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Acta Histochemica
journal homepage: www.elsevier.com/locate/acthis
acta histochemica a Journal of Structural Biochemistry. Cell and Tissue Imaging
Human adrenocortical carcinoma cell line (NCI-H295R): An in vitro screening model for the assessment of endocrine disruptors’ actions on steroidogenesis with an emphasis on cell ultrastructural features
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Hana Duranova ª,*,1, Veronika Fialkovaª, Veronika Valkova ª, Jana Bilcikova ª, Lucia Olexikova b, Norbert Lukac“, Peter Massanyi, Zuzana Knazickad
a AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
b Institute of Farm Animal Genetics and Reproduction, NPPC - Research Institute for Animal Production in Nitra, Hlohovecká 2, 951 41 Lužianky, Slovak Republic
” Institute of Applied Biology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic d Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovak Republic
ARTICLE INFO
Keywords:
NCI-H295R cell line Steroidogenesis Ultrastructure Stereological approaches Endocrine disruption
ABSTRACT
Cell lines as an in vitro model for xenobiotic screening and toxicity studies provide a very important tool in the field of scientific research at the level of molecular pathways and gene expression. Good cell culture practice and intracellular characterization, as well as physiological properties of the cell line are of critical importance for in vitro reproductive toxicity testing of various endocrine-disrupting chemicals. The NCI-H295R, human adreno- carcinoma cell line, is the most widely used in vitro cellular system to study the human adrenal steroidogenic pathway at the level of hormone production and gene expression, as it expresses genes that encode for all the key enzymes for steroidogenesis. In this review, we aim to highlight the information considering the origin, devel- opment, physiological and ultrastructural characteristics of the NCI-H295R cell line. The review also creates a broad overview of the cell line usage in various range of studies related to the steroidogenesis issues. To our best knowledge, the paper provides the first report of quantitative data (ex novo) from stereological estimates of component (volume, surface) densities of nuclei, mitochondria, and lipid droplets of the NCI-H295R cells. Such ultrastructural measurements can be valuable in the assessment of underlying mechanisms of changes in the cell steroid hormone production induced by the action of diverse endocrine disruptors. Thus, they can significantly contribute to complexity of structure-function relationships in association with steroidogenesis.
1. Introduction
In recent years, the concept of environmental contaminants with endocrine disrupting activity has attracted more and more attention of the scientific community. Endocrine-disrupting chemicals (EDCs) are commonly defined as heterogeneous groups of exogenous compounds that may interfere with synthesis, secretion, transport, metabolism, re- ceptor binding, and actions on target tissues or elimination of endoge- nous hormones, altering the endocrine and homeostatic systems (Gore et al., 2015; Lee, 2018; Lauretta et al., 2019; Kňažická et al., 2021). As a consequence, abnormal growth patterns and neurodevelopmental
delays, as well as adverse developmental, reproductive, neurological, and immune issues may occur in children and adult humans, respec- tively (Monneret, 2017). To gain a deeper insight into the underlying machineries behind the negative health effects of the EDCs, several in vitro cell lines or primary cultures with gonadal tissue linked to hor- monal steroidogenesis have been employed (Kňažická et al., 2021).
Generally, in vitro cellular model systems have become progressively more important as a valuable tool for investigation of different cellular processes under various defined conditions. The techniques required to allow for cell growth and maintenance in a controlled environment outside of living organisms (in vitro) have been developed throughout
* Corresponding author. E-mail addresses: hana.duranova@uniag.sk (H. Duranova), veronika.fialkova@uniag.sk (V. Fialkova), veronika.valkova@uniag.sk (V. Valkova), jana.bilcikova@ uniag.sk (J. Bilcikova), lucia.olexikova@nppc.sk (L. Olexikova), norbert.lukac@uniag.sk (N. Lukac), peter.massanyi@uniag.sk (P. Massanyi), zuzana.knazicka@ uniag.sk (Z. Knazicka).
1 https://orcid.org/0000-0002-7274-6210.
the 20th century (Langdon, 2004). According to Segeritz and Vallier (2017), the main advantage of cell cultures used in scientific research is their homogeneity and associated reproducibility in generated data. Among them, cell cultures of the adrenal cortex have been recognized as one of the suitable models to study the biology of the various steroido- genic pathways and processes (including hormonal control, in- terrelationships, and secretion) (Gazdar et al., 1990). However, the use of primary adrenal cells as an in vitro model has some common limita- tions including a constant requirement for fresh tissue and the diffi- culties related to the isolation of adequate cortical cells (Wang and Rainey, 2012). To overcome the problems with tissue accessibility and quality, scientists have attempted to establish cell lines from adreno- cortical carcinomas (ACCs). Out of them, human adrenocortical NCI-H295 cell line was shown to be less sensitive to cytotoxicity (Gazdar et al., 1990; Sanderson and van den Berg, 2003), and thus, it is most widely used as an in vitro model in molecular assessment of adrenal steroidogenesis (Samandari et al., 2007), and in the screening of EDCs effects on steroid hormone synthesis and metabolism (Sanderson and van den Berg, 2003). Further consideration of its usage deals with the study of adrenal cell proliferation (Rainey et al., 2004; Montanaro et al., 2005) and cancer therapies (Parmar et al., 2011).
