Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.