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Analysis of 7-methylbenz[a]anthracene-DNA adducts formed in SENCAR mouse epidermis by 32P-postlabeling
W Baer-Dubowska1, S V Vulimiri, R G Harvey
1The University of Texas M.D. Anderson Cancer Center, Science Park-Research Division, Smithville 78957, USA.
Abstract:
The present study has analysed the DNA adducts formed in SENCAR mouse epidermis following topical application of 7-methylbenz[a]anthracene (7-MBA). Mice were treated with 400 nmol of 7-MBA, which represents an initiating dose of this hydrocarbon for SENCAR mice. DNA adducts were analysed 24 h after topical application of the hydrocarbon by 32P-postlabeling coupled with either HPLC analysis or an improved TLC procedure giving better resolution of DNA adducts through the use of a D6 solvent [isopropanol:4N NH4OH (1:1)] following D5. Twenty-four hours after topical application of 400 nmol 7-MBA, the level of total covalent binding was 0.37 +/- 0.07 pmol/mg DNA as determined by 32P-postlabeling. This level of binding correlated well with the relative tumor initiating activity of this hydrocarbon compared to 7,12-dimethylbenz[a]anthracene (6.4 +/- 0.01 pmol/mg DNA) and dibenz[a,j]anthracene (0.03 +/- 0.01 pmol/mg DNA). Analysis of the 32P-labeled 3',5'-diphosphodeoxyribonucleosides by HPLC and TLC revealed the presence of deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both the anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs). The major DNA adduct derived from 7-MBA in mouse epidermis was tentatively identified as (+) anti-7-MBADE-trans-N2-dGuo. In addition, a minor dGuo adduct derived from the bay-region syn-diol-epoxide of 7-MBA was detected as well as a minor dAdo adduct from this diol-epoxide. Another minor dAdo adduct was also detectably present which arose from either the anti- or syn-diol epoxide. Furthermore, several unidentified DNA adducts were present in both HPLC and TLC chromatograms of DNA samples from 7-MBA-treated mice. These results are discussed in terms of the role of specific 7-MBA-DNA adducts in tumor initiation by this hydrocarbon.
Insights
This study identified specific DNA adducts in mouse skin after exposure to 7-methylbenz[a]anthracene (7-MBA). The major adduct, (+) anti-7-MBADE-trans-N2-dGuo, is linked to the tumor-initiating activity of this chemical carcinogen.
Area of Science:
- Toxicology
- Carcinogenesis
- Molecular Biology
Background:
- 7-methylbenz[a]anthracene (7-MBA) is a polycyclic aromatic hydrocarbon known for its tumor-initiating properties.
- Understanding the specific DNA adducts formed by carcinogens is crucial for elucidating mechanisms of carcinogenesis.
Purpose of the Study:
- To analyze the types and levels of DNA adducts formed in SENCAR mouse epidermis after topical application of 7-MBA.
- To correlate the identified DNA adducts with the known tumor-initiating activity of 7-MBA.
Main Methods:
- SENCAR mice were topically treated with a single initiating dose of 7-MBA (400 nmol).
- DNA adducts were analyzed 24 hours post-treatment using 32P-postlabeling coupled with High-Performance Liquid Chromatography (HPLC) and Thin-Layer Chromatography (TLC).
- Improved TLC methods with a specific solvent system (D6) were employed for better adduct resolution.
Main Results:
- The total covalent binding of 7-MBA to DNA was 0.37 +/- 0.07 pmol/mg DNA, correlating with its tumor-initiating activity.
- Major DNA adducts were identified as deoxyguanosine (dGuo) and deoxyadenosine (dAdo) adducts formed from both anti- and syn-bay-region diol-epoxides of 7-MBA (anti- and syn-7-MBADEs).
- The primary adduct was tentatively identified as (+) anti-7-MBADE-trans-N2-dGuo, with minor adducts from syn-diol-epoxide and other unidentified adducts also detected.
Conclusions:
- The study successfully identified and characterized key DNA adducts formed by 7-MBA in mouse epidermis.
- The findings support the role of specific 7-MBA-DNA adducts, particularly (+) anti-7-MBADE-trans-N2-dGuo, in the initiation of tumors by this hydrocarbon.