2. Origin, development and establishment of the NCI-H295R cell line
Regarding the need to develop appropriate cellular model systems that provide an avenue to study the complex signaling pathways mimicking adrenal steroid production in both adrenal physiology and pathology, different immortalized ACC cell lines have been developed. Establishment of these cell lines from adrenocortical tumors resulted mainly from the limited access to fresh adrenal tissue and technical difficulties in cell isolation and culture (Lo Iacono et al., 2021; Nanba et al., 2021). To investigate the molecular and biochemical mechanisms maintaining and regulating adrenal steroidogenesis and tumorigenesis, current human adrenal cell lines including the original NCI-H295 and its substrains: H295A, H295R, HAC13, HAC15, HAC50, and H295RA, as well as the recently established MUC-1, CU-ACC1, CU-ACC2 (Nanba et al., 2021), SJ-ACC3 (Pinto et al., 2019), and JIL-2266 (Landwehr et al., 2021) are used by many experimental laboratories.
The parental NCI-H295 cell line was isolated in 1980 from a 48-year- old black female patient diagnosed with invasive primary ACC. From the tumor, a surgically resected tissue (14 x 13 x 11 cm) was finely minced, defragmented, and maintained as floating aggregates in various serum- containing and serum-free culture media for a year period (Gazdar et al., 1990). Since the original NCI-H295 cells, proliferating only in medium supplemented with selenium (Se), insulin and transferrin (Tf), had a very long population doubling time (> 96 h) (Gazdar et al., 1990; Rainey et al., 2004), the cells were continuously flushed with fresh growth medium for a period of 3 months to deliberately remove the floating suspended cells and keep the viable attached subtypes (Parmar et al., 2011). In such a way, using different culture conditions, two strains termed as NCI-H295R and NCI-H295A were adapted as derivatives from the original parental NCI-H295 cells (Rainey and Mrotek, 2002), both growing more attached to the surface as a tight monolayer. Besides the monolayer, the results obtained by Lichtenauer et al. (2013) revealed a spheroid growth of the NCI-H295R cells under specific culture condi- tions. In the presence of epithelial growth factor (EGF) and fibroblast growth factor (FGF) in a serum-free medium, the cells readily formed cell clusters progressively adopting the typical spheroid appearance. Essentially, a population doubling time was reduced from five (taking in the parental NCI-H295 cells) to two days in the NCI-H295R cells (Rainey et al., 2004; Nanba et al., 2021).
Currently, the NCI-H295R cell line is commercially available from the American Type Culture Collection (ATCC, Manassas, VA, USA) as ATCC® CRL-2128TM. This cell line is presently cultured in Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 nutrient mixture (DMEM/
F12) supplemented with sodium bicarbonate, ITS+ Premix, and BD Nu- Serum. According to ATCC, the complete growth medium for NCI- H295R cell line (DMEM/F12) contains insulin, Tf, Se, bovine serum albumin (BSA) and linoleic acid, which are part of ITS+ Premix and BD Nu-Serum (Kňažická et al., 2021). Moreover, based on the serum sup- plement used for more rapid growth, three substrains were developed from the NCI-H295R cell line which have been termed as NCI-H295R-S1, NCI-H295R-S2, and NCI-H295R-S3 (Rainey et al., 2004). All three strains grow as adherent monolayer cultures; however, their responses and growth characteristics vary significantly depending on the different growth medium and culture conditions in individual laboratories. In effect, the NCI-H295R-S1 strain grows in a commercially available BD Nu-Serum type I (5 %, Collaborative Biomedical Products, Bedford, MA) supplemented medium, and is commercially available from the ATCC (ATCC® CRL-2128™M) (Hecker and Giesy, 2008; Wang and Rainey, 2012). Second one, the NCI-H295R-S2 grows in the medium with the serum substitute called Ultroser-G (2 %, Pall Corporation, Port Washington, NY). And the last strain, the NCI-H295R-S3, utilizes for growth the serum called Cosmic Calf (10 %, Invitrogen, Grand Island, NY) (Rainey et al., 2004; Wang and Rainey, 2012) not requiring any additional (expensive) ITS+ supplementation, thus, being more advan- tageous than the remaining two strains (Nanba et al., 2021).
3. Physiological properties of the NCI-H295R cell line and biosynthesis of steroid hormones
The NCI-H295R adrenocarcinoma cell line possesses the physiolog- ical characteristics of zonally undifferentiated human fetal adrenal cells (Gazdar et al., 1990; Harvey and Everett, 2003). The cells are pluripo- tent and have the ability to produce steroid hormones of all the three phenotypically distinct zones: (i) the outer zona glomerulosa - regulated by angiotensin II (Ang II) and potassium (K+) to synthesize mineralo- corticoids (aldosterone); (ii) the intermediate zona fasciculata - controlled by ACTH to produce glucocorticoids (cortisol); (iii) and the central zona reticularis - regulated by adrenocorticotropic hormone (ACTH) plus other yet to be determined factors to secrete adrenal an- drogens, dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) (as precursors for androstenediol and testosterone synthesis) (Parker and Rainey, 2004; Vinson, 2016), all of them creating the adult adrenal cortex (Gazdar et al., 1990).
Similar to the parental NCI-H295 cell line, the NCI-H295R cells ex- press genes that encode for most of the key enzymes necessary for ste- roidogenesis (Fig. 1) such as steroidogenic acute regulatory protein (StAR), 30-hydroxysteroid dehydrogenase (HSD3B2), 17ß-hydroxyste- roid dehydrogenase type 1 (HSD17B1), 170-hydroxysteroid dehydro- genase type 2 (HSD17B2), 17ß-hydroxysteroid dehydrogenase type 3 (HSD17B3), and 170-hydroxysteroid dehydrogenase type 4 (HSD17B4). Also, several forms of cytochrome P450 known to be involved in normal adrenal steroidogenesis [P450 side-chain cleavage enzyme (CYP11A1), 21-hydroxylase (P450c21; CYP21A2), 116-hydroxylase (P450c11b; CYP11B1), aldosterone synthase (P450c11AS; CYP11B2), 17a-hydrox- ylase and/or 17,20-lyase of cytochrome P450c17 (CYP17A1), and aro- matase (CYP19A1)] were detected in the cell line (Gazdar et al., 1990; Harvey and Everett, 2003; Rainey et al., 2004; Hecker et al., 2006; Oskarsson et al., 2006; Samandari et al., 2007; Ding et al., 2017; Strajhar et al., 2017).
