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TITLE: EXPRESSION OF CYTOCHROME P450 ISOZYMES IN PRIMARY CORTICAL CULTURES FROM RAT FRONTAL CORTEX
Citation:
Shafer, Tim, E. Winkfield, G. Nelson, Steve Simmons, B. Chorley, AND W. Mundy. TITLE: EXPRESSION OF CYTOCHROME P450 ISOZYMES IN PRIMARY CORTICAL CULTURES FROM RAT FRONTAL CORTEX. Society of Toxicology, San Diego, CA, March 22 - 27, 2015.
Impact/Purpose:
ABSTRACT: The need to screen large numbers of chemicals for developmental neurotoxicity has spurred the development of high throughput/high content assays for neurite outgrowth, synaptogenesis and electrical activity using primary cultures of cortical neural tissue. While these assays are capable of screening large numbers of chemicals, the ability of these cell cultures to metabolize chemicals is unknown, which affects interpretation of results and necessitates metabolite testing in these assays. Therefore, the present study examined the expression of mRNA for cytochrome P450 isozymes in primary cortical neural cultures on Days in Vitro (DIV) 1 and 14. Primary cultures of rat frontal cortex were prepared from 0-24h old rat pups and plated onto 30 mm dishes. On DIV 1 and 14, total RNA was isolated. In addition, total RNA was isolated from 0-24h old rat pup liver tissue for comparison to the neural mRNA expression patterns. mRNA expression levels were quantified using hydrolysis probe real-time qPCR assays for each of the cytochrome P450 gene targets: Cyp1a1, Cyp2c11, Cyp2d4, Cyp2e1, Cyp2s1, Cyp3a2, Cyp3a23/3 and Cyp4x1. Per unit of mRNA, β-actin mRNA expression was the same in liver and brain tissue, thus all values were normalized to β-actin mRNA expression. While readily expressed in rat liver tissue, there was little to no expression of Cyp2e1, Cyp3a2, Cyp3a23/3 or Cyp1a1 in cortical cultures. Cell-based P450 glo assays confirmed the lack of functional Cyp1A and Cyp 3A. When normalized to β-actin, Cyp2d4 and Cyp2s1 were expressed on DIV1 and 14, but at levels much lower than in rat liver tissue. By contrast, normalized gene expression of Cyp2c11 and Cyp4x1 in cortical cultures on DIV 1 was the same, and by DIV 14 was increased, compared to its expression in the liver. While protein levels and functional activity of these Cyps remain to be confirmed, these data indicate that primary cortical cultures may have limited metabolic capability compared to rat liver tissue. (This abstract does not represent Agency Policy).
Description:
TITLE: EXPRESSION OF CYTOCHROME P450 ISOZYMES IN PRIMARY CORTICAL CULTURES FROM RAT FRONTAL CORTEX