba, M .; Yamada, H .; Tominaga, K .; Sato, A .; Yanagida, K. Effects of Various Elements in Seminal Plasma on Semen Profiles. J. Mamm. Ova Res. 2004, 21, 141-148. DOI: 10.1274/jmor.21.141.
[46] Xu, Y .; Xiao, F. L .; Xu, N .; Qian, S. Z. Effect of Intra- Epididymal Injection of Copper Particles on Fertility, Spermatogenesis and Tissue Copper Levels in Rats. Int. J. Androl. 1985, 8, 168-174. DOI: 10.1111/j.1365-2605.1985. tb00830.x.
[47] Skandhan, K. P. Review on Copper in Male Reproduction and Contraception. Rev. Fr. Gynecol. Obstet. 1992, 87, 594-608.
[48] Eidi, M .; Eidi, A .; Pouyan, O .; Shahmohammadi, P .; Fazaeli, R .; Bahar, M. Seminal Plasma Levels of Copper and Its Relationship with Seminal Parameters. Iran. J. Reprod. Med. 2010, 8, 60-65.
[49] Scialli, R. A .; Zinaman, J. M. Reproductive toxicology and infer- tility. McGraw, Hill: New York, 1993.
[50] Roychoudhury, S .; Slivkova, J .; Bulla, J .; Massanyi, P. Copper Administration Alerts Fine Parameters of Spermatozoa Motility in vitro. Folia Vet. 2008, 52, 64-68.
[51] Knazicka, Z .; Tusimova, E .; Bezakova, J .; Miskeje, M .; Bojnanska, T .; Lukac, N. Relationship between Copper in Different Culture Media and Bovine Spermatozoa Motility
[53]
Parametres in vitro. J. Microbiol. Biotech. Food Sci. 2017/2018, 7, 226-234.
[52] Pesch, S .; Bergmann, M .; Bostedt, H. Determination of Some Enzymes and Macro- and Microelements in Stallion Seminal Plasma and Their Correlations to Semen Quality. Theriogenology 2006, 66, 307-313. DOI: 10.1016/j.theriogenol- ogy.2005.11.015.
Zhang, L. H .; Luo, Z .; Song, Y. F .; Shi, X .; Pan, Y. X .; Fan, Y. F .; Xu, Y. H. Effects and Mechanisms of Waterborne Copper Exposure Influencing Ovary Development and Related Hormones Secretion in Yellow Catfish Pelteobagrus Fulvidraco. Aquat. Toxicol. 2016, 178, 88-98. DOI: 10.1016/j.aquatox.2016. 07.014.
[54] Hoseini, S. M .; Rajabiesterabadi, H .; Kordrostami, S. Chronic Exposure of Rutilus Rutilus Caspicus Fingerlings to Ambient Copper: Effects on Food Intake, Growth Performance, Biochemistry and Stress Resistance. Toxicol. Ind. Health 2016, 32, 375-383. DOI: 10.1177/0748233713500825.
[55] Yang, J .; Hu, S .; Rao, M .; Hu, L .; Lei, H .; Wu, Y .; Wang, Y .; Ke, D .; Xia, W .; Zhu, C .- H. Copper Nanoparticle-Induced Ovarian Injury, Follicular Atresia, Apoptosis, and Gene Expression Alterations in Female Rats. Int. J. Nanomed. 2017, 12, 5959-5971. DOI: 10.2147/IJN.S139215.
[56] Klevay, L. M .; Christopherson, D. M. Copper Deficiency Halves Serum Dehydroepiandrosterone in Rats. J. Trace Elem. Med. Biol. 2000, 14, 143-145. DOI: 10.1016/S0946-672X(00)80002-4.
[57] Zheng, G .; Wang, L .; Guo, Z .; Sun, L .; Wang, L .; Wang, C .; Zuo, Z .; Qiu, H. Association of Serum Heavy Metals and Trace Element Concentrations with Reproductive Hormone Levels and Polycystic Ovary Syndrome in Chinese Population. Biol. Trace Elem. Res. 2015, 167, 1-10. DOI: 10.1007/s12011-015- 0294-7.
[58] Hecker, M .; Giesy, J. P. Novel Trends in Endocrine Disruptor Testing: The H295R Steroidogenesis Assay to Identify Inducers and Inhibitors of Hormone Production. Anal. Bioanal. Chem. 2008, 390, 287-291. DOI: 10.1007/s00216-007-1657-5.
[59] Hilscherova, K .; Jones, P. D .; Gracia, T .; Newsted, J. L .; Zhang, X .; Sanderson, J. T .; Yu, R. M. K .; Wu, R. S. S .; Giesy, J. P. Assessment of the Effects of Chemicals on the Expression of Ten Steroidogenic Genes in the H295R Cell Line Using Real- Time PCR. Toxicol. Sci. 2004, 81, 78-89. DOI: 10.1093/toxsci/ kfh191.
[60] Hecker, M .; Newsted, J. L .; Murphy, M. B .; Higley, E. B .; Jones, P. D .; Wu, R. S. S .; Giesy, J. P. Human Adrenocarcinoma (H295R) Cells for Rapid in vitro Determination of Effects on Steroidogenesis: Hormone Production. Toxicol. Appl. Pharmacol. 2006, 217, 114-124. DOI: 10.1016/j.taap.2006.07. 007.
[61] OECD 2011. H295R Steroidogenesis Assay, OECD Guideline for the Testing of Chemicals No. 456, Paris. http://www.oecd;ili- brary.org/environment/test;no;456;h295r;steroidogenesis;assay_ 9789264122642. ; en.
[62] Zhang, X .; Yu, R. M. K .; Jones, P. D .; Lam, G. K. W .; Newsted, J. L .; Gracia, T .; Hecker, M .; Hilscherova, K .; Sanderson, J. T .; Wu, R. S. S .; Giesy, J. P. Quantitative RT-PCR Methods for Evaluating Toxicant-Induced Effects on Steroidogenesis Using the H295R Cell Line. Environ. Sci. Technol. 2005, 39, 2777-2785. DOI: 10.1021/es048679k.
[63] United States Environmental Protection Agency, Endocrine Disruptor Screening Program Test Guidelines OPPTS 890.1550: Steroidogenesis (Human Cell Line - H295R), EPA 640-C-09- 003, 2009 http://www.epa.gov/ocspp/pubs/frs/publications/Test Guidelines/series890.htm.
[64] Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Surrival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55-63. DOI: 10.1016/ 0022-1759(83)90303-4.
[65] Gazdar, A. F .; Oie, H. K .; Shackleton, C. H .; Chen, T. R .; Triche, T. J .; Myers, C. E .; Chrousos, G. P .; Brennan, M. F .; Stein, C. A .; La Rocca, R. V. Establishment and
Characterization of a Human Adrenocortical Carcinoma Cell Line That Expresses Multiple Pathways of Steroid Biosynthesis. Cancer Res. 1990, 50, 5488-5496.
[66] Rainey, W. E .; Saner, K .; Schimmer, B. P. Adrenocortical Cell Lines. Mol. Cell. Endocrinol. 2004, 228, 23-28. DOI: 10.1016/j. mce.2003.12.020.
[67] Gracia, T .; Hilscherova, K .; Jones, P. D .; Newsted, J. L .; Zhang, X. W .; Hecker, M .; Higley, E. B .; Sanderson, J. T .; Yu, R. M. K .; Wu, R. S. S .; Giesy, J. P. The H295R System for Evaluation of Endocrine-Disrupting Effects. Ecotoxicol. Environ. Saf. 2006, 65, 293-305. DOI: 10.1016/j.ecoenv.2006.06.012.
[68] Ding, L .; Murphy, M. B .; He, Y .; Xu, Y .; Yeung, L. W .; Wang, J .; Zhou, B .; Lam, P. K .; Wu, R. S .; Giesy, J. P. Effects of Brominated Flame Retardants and Brominated Dioxins on Steroidogenesis in H295R Human Adrenocortical Carcinoma Cell Line. Environ. Toxicol. Chem. 2007, 26, 764-772. DOI: 10. 1897/06-388R1.1.
[69] Strajhar, P .; Tonoli, D .; Jeanneret, F .; Imhof, R. M .; Malagnino, V .; Patt, M .; Kratschmar, D. V .; Boccard, J .; Rudaz, S .; Odermatt, A. Steroid Profiling in H295R Cells to Identify Chemicals Potentially Disrupting the Production of Adrenal Steroids. Toxicology 2017, 381, 51-53. DOI: 10.1016/j.tox.2017. 02.010.