Of a total of 30 different steroids reportedly synthesized and secreted by the NCI-H295R cells, approximately 20 were identified as known steroid hormones (Wang and Rainey, 2012). Most importantly, the cells differ profoundly in gene expression and steroid profiles as compared to other adrenocortical cell lines including also NCI-H295A. Indeed, the NCI-H295R cells displayed lower expression of HSD3B2 along with higher 17,20 lyase (CYP17) activity explaining their ‘androgenic’ steroid profile, whereas the NCI-H295A cells exhibited higher expression of HSD3B2, sulfonyl transferase, and cytochrome b5 indicating high rates of mineralocorticoids (17a-hydroxyprogesterone and 11-deoxycortisol,
H
H
H
HO
cholesterol
CYP11A
O
O
o
OH
o
OH
OH
Mineralocorticoids
o
o
Zona glomerulosa
36-HSD
CYP21
CYP11B1
HO
CYP11B1/
HO
H
CYP11B2
H
H
H
H
4
H
H
H
H
H
H
H
H
H
z
HO
0
o
o
0
pregnenolone
progesterone
11-deoxycorticosterone
corticosterone
aldosterone
CYP17
0
CYP17
OH
o
o
O
Glucocorticoids
Zona fasciculata
HO
OH
OH
OH
36-HSD
OH
CYP21
CYP11B1
HO
H
H
H
H
E
A
8
H
H
H
H
H
H
H
H
HO
o
0
o
17a-hydroxypregnenolone
17a-hydroxyprogesterone
11-deoxycortisol
cortisol
CYP17
CYP17
o
o
o
36-HSD
CYP19
H
H
H
H
H
H
H
H
H
HO
o
HO
dehydroepiandrosterone
androstendione
estrone
Sex hormones Zona reticularis
17ß-HSD
17ß-HSD
17ß-HSD
OH
OH
OH
OH
3B-HSD
CYP19
H
H
H
H
H
H
H
H
H
H
H
H
HO
0
HO
androstenediol
O
testosterone
17ß-estradiol
dihydrotestosterone
5a-reductase
which are precursors of glucocorticoids) production (Samandari et al., 2007; Kurlbaum et al., 2020). So, it can be seen that the NCI-H295R cells resemble their parental NCI-H295 cells (Gazdar et al., 1990) more than NCI-H295A ones. Furthermore, low basal production of cortisol and aldosterone in the NCI-H295R cells reflects also a low expression of CYP11B1 and CYP11B2 in the absence of inducers (mainly Ang II and K+) (Odermatt et al., 2016). Regarding this fact, a higher aldosterone production and messenger ribonucleic acid (mRNA) expression of CYP11B2 were reported in the NCI-H295R-S2 strain as compared to the other ones (NCI-H295R-S3 and NCI-H295R-S1) (Wang and Rainey, 2012).
Using enzyme-linked immunosorbent (ELISA) assays, the basal pro- duction of progesterone, testosterone, and 17ß-estradiol in the NCI- H295R cells after 48 h has been reported in the range of 7.0 ± 1.20 ng/ml, 1.60 ± 0.40 ng/ml, and 0.51 ± 0.13 ng/ml, respectively (Hecker et al., 2006). After 72 h of cultivation, the production of the hormones (mainly progesterone and testosterone) was lower (0.58 ± 0.18 ng/ml and 0.57 ±0.29 ng/ml, respectively) (Nielsen et al., 2012). With respect to sexual steroid hormones, the results by Mon- tanaro et al. (2005) have demonstrated the ability of the NCI-H295R cells to convert androgens to estrogens by a constitutive expression of active cytochrome P450 aromatase protein. In this way, the estrogens through a short autocrine loop (mediated by their own receptors)
contribute to enhanced NCI-H295R cell proliferation which is under androgen effects (through the androgen receptors) inhibited.
Remarkably, the synthesis of certain zone-specific steroid hormone groups can be selectively promoted in the NCI-H295R cells after treat- ment with various hormonal endogenous regulators (Odermatt et al., 2016). As mentioned above, these cells have been shown to respond to Ang II (as the primary regulator of the renin-angiotensin-aldosterone system; RAAS) and K+ stimulations by significant increasing produc- tion of aldosterone (Nogueira et al., 2007; Lichtenauer et al., 2013) via enhanced transcription of CYP11B2 promoter (from the Ca2+/cAMP-responsive element located in the promoter) (Yarimizu et al., 2015). Also, the expression of 3ß-hydroxysteroid dehydrogenase (HSD3B1) was found to be stimulated by Ang II (Ota et al., 2014); however, the orphan nuclear receptor nerve growth factor IB (NGFI- B)-responsive element (located in the HSD3B1 promoter) failed to respond to K+ stimulation (Yarimizu et al., 2015). According to Nogueira et al. (2007), the NCI-H295R cellular system represents the only human adrenal model responding to Ang II. In these cells, the hormonal regulator binds to type 1 Ang II (AT1) receptors resulting in activation of protein kinase C (PKC) and increased production of aldo- sterone (Parmar et al., 2011). Since the NCI-H295A cells do not express substantial levels of AT1 receptor, they are not responsive to Ang II (Samandari et al., 2007). Moreover, the study by Nishi et al. (2013) have
documented increased secretion of glucocorticoids (from a basal level of 40.1 ± 4.15 pmol/104 cells to 80.8 ±12.8 pmol/104 cells) in the NCI-H295R cells exposed to 100 nM Ang II (with no linkage to cAMP) in the presence of extracellular Ca2+. On the other hand, the production of adrenal androgens [DHEA, DHEAS, androstenedione, 11ß-hydroxyan- drostenedione (11OHA)] and glucocorticoids (cortisol, 11-deoxycorti- sol, corticosterone) can be stimulated in the NCI-H295R cells by treatment with agonist of protein kinase-A (PKA) pathway, such as dibutyryl cAMP (dbcAMP) and forskolin (Oskarsson et al., 2006; Xing et al., 2011), due to increased intracellular cAMP following enhanced CYP17 activity. Also, the steroidogenesis inducer forskolin (10 mM) is suggested as a relevant control in various experiments (Strajhar et al., 2017). The results by Ahmed et al. (2018) have shown that precursor steroids (such as progesterone, pregnenolone, DHEA, and 21-hydroxy- progesterone) reached their highest production point at 24 h after treatment with 1.5 uM forskolin, and end-products (such as estradiol, testosterone, and cortisol) continued to increase until 72 h after the treatment.
Interestingly, ACTH as the primary hormonal regulator of adrenal cortisol production (Wang and Rainey, 2012) has a greater resemblance to a glomerulosa cell phenotype in the NCI-H295R cell line with a transient increase in aldosterone but not in cortisol production (Oder- matt et al., 2016). As in the case of the parental NCI-H295 cell line, ACTH has been found to be less effective than dbcAMP and forskolin in stimulation of the NCI-H295R cells steroidogenesis. This finding is in part a reflection of low levels of ACTH receptor (melanocortin 2 re- ceptor, MC2R) expression in these cells (Nanba et al., 2016, 2021) resulting in only modest ACTH responsiveness (Rainey et al., 2004; Samandari et al., 2007; Parmar et al., 2008; Wang and Rainey, 2012). Since ACTH primarily regulates cortisol production through cAMP signaling, its activation can be mimicked by addition of forskolin (to activate adenylyl cyclase) or cAMP (Rainey et al., 2004; Whitworth and Hinson, 2005) activating the PKA pathway (Ullerås et al., 2008).
4. Ultrastructural appearance of the NCI-H295R cell line as an indicator of steroidogenesis activity
Ultrastructural studies performed on the human adrenocarcinoma cell lines are relatively rare. Characterization of human invasive primary adrenocortical carcinoma and the tumor-derived NCI-H295 cell line from an ultrastructural point of view has been carried out by Gazdar et al. (1990). Their study confirmed the steroidogenic character of both samples investigated. In effect, the original tumor cells were charac- terized by enormous numbers of mitochondria, vesicular smooth endo- plasmic reticulum (SER), epithelioid nuclei with prominent nucleoli, and even rare cytoplasmic structures resembling spironolactone bodies. The cultured tumor cells clearly retained many of the same features including epithelioid nuclei with single prominent nucleolus, large numbers of mitochondria, moderate vesicular SER, and prominent Golgi apparatus (GA). Also, microvilli were quite conspicuous.
At the ultrastructural level, the steroidogenic phenotype can also be seen in the NCI-H295R cell line. In agreement with our previously un- published (ex novo) results, these cells of oval to cuboidal shape have infrequent microvilli on their surfaces, and they are characterized by round to oval nuclei with irregular contours (the nuclei are only occa- sionally deeply lobulated or indented). Nuclear volume and surface densities are estimated to be 38.24 ± 8.53 % and 0.11 ± 0.03 um-1, respectively. The nuclei possess one or two prominent conspicuous nucleoli and clumps of condensed chromatin (heterochromatin) scat- tered throughout the lighter staining euchromatin; heterochromatin is also concentrated at the nuclear periphery. In the nucleus interior, un- usual membranous inclusion bodies can also be occasionally observed (Fig. 2). As other malignant cells, the NCI-H295R cell line displays an increased N/C ratio which is found to be 0.63 ± 0.25. Moreover, they exhibit ultrastructural features of steroid-producing cells such as mod- erate rough endoplasmic reticulum (RER) and GA with many vesicles
SER
9
M
GA
M
N
LD
Nu
RER
um
and granules. However, the occurrence of SER (forming an anasto- mosing network of slender tubules in the cytoplasm) as a typical feature of steroid-secreting cells is in our cells observed to be rather scarce. Besides the indicators of cellular secretory activity, cytoplasm of the cells also contains large numbers of rounded (ovoid), rod-shaped and/or elongated mitochondria with lamellar cristae and dense matrix (Fig. 3). These double-bounded organelles are found to be randomly dispersed in the cytoplasm or to form clusters of various sizes. They occupy approximately 5.38 ± 1.83 % of the cell fraction, and their surface density ranges from 0.03 to 0.36 um-1 with an average of 0.20 ± 0.09 um-1. In the cytoplasm, a small number of lysosomes, many free ribosomes and frequent vacuoles of various sizes are present, as well. In some cells, cytoplasmic inclusions composed of concentric whorls of tightly packed membranes can also be seen. Electron-dense LDs (Fig. 3) are, however, present in small amounts, and our estimations of their volume and surface fractions have shown their values of 0.60 ± 1.05 % and 0.008 ± 0.016 um-1, respectively.
