Toxicogenomic outcomes predictive of forestomach carcinogenesis following exposure to benzo(a)pyrene: Relevance to human cancer risk

Abstract

Forestomach tumors are observed in mice exposed to environmental carcinogens. However, the relevance of this data to humans is controversial because humans lack a forestomach. We hypothesize that an understanding of early molecular changes after exposure to a carcinogen in the forestomach will provide mode-of-action information to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present study we exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors of the rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach. Adult Muta™Mouse males were orally exposed to 25, 50, and 75 mgBaP/kg-body-weight/day for 28 consecutive days. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, and protein analyses were employed to characterize responses in the forestomach. Microarray results showed altered expression of 414 genes across all treatment groups (± 1.5 fold; false discovery rate adjusted P ≤ 0.05). Significant downregulation of genes associated with phase II xenobiotic metabolism and increased expression of genes implicated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiation were observed in treated groups in a dose-dependent manner. A systematic comparison of the differentially expressed genes in the forestomach from the present study to differentially expressed genes identified in human diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showed significant commonalities between the two models. Our results provide molecular evidence supporting the use of the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans.

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

“…Indeed, BPDE–DNA adducts correlate well with TP53 induction in cultured human lymphocytes as measured by immunocytochemical staining (Godschalk et al, 2001). This finding is in agreement with the robust activation of p53 target genes that we observed in the lung, liver and forestomach of BaP-treated mice (Labib et al, 2012, 2013; Malik et al, 2012). Similarly, BaP exposure of human TK6 cells increased DNA damage and apoptosis (Figure 4) in parallel with changes in the expression of TP53-regulated genes (e.g.…”

“…We previously reported a robust response in the p53 signaling pathway in gene expression profiles from the livers (Malik et al, 2012), lungs (Labib et al, 2012) and forestomachs (Labib et al, 2013) of mice treated for 28 days with BaP. Multiple p53-dependent genes (including Cdkn1a, Ccng1 and Ccnd1 ) were similarly dose-dependently upregulated in mouse liver and lung at the 3-day time point.…”

“…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). …”

“…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).…”

“…Circadian rhythm and lipid metabolism are examples of BaP-inducible pathways (also affected by BaP in other toxicogenomics studies; Hockley et al, 2006; Van Delft et al, 2010) that are difficult to fit into a carcinogenic MOA. In addition, gene signatures related to immunotoxicity were prevalent in the lung and forestomach datasets, indicative of immunosuppression and proinflammatory responses, respectively (Halappanavar et al, 2011; Labib et al, 2012; Labib et al, 2013). Therefore, our genomics data identify immunosuppression as a possible hazard that is a plausible modulating factor in the MOA for BaP-induced carcinogenicity in the lung and the recognition of excessively accumulated metabolites as potential pathogens in the forestomach.…”