Dysregulation of RORA, a Risk Gene for ASD, By “Low-Dose” Exposure to the Herbicide Atrazine, an Endocrine Disrupting Compound

Background:  We have previously shown that retinoic acid-related orphan receptor alpha (RORA) is deficient in a subgroup of individuals with ASD and that it potentially regulates the transcription of hundreds of ASD-associated genes. Because RORA is in turn regulated in opposite directions by male and female sex hormones, we hypothesize that RORA may be dysregulated by endocrine disrupting compounds (EDCs), like the herbicide atrazine, which may result in downstream or genome-wide changes in the expression of genes relevant to the pathobiology of ASD. 

Objectives:  The goals of this study were to: 1) investigate changes in RORA expression induced by low-dose exposure to atrazine; 2) investigate the potential for genome-wide alterations of biochemical pathways relevant to ASD; and 3) develop a high-throughput assay for screening other EDCs that may elevate risk for ASD based on dysregulation of RORA expression.

Methods:  

Treatment of neuronal cells with low-dose atrazine. The human neuronal cell model (SH-SY5Y) was exposed to 10-fold dilutions of atrazine solubilized in DMSO (range 0.1nM-1000nM) for 2 hours.

RORA expression and transcriptome analyses. RORA expression as a function of atrazine concentration was determined by RT-qPCR analyses.  Transcriptome analyses of atrazine-treated SH-SY5Y samples at selected doses were also performed using the Affymetrix GeneChip Human Transcriptome Array 2.0. Genome-wide expression changes were analyzed using Transcriptome Array Console (TAC) and Expression Console software, as well as Partek Genomics Suite. Functional and network prediction analyses were performed using QIAGEN Ingenuity Pathway Analysis (IPA).

Development of a high-throughput screening assay. SH-SY5Y cells were co-transfected with a construct containing the RORA promoter-driven Firefly luciferase reporter gene and a separate construct containing the Renilla reniformis luciferase gene under the control of a constitutive promoter. Following transfection, the cells were exposed to varying levels of atrazine for 2 hours. Firefly luciferase activity was normalized to that of Renilla luciferase in each well to determine the relative levels of RORA promoter activity in each sample. 

Results:  Preliminary results show that RORA is dysregulated biphasically in a neuronal cell model by low-dose exposure to atrazine. Additionally, using microarrays for expression profiling, we were able to observe that exposure to subnanomolar and nanomolar concentrations of atrazine induce differential gene expression at the whole genome level, notably affecting genes with ASD-associated neurological functions. Furthermore, a number of genes impacted by atrazine exposure are among the annotated genes within the SFARI database of ASD risk genes. The high-throughput dual-luciferase reporter assay replicates in part the atrazine-induced changes in RORA expression seen by RT-qPCR analyses. 

Conclusions:  Low, subtoxic doses of EDCs, such as atrazine, may increase risk for ASD by dysregulating RORA, a gene that we have validated as a transcriptional regulator of multiple genes associated with the pathobiology of autism. We anticipate that our high-throughput screen will provide additional insight into gene-environment interactions that impact neurodevelopment as well as increased awareness of the biological consequences of low-dose levels of EDCs that are below current EPA-approved limits.