Our preliminary results from the qualitative ultrastructural charac- teristics of the NCI-H295R cell line are in agreement with the studies by Junquero et al. (2001), Thomopoulos et al. (2001), and Cerquetti et al. (2021). However, the data from stereological estimation, obtained ac- cording to the methodology described in detail by Duranova et al. (2022), is not able to compare with other one because of its absence prior to our report. So, to our best knowledge, the findings are pio- neering in a connection of ultrastructural features of the human adre- nocarcinoma NCI-H295R cell line with the quantitative parameters (related to volume and surface densities) of their nuclei, mitochondria, and LDs. Hence, they can provide new insights into the complex pro- cesses of steroidogenesis related to structure-function coupling, and also contribute to a promising platform for various biological and biomedical applications.
In our study, not only volume fractions of selected organelles of in- terest but also their surface densities were estimated. This quantitative
RER
LD
LD
M
2 um
M
N
C
☐
2 um
B
N
SER
5 cum
A
500 nm
D
parameter, defined as the ratio of the total surface area to the volume of the reference space (Wagner et al., 2015), gives a direct information considering the surface area of the cellular components as another important aspect from the functional point of view (Duranova et al., 2020). According to Howard and Reed (2005), the surface area of an object is often a valuable indicator of the capacity of surface-limited processes to function; thus, such measurement can contribute to complexity of structure-function relationships. For instance, estimation of the nuclear component densities may be theoretically indicative of rates of DNA and RNA synthetic processes, and also nucleocytoplasmic shuttling (through nuclear pore complexes) when taking into consider- ation the function of cellular nucleus (Pavelka and Roth, 2015).
The N/C ratio, expressing the amount of space occupied by the nu- cleus in relation to that occupied by the cytoplasm (Turgeon, 2005), has proven to be helpful for assessment of atypia and malignant lesions. In effect, the high N/C ratio of the cells usually exhibiting a low to mod- erate N/C ratio is typically a sign of their atypia or even malignancy (Vaickus and Tambouret, 2015). For example, the values for N/C ratio of two different human breast cancer cell lines (MCF-7 and MDA-MB-231), and PC-3 prostate cancer cells have been calculated (using a different approach) to be 0.68, 0.66, and 0.54, respectively (Moore et al., 2019). Taking into account just the aspect of malignancy (not relationship to the steroid hormone sensitivity and/or steroid hormone receptor expression), the data is similar to that estimated in our adrenocarcinoma NCI-H295R cell line (0.63 ± 0.25).
Mitochondria as semi-autonomous organelles possess ultrastructural features (i.e., size, number, and volume density) which are very dynamic and reflect their functional state (Duranova et al., 2020). In effect, mitochondrial volume density as an index of cellular aerobic (oxidative) capacity is well matched to metabolic needs of the cell. Hence, any changes in the cellular oxidative capacity can be met by corresponding increases or decreases in volumetric proportion of the organelles (Andrade, 2010). Additionally, numerous interactions between the mitochondrial metabolic and genetic systems, and the rest of the eukaryotic cell is mediated by the outer mitochondrial membrane (OMM). The membrane consists of many proteins (including a diverse
set of enzymes, components of precursor protein translocation and insertion machineries, pore forming proteins, and proteins mediating mitochondrial fusion and fission) playing a crucial role in the biogenesis, inheritance, and morphology of the organelle (Walther and Rapaport, 2009). Among them, a complex multi-component ‘transduceosome’ [including the 18 kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC-1), TSPO-associated protein 7 (PAP7, ACBD3 for acyl-CoA-binding-domain 3), and protein kinase A regulatory subunit 1« (PKAR1A)] which interacts and moves cholesterol (for steroid-hormone production) from the OMM to mitochondrial P450scc is also present (Papadopoulos and Miller, 2012). Therefore, one could speculate that an estimation of surface area of mitochondrial profile envelope in the cellular volume might reveal e.g., the density of channel-forming membrane transport proteins (i.e., porins) or rate of mitochondrial lipid synthesis (provided by the enzymes involved in the OMM) (Alberts et al., 2002) or rate of cholesterol loading into OMM and its moving to mitochondrial steroidogenic enzyme P450scc (Papado- poulos and Miller, 2012).
