Quantitative RT-PCR Methods for Evaluating Toxicant-Induced Effects on Steroidogenesis Using the H295R Cell Line

Abstract

Gene expression profiles show considerable promise for the evaluation of the toxic potential of environmental contaminants. For example, any alterations in the pathways of steroid synthesis or breakdown have the potential to Cause endocrine disruption. Therefore monitoring these pathways can provide information relative to a chemical's ability to impact endocrine function. One approach to monitoring these pathways has been to use a human adrenocortical carcinoma cell line (H295R) that expresses all the key enzymes necessary for steroidogenesis. In this study we have further developed these methods using accurate and specific quantification methods utilizing molecular beacon-based quantitative RT-PCR (Q-RT-PCR). The assay system was used to analyze the expression patterns of 11 steroidogenic genes in H295R cells. The expression of gene transcripts was measured using a real-time PCR system and quantified based on both a standard curve method using a dilution series of RNA standards and a comparative Ct method. To validate the optimized method, cells were exposed to specific and nonspecific model compounds (inducers and inhibitors of various steroidogenic enzymes) for gene expression profiling. Similar gene expression profiles were exhibited by cells treated with chemicals acting through common mechanisms of action. Overall, our findings demonstrated that the present assay can facilitate the development of compound-specific response profiles, and will provide a sensitive and integrative screen for the effects of chemicals on steroidogenesis.

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

“…The HMGR, StAR, and CYP11A expressions were not markedly altered. This may be due to the basal mRNA abundance of HMGR, StAR, and CYP11A [22]. In the present study, PFOS inhibited the conversion of progesterone to testosterone through inhibition of CYP17.…”

“…The H295R cell line can express most of the key enzymes and functional proteins in the steroidogenesis pathway [21]. This cell line has been shown to be a potential in vitro model for screening adverse effects of chemicals on steroidogenesis [22]. The results from in vitro studies should be verified by in vivo studies.…”

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

“…Quantitative real-time PCR (qRT-PCR) was analyzed on the ABI 7300 real time PCR system (Applied Biosystems) using SYBR® Green PCR master mix (Applied Biosystems). The primer design and concentrations, thermal cycle profiles and quantification of target genes were performed as described previously (Zhang et al, 2005). The housekeeping gene prophobilinogen deaminase (PBGD) did not vary under experimental conditions in the present study (data not shown) and was used as an internal control.…”

“…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.…”

Classification of Chemicals Based on Concentration-Dependent Toxicological Data Using ToxClust

“…Cells were exposed to different concentrations of chemicals for 48 h in 24-well plates (COSTAR, Bucks, U.K.). DMSO was used as a carrier solvent and did not exceed 0. on an ABI Prism high throughput 7900HT system using the primers previously described (8). Chemicalinduced concentration-dependent response curves of hormone production and mRNA levels are provided in Tables S1 and S2 of the Supporting Information.…”