Class-specific immunoadsorption purification for polycyclic aromatic hydrocarbon-DNA adducts.
An immunoenrichment procedure has been developed for applications in the detection and identification of a broad range of polycyclic aromatic hydrocarbon (PAH)-DNA adducts at very low abundance. The procedures are based on a monoclonal antibody raised to r-7,trans-8,trans-9-trihydroxy-cis-10-(N2-deoxyguanosyl-5'-phospha te)- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE-N2-dG) which has been tested for cross-reactivity towards DNA and proteins (bovine serum albumin, chicken gamma globulin and human globin) covalently modified with a range of PAH diol-epoxides. The antibody recognised DNA adducted with the diol-epoxides of benzo[a]pyrene, benz[a]anthracene, chrysene, dibenz[a,h]anthracene or picene. The antibody also cross-reacted with the 7,8,9,10-tetraol derived from benzo[a]pyrene and the 1,2,3,4-tetraols of benz[a]anthracene and chrysene. The degree of cross-reactivity was greatest for PAH adducts with structural features similar to anti-BPDE-N2-dG proximate to the base attachment. The antibody also recognised a range of PAHs adducted to human globin; these included adducts of benzo[a]pyrene, benz[a]anthracene and chrysene diol-epoxides. This wide range of recognition provides good evidence for the class-specific recognition of PAH adducts by the antibody. When immobilized on Sepharose 4B and used in the immunoadsorption purification of adducted nucleotides, the antibody selectively enriched adducts of benzo[a]pyrene, benz[a]anthracene and chrysene from normal nucleotides. Quantitative measurements with [14C]benzo[a]pyrene-DNA adducts showed that the immobilized antibody was able to enrich benzo[a]pyrene adducts from a DNA hydrolysate containing adducts at a level of 1 adduct/10(10) normal nucleotides. In addition, this immunoadsorption technique was effective in enriching a mixture of DNA adducts formed in the skin of CF1 mice treated cutaneously with a mixture of [3H]benzo[a]pyrene and [3H]chrysene. Class-specific immunoenrichment procedures for DNA adducts are important in assisting the identification of genotoxic components in complex mixtures. The performance characteristics of this immobilized antibody suggest that it may be suitable for application in the detection, identification and monitoring of human exposures to low levels of PAHs.
Shell Research Ltd, Sittingbourne Research Centre, Kent, UK.
E D
ED
Booth
J P
JP
Aston
P T
PT
van den Berg
R A
RA
Baan
D A
DA
Riddick
L T
LT
Wade
A S
AS
Wright
W P
WP
Watson
D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods; D003429:Cross Reactions; D018736:DNA Adducts / Q000302:isolation & purification*; D005260:Female; D007163:Immunosorbent Techniques; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D011083:Polycyclic Compounds / Q000302:isolation & purification*; D012015:Reference Standards; D012474:Salmon; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D018736:DNA Adducts; D011083:Polycyclic Compounds