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Transcriptomics Analysis and Hormonal Changes of Male and Female Rats Treated Chronically with a Low Dose of Acrylamide in their Drinking Water

ABSTRACT: Acrylamide is known to produce follicular cell tumors of the thyroid in rats. RccHan Wistar rats were exposed in utero to a carcinogenic dose of acrylamide (3 mg/Kg bw/day) from gestation day 6 to delivery and then through their drinking water to postnatal day 35. In order to identify potential mechanisms of carcinogenesis in the thyroid glands, we used a transcriptomics approach. Thyroid glands were collected from male pups at 10 PM and female pups at 10 AM or 10 PM in order to establish whether active exposure to acrylamide influenced gene expression patterns or pathways that could be related to carcinogenesis. While all animals exposed to acrylamide showed changes in expected target pathways related to carcinogenesis, such as DNA repair, DNA replication, chromosome segregation, among others, animals that were sacrificed while actively drinking acrylamide-laced water during their active period at night showed increased changes in pathways related to oxidative stress, detoxification pathways, metabolism, and activation of checkpoint pathways, among others. In addition, thyroid hormones, T3 and T4, were increased in acrylamide-treated rats sampled at night, but not in quiescent animals, compared to controls. The data clearly indicate that time of day for sample collection is critical to identifying molecular pathways that are altered by the exposures. These results suggest that carcinogenesis in the thyroids of acrylamide treated rats may ensue from several different mechanisms such as hormonal changes and oxidative stress and not only from direct genotoxicity, as has been assumed to date.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE62026 | GEO | 2014/10/06

SECONDARY ACCESSION(S): PRJNA263137

REPOSITORIES: GEO

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Project description:Acrylamide is a type-2 alkene monomer with established human neurotoxic effects. While the primary source of human exposure to acrylamide is occupational, other exposure sources include food, drinking water, and smoking. In this study, neurobehavioral assays coupled with transcriptional profiling analysis were conducted to assess both behavioral and gene expression effects induced by acrylamide neurotoxicity in rats when administered during early postnatal life. Acrylamide administration in rat pups induced significant characteristic neurotoxic symptoms including increased heel splay, decrease in grip strength, and decrease in locomotor activity. Transcriptome analysis with the Affymetrix Rat Genome 230 2.0 array indicated that acrylamide treatment caused a significant alteration in the expression of genes involved in muscle contraction, pain regulation, and dopaminergic neuronal pathways. First, in agreement with the observed behavioral effects, expression of the Mylpf gene involved in muscle contraction was downregulated in the spinal cord in response to acrylamide. Second, in sciatic nerves, acrylamide repressed the expression of the opioid receptor gene Oprk1 that is known to play a role in neuropathic pain regulation. Finally, in the cerebellum, acrylamide treatment caused a decrease in the expression of the nuclear receptor gene Nr4a2 that is required for development of dopaminergic neurons. Thus, our work examining the effect of acrylamide at the whole-genome level on a developmental mammalian model has identified novel genes previously not implicated in acrylamide neurotoxicity that can be further developed into biomarkers for assessing the risk of acrylamide exposure. Three-week-old male Wistar rat pups were treated with either acrylamide or saline daily (30 mg/kg) for 21 days, then tissues (cerebellum, spinal cord, and sciatic nerve) were harvested and frozen. Two biological replicate samples, each sample consisting of pooled tissue from 2 rats, were analyzed for each treatment.

Project description:One µg of total RNA from either an individual rat or from a pooled sample was amplified and labeled with a fluorescent dye (either Cy3 or Cy5) using the Low RNA Input Linear Amplification Labeling kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s protocol. The amount and quality of the resulting fluorescently labeled cRNA was assessed using a Nanodrop ND-100 spectrophometer and an Agilent Bioanalyzer. Equal amounts of Cy3- or Cy5-labeled cRNA were hybridized to the Agilent Rat Oligo Microarray (Agilent Technologies, Inc., Palo Alto, CA) for 17 hrs, prior to washing and scanning. Data was extracted from the resulting images using Agilent’s Feature Extraction Software (Agilent Technologies, Inc., Palo Alto, CA). For day/night comparisons, two types of complementing hybridizations were performed: hepatic RNA from individual day rats was hybridized against a pool made up of RNA from 12 hour offset night rats, and RNA from individual night rats was hybridized against a pool made up of RNA from 12-hour offset day rats. Specifically, hepatic RNA samples from three individual day rats collected ten hours after light on (CT10) were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six night rats collected ten hrs after lights off (CT22); and RNA samples from three individual night rats collected at CT22 were hybridized against a pooled RNA sample composed of equal aliquots of RNA from the livers of six day rats collected at CT10. This was replicated in a second study for a total of 24 hybridizations of 12 hour offset samples. For time of day comparisons, equal aliquots of RNA from the livers of six rats collected at each of four different times of the circadian day (CT4, CT10, C16 and CT22) were pooled. The two replicate studies thus resulted in 8 pools (two replicate pools/time point times four time points); each pool was hybridized against a Universal Rat Reference RNA Standard (Stratagene, La Jolla, CA). Keywords: time-course

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Transcriptomics Analysis and Hormonal Changes of Male and Female Rats Treated Chronically with a Low Dose of Acrylamide in their Drinking Water

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