Besides mitochondria, LDs also have a substantial role in cellular lipid metabolism and energy homeostasis (Cui and Liu, 2020). In ste- roidogenic cells, the organelles are the main storage of cholesterol (in the form of cholesterol esters, CEs), as the starting material (precursor) for the biosynthesis of steroid hormones, from which it is mobilized to the initiating rate-limiting enzyme complex located on the inner mito- chondrial membrane (IMM) (Bollag et al., 2008). Additionally, choles- terol is derived from (i) de novo synthesis in the ER [through 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMGCoA) reductase (HMGCR) activity, also known as the mevalonate pathway], (ii) plasma lipoprotein-derived CEs obtained by either low density lipoprotein (LDL) receptor-mediated endocytic selective uptake or that mediated via the scavenger receptor class B type I (SR-BI), (iii) and from plasma membrane-associated free cholesterol in some cultured cell systems (Hu et al., 2010; Shen et al., 2016; Boulate et al., 2019). Although the LDs are a prominent feature of normal adrenocortical cells (Walther and Farese, 2012), their less number was observed in ACC cells (Urushibata, 1971) which corresponds to our results showing their low volume density in
the NCI-H295R cells. This fact can be associated with the maximum secreting activity of the cells when LDs diminish in size and often even disappear completely (Brenner, 1966). Inconspicuous LDs (perhaps demonstrating the entirely endogenous production of cholesterol re- quirements) were also seen in the NCI-H295 cells (Gazdar et al., 1990). For many cultured cell lines (especially for steroidogenic cells), the growth and reproduction requirement to receive lipids from external culture media components is desirable (Shen et al., 2016; Talbott et al., 2020). In general, commercially available fractions of animal serum contain a number of serum lipids (including cholesterol, fatty acids, and phospholipids) bound to select serum proteins such as BSA which is a common carrier for fatty acids and lipids (Van der Valk et al., 2004). In effect, supplementation of cell culture systems with polyunsaturated fatty acids including linoleic acid or linolenic acid, phospholipids or sterols significantly improves the cell performance. Providing cells with appropriate external preformed lipids (even when not essential) lessens their biosynthetic load, and as a consequence is more efficient cell metabolism (Whitford and Manwaring, 2004; Yao and Asayama, 2017). Taking notice of the culture medium, our NCI-H295R cells were grown in a 1: 1 mixture of DMEM/F12 supplemented with 1.20 g/L of sodium bicarbonate, 5.00 ml/L of ITS+ Premix, and 12.50 ml/L of BD Nu-Serum. This complete medium containing (according to ATCC) 0.00625 mg/ml of insulin, 0.00625 mg/ml of Tf, 6.25 ng/ml of Se, 1.25 mg/ml of BSA, and 0.00535 mg/ml of linoleic acid has been employed in several our experiments (Knazicka et al., 2013, 2021, 2015; Bilcikova et al., 2020; Lukac et al., 2020; Kňažická et al., 2021). Since the levels of progesterone and testosterone produced by the NCI-H295R cells (following 48 h culture) have been determined to be in the range of 15.47-21.05 ng/ml and 8.28-10.75 ng/ml, respectively (Bilcikova et al., 2020; Lukac et al., 2020), which is even higher as compared to the study performed by Hecker et al. (2006), we can consider the internal and external cholesterol sources to be sufficient for the process of ste- roidogenesis in our NCI-H295R cells in spite of their lower occurrence of LDs. Therefore, the relatively low volume fraction of the organelles in these steroidogenic cells could be rather attributed to their maximum secreting capacity than to other factors.
Generally, the principal framework of the biosynthetic process is similar in various steroidogenic cells; in particular, an initial mito- chondrial step related to the START domain (which contains protein- mediated cholesterol transport to the mitochondria and its conversion to pregnenolone by the P450scc) is conserved among the cells (Bassi et al., 2021). In effects, the steroid hormones are synthesized de novo from cholesterol not only in mitochondria as a central in steroidogenesis but also in above-mentioned ER (Shen et al., 2016) which is another subcellular site of the enzymes involved in this biological process (Gazdar et al., 1990). Hence, the interactions between the mitochondria, ER, cytosol, and LDs play an undoubtedly fundamental role in hormone-induced steroidogenesis. Taking into account this consider- ation, any changes in the organelles ultrastructure reflect the functional status of the steroid-producing cells. Especially, the SER and mito- chondria was revealed to have an astonishing capacity to adapt to the varying demands for steroidogenesis (Bornstein et al., 1998) and to provide adequate amounts of adenosine triphosphate (ATP) necessary for the proper response of the cell to environmental challenges (Berto- ni-Freddari et al., 2006). Regarding this fact, the ultrastructural char- acterization of the NCI-H295R cell line, especially related to SER, mitochondria and LDs, by means of qualitative and quantitative (ste- reological approaches) characteristics can serve as an important tool to reveal alterations in steroidogenesis of the cell line induced by the ef- fects of various EDCs. Together with other molecular methods, a more detailed picture of their action machinery on steroid production of the cell can be so drawn.
5. The NCI-H295R cell line as model for in vitro screening of EDCs action
Owning the multiple pathways of steroidogenesis, the NCI-H295R cell line is a widely accepted model of hormonally active ACC (Fior- entini et al., 2016). Moreover, the results obtained by Oskarsson et al. (2006) have shown that the steroidogenic gene expression in the cell line well correlates to the expression in normal human adult adrenal. Hence, these unique features together with less sensitivity to cytotoxicity in comparison with other cell lines (Gazdar et al., 1990) make the NCI-H295R cells the most an appropriate in vitro model to study adre- nocortical function, regulation of steroidogenesis (Hecker et al., 2006; Samandari et al., 2007; Nishi et al., 2013), and enzymatic activities of steroidogenic genes (Hilscherova et al., 2004; Hecker et al., 2006), as well as for in vitro testing of EDCs (Hilscherova et al., 2004; Knazicka et al., 2013, 2015, 2021; Maglich et al., 2014; Háhn et al., 2016; Nakano et al., 2016; Kristensen et al., 2018; Kwak et al., 2018; Munkboel et al., 2018; Bilcikova et al., 2020; Lukac et al., 2020; Kňažická et al., 2021). Furthermore, unlike tissue-based assays, the NCI-H295R cell line per- mits evaluation of EDC potential action on cell viability and/or cyto- toxicity. This important attribute allows for the discrimination of impacts caused by cytotoxicity from those induced by the direct inter- action of chemicals with steroidogenic pathways. In tissue explants systems (consisting of multiple cell types with different functionalities and sensitivities), it is not possible (Hecker and Giesy, 2008; OECD Organization for or Economic Cooperation and Development, 2011).
