Testing an Aflatoxin B1 Gene Signature in Rat Archival Tissues

Abstract

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c and C8orf46 homolog. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p≤0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25–500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.

Transcriptomic alterations induced by Monuron in rat and human renal proximal tubule cells in vitro and comparison to rat renal-cortex in vivo

“…27 In the rat, Fhit is located on chromosome 15 and many studies suggest that its lost is an early event in carcinogenesis. [28][29][30][31] Although we saw a decrease in expression of Fhit gene in both kidney and the liver, the decrease was only statistically significant in the liver at the protein level (Fig. 4).…”

Laboratory to Community: Chemoprevention Is the Answer

“…The concept of gene expression profiling is based on the assumption that environmental toxicants will exert their effects directly or indirectly by perturbing normal cell signaling processes, consistent with the findings of Johnson and colleagues (1). It was predicted that by perturbing normal cell signaling processes, a cascade of events would be elicited that culminate in gene or protein expression patterns unique for specific toxicants (12,19,20).…”

“…They took advantage of toxicogenomic microarray technology to examine the effect of a triterpenoid imidazole derivative, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) on the expression of genes known to be turned "-on or -off" by exposure to aflatoxin B 1 (12). Using the relatively inexpensive, high-throughput platform developed by the National Institute of Environmental Health Sciences (19,20), they evaluated transcript profiles or gene signatures previously shown to be predictive of aflatoxin B 1 -induced hepatocellular toxicity (21).…”

Simpler alternative to CARCINOscreen^® based on quantitative PCR (qPCR)

“…With regard to the four selected predictive genes for qPCR, Abcb1b is a member of the ATP-binding cassette (ABC) protein superfamily and multi-drug resistanceassociated protein, and the protein transporter is reported as being functional in B-13/H cancer cells (Probert et al, 2014). Merrick et al (2012) reported that administration of the hepatocarcinogen aflatoxin B1 (AFB1) to male F344 rats significantly increased the expression of Abcb1b in the rat liver. This observation suggests that the xenosensor activity of Abcb1b was likely responding to AFB1 or its metabolites (Merrick et al, 2012).…”

“…Merrick et al (2012) reported that administration of the hepatocarcinogen aflatoxin B1 (AFB1) to male F344 rats significantly increased the expression of Abcb1b in the rat liver. This observation suggests that the xenosensor activity of Abcb1b was likely responding to AFB1 or its metabolites (Merrick et al, 2012). In this study, the expression level of Abcb1b detected by qPCR was drastically increased by carcinogens of in the training phase, such as diethylnitrosamine, N-nitrosomorpholine, 2-acetylaminofluorene, etc.…”