Quantitative RT-PCR methods for evaluating toxicant-induced effects on steroidogenesis using the H295R cell line.
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.
Zhang
Xiaowei
X
Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824, USA. zhangx22@msu.edu
Yu
Richard M K
RM
Jones
Paul D
PD
Lam
Gabriel K W
GK
Newsted
John L
JL
Gracia
Tannia
T
Hecker
Markus
M
Hilscherova
Klara
K
Sanderson
Thomas
T
Wu
Rudolf S S
RS
Giesy
John P
JP
D045744:Cell Line, Tumor; D004703:Endocrine System / Q000187:drug effects*; D004784:Environmental Monitoring / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D010750:Phosphoproteins / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013256:Steroids / Q000378:metabolism / Q000633:toxicity*; D014057:Tomography, X-Ray Computed / Q000379:methods; D000098682:Steroidogenic Acute Regulatory Protein
D010750:Phosphoproteins; D013256:Steroids; D000098682:Steroidogenic Acute Regulatory Protein; D012313:RNA