Generally, the H295R assay integrates the effects of direct-acting hormone agonists and antagonists, as well as chemicals affecting signal transduction pathways for steroid hormone production, and provides data on both gene expression and hormone secretion. These properties suggest the cell line as a valuable instrument for screening assay to detect and evaluate impacts of EDCs on the process of ste- roidogenesis (Gracia et al., 2006). In fact, the H295R Steroidogenesis Assay has been included in the Tier1 Screening Battery of the United States Environmental Protection Agency (EPA) Endocrine Disruptor Screening Program (EDSP). The test guideline of the H295R Steroido- genesis Assay (TG 456) was further validated by the Organization for Economic Cooperation and Development (OECD) (OECD Organization for or Economic Cooperation and Development, 2011; Knazicka et al., 2013). Moreover, these cells have been widely employed in other testing efforts such as ToxCast and European REACH, as well as OECD (Gracia et al., 2006; Breen et al., 2010; Rotroff et al., 2013), all of them using simultaneous hormone measurements in the NCI-H295R cell model for identification of diverse endocrine active environmental pollutants. In this view, a multi-parameter assay in the NCI-H295R cells can have a considerable predictive performance for male and female reprotoxicity (Maglich et al., 2014).
Currently, treatments with a variety of agents (with beneficial or adverse impact on human health) have been shown to alter steroid hormone production in the NCI-H295R cells. Most importantly, it is necessary to keep in mind that steroid hormone secretion by the cell line is strongly affected by culture conditions (such as cell line passage and growth conditions) themselves (Kempná et al., 2010; Udhane et al., 2015; Kurlbaum et al., 2020), and this aspect still should be taken into consideration in screening activity of diverse endocrine disruptors. In this line, Kempná et al. (2010) have analyzed androgen production and P450c17 activities in the H295R cells grown under normal and serum-free conditions. Their findings revealed higher androgens pro- duction (induced via increased CYP17A1-17,20-lyase activity and sup- pressed HSD3B2 expression and activity) in the cells growing in starvation medium.
Since numerous phytochemicals, mainly polyphenols, exert anti- neoplastic activity, their use in the treatment of ACC is still a matter of debate (Manso et al., 2021). Taking into account the steroidogenesis, Ohno et al. (2002) have found reduced cortisol production (by 6.3, 69.6, 47.5, 26.6, 13.8, 11.3, 47.3 %) in the dbcAMP-stimulated NCI-H295R
after treatment with 12.5 or 25 uM of a range of flavonoid phyto- chemicals such as 6-hydroxyflavone, 4’-hydroxyflavone, apigenin, daidzein, genistein, formononetin, and biochanin A, respectively. Dose-dependent decreased levels of androstenedione and deoxy- corticosterone (suggesting inhibition of 3ß-HSD) were observed in the forskolin-stimulated NCI-H295R cells treated with 1, 3, 10, and 30 µM of daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol (Hasegawa et al., 2013). Additionally, the findings by Sanderson et al. (2004) showed that the natural flavonoids 7-hydroxyflavone, chrysin, and to lesser extent apigenin inhibited aromatase activity in the NCI-H295R cells in concentration-dependent manner. Nonetheless, flavone quercetin and isoflavone genistein at 10 µM stimulated the ac- tivity of the enzyme investigated (by 4- and 2.5-fold induction, respec- tively) that is coincided with increased intracellular cAMP concentrations, and increased levels of the cAMP-dependent pII, and to a lesser extent 1.3 promoter-specific aromatase transcripts.
Besides the flavonoids, numerous other aromatase inhibitors such as letrozole, ketoconazole, and aminoglutethimide (Higley et al., 2010) using in the treatment of breast cancer (Nabholtz, 2008; Lønning, 2011) have been applied to evaluate their endocrine disruptor effects in the NCI-H295R cells. In this regard, strong inhibitory action of ketoconazole with an EC50 value of 0.15 umol/L (Ohlsson et al., 2010), and 0.24 umol/L (Winther et al., 2013) on cortisol secretion by these cells have been found.
The aromatase activity, as well as the production of sexual steroid hormones in the NCI-H295R cell line has also been assessed in connec- tion with the impacts of various toxic environmental contaminants. Considering pesticides, the studies have demonstrated that imazalil, prochloraz, and epoxiconazole are potent inhibitors (Sanderson et al., 2002; Heneweer et al., 2004; Higley et al., 2010) while atrazine, sima- zine, propazine, and vinclozolin are (through inhibition of phosphodi- esterase activity) aromatase inducers (Sanderson et al., 2000, 2002; Higley et al., 2010). In addition to a dose-dependent inhibition of cortisol secretion, the findings by Ohlsson et al. (2009) showed a biphasic secretion pattern of aldosterone in the NCI-H295R cells exposed to prochloraz with stimulatory (a 2-fold increase) and inhibi- tory (reduction to 23 % of control level) effects on the secretion at low (0.1 µM) and high (10 uM) concentrations, respectively. This striking observation was attributed to a dose-dependent prochloraz-induced in- hibition of the CYP17A1 and CYP21A2 enzymes, resulting in shunting of precursors from the cortisol to the aldosterone pathway. The similar biphasic dose-response impact on aldosterone secretion in the NCI-H295R cells was also reported for ketoconazole but not for imazalil indicating that the biphasic response is not general for all imidazole fungicides (Ohlsson et al., 2010). Reduced concentrations of proges- terone/pregnenolone and testosterone in the H295R cell line treated with vinclozolin (30 and 100 uM) were observed by Hecker et al. (2006). In this study, concentration of progesterone/pregnenolone was also increased by prochloraz (1 uM) treatment; however, the concentration of testosterone was due to the action of the pesticide decreased. Dose-dependent significant increases in the concentration of progester- one with dramatic decreases in the levels of testosterone (as well as DHEA and cortisol) in the NCI-H295R cells treated with prochloraz were also reported by Maglich et al. (2014) and Nakano et al. (2016). This fact demonstrates the ability of the cellular model system to identify com- pounds that both increase and/or decrease hormone levels.
