Hepatic mRNA, microRNA, and miR‐34a‐Target responses in mice after 28 days exposure to doses of benzo(a)pyrene that elicit DNA damage and mutation

Abstract

Benzo(a)pyrene (BaP) is a mutagenic carcinogen that is ubiquitous in our environment. To better understand the toxic effects of BaP and to explore the relationship between toxicity and toxicogenomics profiles, we assessed global mRNA and microRNA (miRNA) expression in Muta™Mouse. Adult male mice were exposed by oral gavage to 25, 50, and 75 mg/kg/day BaP for 28 days. Liver tissue was collected 3 days following the last treatment. Initially, we established that exposure to BaP led to the formation of hepatic DNA adducts and mutations in the lacZ transgene of the Muta™Mouse. We then analyzed hepatic gene expression profiles. Microarray analysis of liver samples revealed 134 differentially expressed transcripts (adjusted P < 0.05; fold changes > 1.5). The mRNAs most affected were involved in xenobiotic metabolism, immune response, and the downstream targets of p53. In this study, we found a significant 2.0 and 3.6-fold increase following exposure to 50 and 75 mg/kg/day BaP, respectively, relative to controls for miR-34a. This miRNA is involved in p53 response. No other significant changes in miRNAs were observed. The protein levels of five experimentally confirmed miR-34a targets were examined, and no major down-regulation was present. The results suggest that liver miRNAs are largely unresponsive to BaP doses that cause both DNA adducts and mutations. In summary, the validated miRNA and mRNA expression profiles following 28 day BaP exposure reflect a DNA damage response and effects on the cell cycle, consistent with the observed increases in DNA adducts and mutations. Environ. Mol. Mutagen., 2012. © 2011 Crown in the right of Canada

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water

“…In addition, ROS produced as by-products of BaP metabolism (from BaP catechol–quinone redox cycling) may oxidize DNA (Lan et al, 2004). BPDE–DNA adducts can be measured directly, and their levels are markedly elevated by BaP treatment in lung, liver and glandular stomach tissues in our studies (Figure 2; Halappanavar et al, 2011; Labib et al, 2012; Lemieux et al, 2011; Malik et al, 2012). Previous studies have established that levels of DNA adducts in the forestomach from mice orally exposed to BaP for 24 hours and five days are comparable to those formed in the glandular stomach (Arlt et al 2008).…”

“…Using our mouse data, we noted that the transcription of many P450 genes (e.g. Cyp1a1, Cyp7a1 Cyp1b1, Cyp2b10, Cyp2b13, Cyp3a44, Cyp2b9, Cyp2c38 and Cyp2c40 ) increased following treatment with BaP treatment in mouse liver and lung (Halappanavar et al, 2011; Malik et al, 2012; Yauk et al, 2011). Of these, Cyp1a1 responded at the earliest time points and with the highest fold change compared with controls.…”

“…All samples were analyzed using Agilent 4×44K or 8×60K gene expression microarrays. The full DNA microarray and real-time quantitative PCR array analyses are published for liver (Malik et al, 2012), lung (Labib et al, 2012) and forestomach (Labib et al, 2013); the reader should refer to these publications for details relating to microarray data processing and normalization. Data for liver, lung and forestomach are available in the Gene Expression Omnibus (accession numbers GSE24910, GSE35718 and GSE43438, respectively).…”

“…The results in the lung and liver are consistent with expected changes in gene expression in response to a genotoxic carcinogen, whereas the forestomach data point to additional modulating factors at work (a pro-immune, inflammatory response) (Hochstenbach et al, 2012). Please note that BPDE-DNA adducts and lacZ transgene mutant frequency were also assessed in the lung, liver, and glandular stomach tissues from the same mice (Lemieux et al, 2011, Malik et al, 2012, Labib et al, 2012) (Figure 2). Adducts were detected via the 32P-postlabeling method and were found in all three dose groups in all tissues, with the highest relative adduct labeling in the lung tissue at three days post-exposure.…”

“…Many phase II enzymes are activated at the mRNA level by the transcription factor Nrf2 (Nfe2l2) in response to the presence of reactive metabolites and oxidative stress (Kaspar et al, 2009). We observed the transcriptional induction of Nrf2 and its target genes (Nqo1, Ugdh, Srxn1, Akr1b15, Ugt2b15, Gsta1, Gsta2, Gstm3, Gstm4, Gstm7 and Gsto1 ) in mouse lung, liver and forestomach in response to BaP, in at least one time point (3- or 28-day regimens), suggesting that detoxication mechanisms were activated in order to cope with BaP toxicity (Labib et al, 2013; Malik et al, 2012; Yauk et al, 2011). …”