Lung genotoxicity of benzo(a)pyrene in vivo involves reactivation of LINE-1 retrotransposon and early reprogramming of oncogenic regulatory networks

Abstract

Several lines of evidence have implicated long interspersed nuclear element-1 (LINE-1) retroelement in the onset and progression of lung cancer. Retrotransposition-dependent mechanisms leading to DNA mobilization give rise to insertion mutations and DNA deletions, whereas retrotransposition-independent mechanisms disrupt epithelial programming and differentiation. Previous work by our group established that tobacco carcinogens such as benzo(a)pyrene (BaP) reactivate LINE-1 in bronchial epithelial cells through displacement of nucleosome remodeling and deacetylase (NuRD) corepressor complexes and interference with retinoblastoma-regulated epigenetic signaling. Whether LINE-1 in coordination with other genes within its regulatory network contributes to the in vivo genotoxic response to BaP remains largely unknown. Evidence is presented here that intratracheal instillation of ORFeus^LSL mice with BaP alone or in combination with adenovirus (adeno)-CRE recombinase is genotoxic to the lung and associated with activation of the human LINE-1 transgene present in these mice. LINE-1 reactivation modulated the expression of genes involved in oncogenic signaling, and these responses were most pronounced in female mice compared with males and synergized by adeno-CRE recombinase. This is the first report linking LINE-1 and genes within its oncogenic regulatory network with early sexually dimorphic responses of the lung in vivo.

Keywords: LINE-1; ORFeusLSL; benzo(a)pyrene; lung; oncogenic reprogramming.

Fig. 1.

Histology of lung tissues in ORFeus^LSL mice treated with benzo(a)pyrene (BaP) alone or in combination with CRE recombinase. A–I: representative results for all treatment groups at ×20. Blue arrows indicate the pulmonary edema evidenced by vascular dilation, black arrows denote pulmonary vessel congestion, and the red arrow denotes emphysematous changes evidenced by large empty spaces with variable preservation of alveolar structures. Here, n = 3 animals examined.

Fig. 2.

A: schematic of the ORFeus^LSL transgene: 1) the floxed allele of the ORFeus^LSL transgene, 2) the excised allele of the ORFeus^LSL transgene following Cre-mediated excision of the floxed β-geo-stop cassette (LSL), and 3) putative insertion upon retrotransposition. B: long interspersed nuclear element-1 (LINE-1) mRNA in lungs of ORFeus^LSL mice treated with benzo(a)pyrene (BaP) alone or in combination with CRE recombinase. LINE-1 mRNA was measured by RT-qPCR using primers specific for ORFeus^LSL. A (upright), polyadenylation signal; gfp, green fluorescent protein; LTR, long terminal repeat; ORF, open reading frame; RQ, relative quantification; n = 3 animals examined. Different letters denote differences between control, BaP-treated, and CRE-BaP-treated groups at P ≤ 0.05.

Fig. 3.

mRNA abundance of genes within the long interspersed nuclear element-1 oncogenic regulatory network in lungs of ORFeus^LSL mice. A–I: results for different genes within the network: AhR, aryl hydrocarbon receptor; CCL2, chemokine (C-C motif) ligand 2; CYP2A4, cytochrome P450, family 2, subfamily a, polypeptide 4; MGST, microsomal glutathione S-transferase 1; PAH, phenylalanine hydroxylase; POSTN, periostin, osteoblast-specific factor; PTPRB, protein tyrosine phosphatase, receptor type B; TGFB1, transforming growth factor-β1; VCAM, vascular cell adhesion molecule 1. BaP, benzo(a)pyrene; RQ, relative quantification; n = 3 animals examined. Different letters denote differences between control, BaP-treated, and CRE-BaP-treated groups at P ≤ 0.05.