Also, widely industrially used phenolic substances such as bisphenols and 4-nonylphenol have been reported to possess an endocrine dis- rupting potential. In effect, dual actions of bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BSS) on progesterone and testosterone biosynthesis in the NCI-H295R cells in a dose-dependent manner (0.1, 0.5, 1, 10, 25, 50, 75, 100 µM concentrations used) are well documented in the study by Knížatová et al. (2021). These cells exposed to the highest concentration (5 µg/ml) of 4-nonylphenol exhibited signifi- cantly decreased secretion of progesterone and androstenedione, and considerable increased production of 17ß-estradiol and testosterone
(Bistakova et al., 2017).
Furthermore, some heavy metals have also been recognized as endocrine disruptors in humans and animals (Iavicoli et al., 2009; Li et al., 2017; Rattan et al., 2017). In various studies (Knazicka et al., 2013, 2021, 2015; Bilcikova et al., 2020; Lukac et al., 2020; Kňažická et al., 2021), dose-dependent disruptive effects of mercury (Hg), iron (Fe), cadmium (Cd), nickel (Ni), and copper (Cu) on steroidogenesis in the NCI-H295R cells have been investigated. Obtained results have clearly evidenced a dose-dependent depletion in testosterone and pro- gesterone production of the cells following a 48 h in vitro HgCl2 (Kna- zicka et al., 2013), FeSO4.7H2O (Knazicka et al., 2021; Kňažická et al., 2021), NiCl2 (Lukac et al., 2020), CdCl2 (Knazicka et al., 2015), and CuSO4.5H2O (Bilcikova et al., 2020) exposures.
Despite these intensive researches, the studies connecting the im- pacts of EDCs on steroidogenesis with ultrastructural alterations in the NCI-H295R cells are very rare. For instance, the paper of Poli et al. (2013) provides an evidence of the intracellular events (related to the qualitative and quantitative alterations in mitochondrial morphology) underlying the toxic effect of 10-50 µM of mitotane (as a first-line pharmacological approach for the treatment of advanced ACC) in the NCI-H295R cells. Morphological and ultrastructural characteristics in the cells treated with10-7, 10-6 and 3 x 10-6 M of F 12511 (S)-2’,3’, 5’-trimethyl-4’-hydroxy-alpha-dodecylthio-phenylacetanilide (i.e., acyl-coenzyme A: cholesterol O-acyltransferase inhibitor) for 24 or 48 h in relationship with steroid production, transcription of genes involved in steroidogenesis, and lipoprotein uptake are documented in the research by Junquero et al. (2001). The machinery of antiproliferative action of 5 uM of sorafenib (tyrosine kinase inhibitor) in the NCI-H295R cells (after 72 h of cultivation) linked to ultrastructural characterization has been investigated by Cerquetti et al. (2021).
In our next experiments, the influence of various EDCs on the pro- duction of sexual steroid hormones in the NCI-H295R cell line will be also studied from the ultrastructural point of view. Moreover, not only qualitative intracellular characterization but also the estimation of quantitative parameters (volume and surface densities) of the organelles associated with steroidogenic secretory activity of the cells will be performed in this concept. Such studies will be helpful to understand and bring a complex insight into the structure-function relationship of the cell line, and can offer sufficient framework for other investigations of reproductive toxicity.
6. Conclusions
In general, the use of in vitro cell model systems has become increasingly popular research tool for investigation of various biological processes and signaling pathways at molecular level. In comparison with other biological model systems, the employment of in vitro cell cultures has overcome problems with tissue quality, cytotoxicity and homoge- neity, which can be impeded to properly evaluate obtained data.
The human ACC cell lines represent a very good biological object for reproductive toxicity testing. According to recent research reports, there were developed several cell substrains derived from the pluripotent adrenocortical NCI-H295 cell line as model systems for assessment of adrenal cell proliferation, and for anticancer therapy purposes. In this review we provide information considering the NCI-H295 cell line origin, development, physiological properties, ultrastructural features, and derived strains focused on the NCI-H295R one. Currently, the human adrenocortical NCI-H295R cell line is the most widely used in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression as it expresses genes encoding for all the key enzymes for steroidogenesis. It has been used mainly for screening assays to detect and assess impacts of various endocrine disrupting agents introduced to the environment which may adversely affect cell differentiation, growth, and development with po- tential to disrupt the endocrine system. The added value of our study arises from detailed ultrastructural characterization of the NCI-H295R
cell line by means of TEM and stereological principles which can be used as a standard approach contributing to understanding molecular ma- chinery of EDCs action on the cell steroidogenesis.
Funding
This research was supported by the Scientific Agency of the Slovak Republic VEGA Nos. 1/0163/18, APVV-20-0218, APVV-16-0289, SUA Grants nos. 33/2019 and 35/2019, and by the Operational Program Integrated Infrastructure within the project: Demand-driven Research for the Sustainable and Innovative Food, Drive4SIFood 313011V336, cofinanced by the European Regional Development Fund.
CRediT authorship contribution statement
Hana Duranova (H.D.): Conceptualization, Validation, Methodol- ogy, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project admin- istration, Funding acquisition. Veronika Fialkova (V.F.): Methodology, Investigation, Resources, Writing - original draft, Writing - review & editing, Project administration, Funding acquisition. Veronika Valkova (V.V.): Methodology, Resources, Writing - original draft. Jana Bilci- kova (J.B.): Methodology, Investigation, Writing - original draft. Lucia Olexikova (L.O.): Methodology, Resources. Norbert Lukac (N.L.): Methodology, Supervision, Project administration, Funding acquisition. Peter Massanyi (P.M.): Methodology, Supervision, Project adminis- tration, Funding acquisition. Zuzana Knazicka (Z.K.): Methodology, Resources, Writing - original draft, Supervision, Project administration, Funding acquisition.
Declarations of interest
None.
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