Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate.
The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O(6)-methyl-2'-deoxyguanosine (O(6)-mdGuo) by LC-MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10(9) nucleotides while O(6)-mdGuo was almost 50-fold lower (32 adducts per 10(9) nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the "doubling dose", i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7microM MMS were deduced for 7-mG and O(6)-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66microM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose-response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose-response relationships and for the setting of tolerable exposure levels are discussed.
Brink
Andreas
A
Department of Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany.
Schulz
Berta
B
Stopper
Helga
H
Lutz
Werner K
WK
D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D018736:DNA Adducts / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism*; D053960:DNA Breaks / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D007940:Leukemia L5178; D008741:Methyl Methanesulfonate / Q000008:administration & dosage / Q000633:toxicity*; D051379:Mice; D008954:Models, Biological; D009153:Mutagens / Q000008:administration & dosage / Q000633:toxicity*; D053719:Tandem Mass Spectrometry
D018736:DNA Adducts; D009153:Mutagens; D006147:Guanine; C008450:7-methylguanine; C015558:O(6)-methyl-2'-deoxyguanosine; D008741:Methyl Methanesulfonate; D003849:Deoxyguanosine