Toxicogenomic Analysis of Gender, Chemical, and Dose Effects in Livers of TCDD- or Aroclor 1254-Exposed Rats

Chronic exposure of Sprague-Dawley (SD) rats to either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or Aroclor 1254 results in female-selective induction of hepatic tumors. The relative potency of dioxins and PCB mixtures, such as Aroclor 1254, is often estimated using the internationally endorsed toxic equivalency (TEQ) approach. Comparing the genome wide changes in gene expression in both genders following exposure to toxic equivalent doses of these chemicals should identify critical sets of early response genes while further defining the concept of the TEQ of halogenated aromatic hydrocarbons. Aroclor 1254 at 0.6, 6.0 and 60 mg/kg body weight and TEQ doses of TCDD (0.3 and 3.0 µg/kg), calculated to match the top two Aroclor 1254 doses, were orally administered to SD rats for three consecutive days. Day 4 gene expression in hepatic tissue was determined using microarrays. A linear mixed-effects statistical model was developed to analyze the data in relation to treatment, gender, and gender*treatment (G*T) interactions. The genes most changed included 54 genes with and 51 genes without a significant model G*T term. The known aryl hydrocarbon receptor (AHR) battery genes (Cyp1a1, Cyp1a2, Cyp1b1, Aldh3a1), and novel genes, responded in a TEQ dose-dependent manner in both genders. However, an important observation was the apparent disruption of sexually dimorphic basal gene expression, particularly for female rats. Since many of these genes are involved in steroid metabolism, exposure to either TCDD or Aroclor 1254 could disrupt proliferative signals more in female rats as a possible consequence of altered estrogen metabolism. This study extends the findings of previous rodent bioassays by identifying groups of genes, other than the well-characterized AHR response genes, whose disruption may be important in the tumorigenic mechanism in this rat strain. Keywords: dose response, Multifactor Linear Model Twenty four female (171-194 g) and 24 male (190-207 g) S-D rats (Crl:CD(SD)IGS BR, Charles River Laboratories, Wilmington, MA) were randomly assigned to 6 groups of four rats per sex. Test chemicals were administered by gastric intubation in corn oil (5 ml/kg) at dose levels of 0.6, 6.0, or 60 Aroclor 1254 mg/kg, or 0.3 or 3.0 TCDD ug/kg for 3 consecutive days. Control groups for each gender received corn oil alone. On Day 4 the animals were euthanized by CO2 overdose and the livers were removed and frozen. Total RNA was isolated from liver tissues and used to hybridize Affymetrix RG-U34A arrays. A total of 68 chips were used in the downstream analyses. These chips include biological and technical replicates (full details are given in the supplementary file Experimental_Design.xls). A probe-level linear mixed-effects statistical model was developed to analyze the data in relation to treatment, gender and gender*treatment interactions.