Furan is a chemical hepatocarcinogen in mice and rats. Its previously postulated cancer mode of action (MOA) is chronic cytotoxicity followed by sustained regenerative proliferation; however, its molecular basis is unknown. To this end, we conducted toxicogenomic analysis of B3C6F1 mouse livers following three week exposures to non-carcinogenic (0, 1, 2mg/kgbw) or carcinogenic (4 and 8mg/kgbw) doses of furan. We saw enrichment for pathways responsible for cytotoxicity: stress-activated protein kinase (SAPK) and death receptor (DR5 and TNF-alpha) signaling, and proliferation: extracellular signal-regulated kinases (ERKs) and TNF-alpha. We also noted the involvement of NF-kappaB and c-Jun in response to furan, which are genes that are known to be required for liver regeneration. Furan metabolism by CYP2E1 produces cis-2-butene-1,4-dial (BDA), which is required for ensuing cytotoxicity and oxidative stress. NRF2 is a master regulator of gene expression during oxidative stress and we suggest that chronic NFR2 activity and chronic inflammation may represent critical transition events between the adaptive (regeneration) and adverse (cancer) outcomes. Another objective of this study was to demonstrate the applicability of toxicogenomics data in quantitative risk assessment. We modeled benchmark doses for our transcriptional data and previously published cancer data, and observed consistency between the two. Margin of exposure values for both transcriptional and cancer endpoints were also similar. In conclusion, using furan as a case study we have demonstrated the value of toxicogenomics data in elucidating dose-dependent MOA transitions and in quantitative risk assessment.
The mammalian genome is transcribed into mRNAs that code for protein and a broad spectrum of other noncoding (nc) RNA products. Long ncRNAs (lncRNA), defined as ncRNA species > 200 nucleotides long, are emerging as important epigenetic regulators of gene expression that are involved in a spectrum of biological processes of relevance to toxicology. We conducted a gene expression profiling study in the livers of female B6C3F1 mice exposed to the carcinogen furan at 0.0, 1.0, and 2.0mg/kg (noncarcinogenic doses) and at 4.0 and 8.0mg/kg (carcinogenic doses) for 3 weeks. LncRNA differential expression showed a nonlinear dose response with none differentially expressed at 1.0 or 2.0mg/kg, 2 lncRNAs at 4.0mg/kg furan, and 83 at 8mg/kg, representing 13.3% (83/632) of the total number of differentially expressed transcripts. Among the lncRNAs observed, two lncRNAs examined showed transcriptional clustering with nearby protein-coding genes. LincRNA-p21 is an antisense transcript that is 15kb downstream from Cdkn1a locus and appears to be cotranscribed with the protein coding gene Cdkn1a at 8.0mg/kg furan. In a separate independent study, RNA samples from the livers of mice administered benzo(a)pyrene also demonstrated increased levels of Cdkn1a and the antisense lincRNA-p21 transcript. These data demonstrate that lncRNAs are transcriptional targets of furan exposures associated with levels of furan that are cytotoxic and induce cell proliferation. In addition, certain lncRNA transcripts are associated with the expression of nearby coding protein genes. We hypothesize that lncRNAs have potential as epigenetic biomarkers of carcinogenic exposures.
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