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. 2008 Jan 8;649(1-2):161-78.
doi: 10.1016/j.mrgentox.2007.08.007. Epub 2007 Sep 7.

The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

Dhrubajyoti Chakravarti  1 , Divya Venugopal, Paula C Mailander, Jane L Meza, Sheila Higginbotham, Ercole L Cavalieri, Eleanor G Rogan
Affiliations

The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

Dhrubajyoti Chakravarti et al. Mutat Res. .

Abstract

Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N(2)dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early preneoplastic mutations. In conclusion, this study suggests that depurinating adducts play a major role in PAH mutagenesis.

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Figures

Figure 1
Figure 1. H-ras mutations induced in mouse skin 24 h after treatment with various PAH
The nucleotide numbers correspond to GenBank U89950. The cartoons depict the location of the mutations in the H-ras gene, the wild-type bases are shown underneath and the mutations above. In vitro control: pWT, pWT + acid, pWT (4 μg) + anti-BPDE (1 μg in acetonitrile). Experimental: acetone, BP, BPDHD, anti-BPDE, DMBA, DB[a,l]P.
Figure 2
Figure 2. H-ras mutations in mouse skin papillomas induced by DB[a,l]P
The two papillomas (A43 and B32) were obtained by treating the dorsal skin of SENCAR mice with DB[a,l]P (200 nmol) and promoting with chronic twice-weekly treatments with 3 nmol phorbol ester. No. of mutations/Total No. of plasmids: 15/17 (B32) and 25/26 (A43). ▲, deletion. Control: A single treatment with TPA resulted in seven mutations in fortyeight clones (50% A.T to G.C transitions), suggesting that some of the mutations in the tumors may be due to the chronic TPA treatment.
Figure 3
Figure 3. H-ras mutations after treatment of mouse skin DNA with T.G-DNA glycosylase
Incubation of PAH-treated skin DNA with this glycosylase (TDG) results in drastic reduction of A.T to G.C mutations present as G.T heteroduplexes. Mutations/total No. of plasmids: Controls: pWT + anti-BPDE + TDG, acetone-treated skin + TDG. Experimental: DMBA + TDG (2 ins/35), DB[a,l]P + TDG (5/35). ▼, insertion.
Figure 4
Figure 4. Sequence contexts of PAH-induced A.T to G.C mutations
Alignment of the sequences that showed A.T to G.C mutations in mouse skin treated with DMBA or DB[a,l]P. Some of the mutations were scored as A to G and others as T to C, the latter are also presented as A to G (italicized). The results show a frequent presence of doublet sequences flanking the mutated Adenines (underlined) either on the 5′, 3′ or both sides.
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