Determination of stereospecificity of benzo[a]pyrene diolepoxide-DNA antisera with site-specifically modified oligonucleotides.
Antisera developed against benzo[a]pyrene diolepoxide (BPDE)-DNA adducts are sensitive tools for detection of DNA adducts in human samples. All antisera currently used for biomonitoring studies were produced against DNA or guanosine modified with racemic anti-BPDE. Using a non-competitive enzyme-linked immunosorbent assay (ELISA), Venkatachalam and Wani (Carcinogenesis, 15, 565-572, 1994) recently tested polyclonal and monoclonal (5D2) antisera for cross-reactivity against oligonucleotides containing (+)-and (-)-trans-anti-BPDE-N2-guanine or N6-adenine adducts and showed different stereospecificity for the two antisera. Because of the importance of antiserum specificity in human biomonitoring studies, we have tested several monoclonal (Mab 5D11 and 5D2) and polyclonal (Pab #29) antisera developed against racemic anti-BPDE-DNA adducts, and Mab 8E11 developed against anti-BPDE-guanosine adducts. Stereoisomeric anti-BPDE-modified oligonucleotide adducts in the sequence 5'-d(CC-AT-CG*CTACC)-3' where G* = anti-BPDE-N2-dG with (+)-trans, (-)-trans, (+)-cis and (-)-cis adduct stereochemistry at the C10 position of anti-BPDE were tested by competitive ELISA. Two structurally related 5-methylchrysene diolepoxide adducts with G* = (+)- and (-)-trans-anti-5-MeCDE-N2-dG in the same oligonucleotide were also tested. While Mab5D2 had the highest affinity for the (-)-trans-anti-BPDE-modified oligomer, Mab 5D11 and 8E11 and Pab #29 recognized the (+)-trans-anti-BPDE-modified oligomer better than the (-)-trans-anti-BPDE modified oligomer. Mab 5D11 and Pab #29 recognized racemic anti-BPDE-modified DNA adducts better than trans-anti-BPDE-modified oligonucleotides; however, Mab 8E11 showed similar sensitivity to racemic anti-BPDE-DNA adducts and (+)- and (-)-trans-anti-BPDE-modified oligomers. All antisera exhibited lower reactivities with both 5-MeCDE modified oligomers. Because of their sensitive detection of (+)-trans-anti-BPDE-dG adducts, the primary adduct produced in vivo, Mab 8E11 and 5D11 and Pab #29 are appropriate for measurement of most adducts formed in humans.
Hsu
T M
TM
Division of Environmental Sciences/Cancer Center, Columbia University, New York, NY 10032, USA.
Liu
T M
TM
Amin
S
S
Geacintov
N E
NE
Santella
R M
RM
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis*; D001483:Base Sequence; D001665:Binding Sites; D018736:DNA Adducts / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000032:analysis / Q000276:immunology*; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D018736:DNA Adducts; D007106:Immune Sera; D009841:Oligonucleotides; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide