Assessment of the Effects of Chemicals on the Expression of Ten Steroidogenic Genes in the H295R Cell Line Using Real-Time PCR

Abstract

The potential for a variety of environmental contaminants to disturb endocrine function in wildlife and humans has been of recent concern. While much effort is being focused on the assessment of effects mediated through steroid hormone receptor-based mechanisms, there are potentially several other mechanisms that could lead to endocrine disruption. Recent studies have demonstrated that a variety of xenobiotics can alter the gene expression or activity of enzymes involved in steroidogenesis. By altering the production or catalytic activity of steroidogenic or steroid-catabolizing enzymes, these chemicals have the potential to alter the steroid balance in organisms. To assess the potential of chemicals to alter steroidogenesis, an assay system was developed using a human adrenocortical carcinoma cell line, the H295R cell line, which retains the ability to synthesize most of the important steroidogenic enzymes. Methods were developed, optimized, and validated to measure the expression of 10 genes involved in steroidogenesis by the use of real-time quantitative reverse transcriptase PCR. The effects of several model chemicals known to alter steroid metabolism, both inducers and inhibitors, were assessed. Similar expression patterns were observed for chemicals acting through common mechanisms of action. Time-course studies demonstrated distinct time-dependent expression profiles for chemicals able to modulate steroid metabolism. The assay, which allows simultaneous analysis of the expression of numerous steroidogenic enzymes, would be useful as a sensitive and integrative screen for the many effects of chemicals on steroidogenesis.

Effects of fluorotelomer alcohol 8:2 FTOH on steroidogenesis in H295R cells: Targeting the cAMP signalling cascade

“…The transcriptional profiles of most steroidogenic genes (e.g., StAR, CYP11A, CYP11B, CYP17 and CYP21) are cAMP-dependent (Sewer and Watermen, 2001). Many chemicals have been hypothesized to disrupt steroidogenesis via the cAMP pathway, including triazines, forskolin, vinclozolin, isobutyl, and methylxanthine (Hilscherova et al, 2004;Sanderson et al, 2000Sanderson et al, , 2002. To test the hypothesis that 8:2 FTOH exposure inhibits steroidogenesis (including decreased mRNA expression, protein abundance and hormones levels) by reducing cellular cAMP levels, cellular cAMP content was examined.…”

“…Upon cAMP stimulation, these cells produce all the adrenal steroid hormones found in the adult adrenal cortex (Gazdar et al, 1990;Sanderson, 2006). H295R cells have been employed for studying the biological effects and mechanisms of action of a variety of chemicals (e.g., Ding et al, 2007;Hecker et al, 2006Hecker et al, , 2007Hilscherova et al, 2004;Li and Wang, 2005;Sanderson, 2006;Zhang et al, 2005). In the present study, HPLC-MS/MS was used to examine the effects of 8:2 FTOH on the production of seven steroid hormones: progesterone, 17α-OH-progesterone, androstenedione, testosterone, deoxycorticosterone, corticosterone and cortisol.…”

Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine‐related genes in vitro and in vivo

“…The modulation of 10 major genes involved in the synthesis of steroid hormones (CYP11A, CYP11B2, CYP17, CYP19, CYP21, 17bHSD1, 17bHSD4, 3bHSD2, HMGR, and StAR) after exposure was investigated using Q-RT-PCR. The PCR conditions, including sense and antisense primers, have been described by Hilscherova et al [26]. See Table 1 for primer sequences.…”

Effects of tris(2,3-dibromopropyl) isocyanurate on steroidogenesis in H295R cells

“…Hormones analyzed are typed in bold orange, whereas gene expression analyzed is typed in blue. Upwards arrow up-regulation; downwards arrow down-regulation; asterisk statistical significance (P \ 0.05) inhibited by some PCBs (Hilscherova et al 2004). However, polybrominated biphenyls (PBBs 169) induced the StAR gene expression at low concentration (Ding et al 2007).…”

“…Melting curve analyses were performed immediately following the final PCR cycle to differentiate between the desired products and any primer-dimers or DNA contaminants. Specific details of the assay parameters such as primers used and annealing temperatures were derived from a previous study (Hilscherova et al 2004). Relative expression of mRNA was quantified by the comparative C T method described previously (Ji et al 2010;Livak and Schmittgen 2001).…”