[70] Jumhawan, U .; Yamashita, T .; Ishida, K .; Fukusaki, E .; Bamba, T. Simultaneous Profiling of 17 Steroid Hormones for the Evaluation of Endocrine-Disrupting Chemicals in H295R Cells. Bioanalysis 2017, 9, 67-69. DOI: 10.4155/bio-2016-0149.
[71] Chattopadhyay, A .; Sarkar, M .; Biswas, N. M. Dose-Dependent Effect of Copper Chloride on Male Reproductive Function in Immature Rats. Kathmandu Univ. Med. J. 2005, 3, 392-400.
[72] Khushboo, M .; Murthy, M. K .; Devi, M. S .; Sanjeev, S .; Ibrahim, K. S .; Kumar, N. S .; Roy, V. K .; Gurusubramanian, G. Testicular Toxicity and Sperm Quality Following Copper Exposure in Wistar Albino Rats: Ameliorative Potentials of L- Carnitine. Environ. Sci. Pollut. Res. Int. 2018, 25, 1837-1862. DOI: 10.1007/s11356-017-0624-8.
[73] Chattopadhyay, A .; Sarkar, M .; Biswas, N. M. Effect of Copper Chloride on Adrenocortical Activities in Adult and Immature
Male Rats. Environ. Toxicol. Pharmacol. 2002, 11, 79-84. DOI: 10.1016/S1382-6689(01)00107-7.
[74] Bhardwaj, J. K .; Sharma, P. K. Changes in Trace Elements dur- ing Follicular Atresia in Goat (Capra hircus) Ovary. Biol. Trace Elem. Res. 2011, 140, 291-298. DOI: 10.1007/s12011-010-8700- 7.
[75] Kendall, N. R .; Marsters, P .; Guo, L .; Scaramuzzi, R. J .; Campbell, B. K. Effect of Copper and Thiomolybdates on Bovine Theca Cell Differentiation in vitro. J. Endocrinol. 2006, 189, 455-463. DOI: 10.1677/joe.1.06278.
[76] Kendall, N. R .; Marsters, P .; Scaramuzzi, R. J .; Campbell, B. K. Expression of Lysyl Oxidase and Effect of Copper Chloride and Ammonium Tetrathiomolybdate on Bovine Ovarian Follicle Granulosa Cells Cultured in Serum-Free Media. Reproduction 2003, 125, 657-665. DOI: 10.1530/reprod/125.5.657.
[77] Sun, Y .; Wang, W .; Guo, Y .; Zheng, B .; Li, H .; Chen, J .; Zhang, W. High Copper Levels in Follicular Fluid Affect Follicle Development in Polycystic Ovary Syndrome Patients: Population-Based and in vitro Studies. Toxicol. Appl. Pharmacol. 2019, 365, 101-111. DOI: 10.1016/j.taap.2019.01. 008.
[78] Chang, C. S .; Park, S. B .; Choi, J. B .; Kim, H. J. Correlation between Serum Testosterone Level and Concentrations of Copper and Zinc in Hair Tissue. Biol. Trace Elem. Res. 2011, 144, 264-271. DOI: 10.1007/s12011-011-9085-y.
[79] Laskey, J. W .; Phelps, P. V. Effect of Cadmium and Other Metal Cations on in vitro Leydig Cell Testosterone Production. Toxicol. Appl. Pharmacol. 1991, 108, 296-306. DOI: 10.1016/ 0041-008X(91)90119-Y.
[80] Ng, T. B .; Liu, W. K. Toxic Effect of Heavy Metals on Cell Isolated from the Rat Adrenal and Testis. In Vitro Cell. Dev. Biol. 1990, 26, 24-28. DOI: 10.1007/BF02624150.
[81] Nishiyama, S .; Nakamura, K .; Ogawa, M. Effects of Heavy Metals on Corticosteroid Production in Cultured Rat Adrenolcortical Cells. Toxicol. Appl. Pharmacol. 1985, 81, 174-176. DOI: 10.1016/0041-008X(85)90132-2.