Immunoaffinity concentration of human lung DNA adducts using an anti-benzo[a]pyrene-diol-epoxide-DNA antibody. Analysis by 32P-postlabelling or ELISA.
DNA, isolated from 15 human lung autopsy samples, was examined for the presence of polycyclic aromatic hydrocarbon (PAH) DNA adducts. Using the nuclease P1 modification of the 32P-postlabelling technique, between 1 and 12 adducts/10(8) nucleotides were detected prior to immunoconcentration. Autoradiograms from most of the samples revealed a diagonal smear of radioactivity consistent with complex mixture (cigarette smoking) DNA damage. The DNA samples were digested to oligonucleotides, made single-stranded and subsequently applied to immunoaffinity columns containing immobilised anti-benzo[a]pyrene (B(a)P)-7,8-diol-9,10-epoxide (BPDE) DNA polyclonal rabbit antibody. The material remaining bound to the column, in addition to that passing through, was analysed using both ELISA and 32P-postlabelling techniques. Column-bound adducts comprised between 0% and 78% of any particular sample. Immunoconcentration, followed by 32P-postlabelling of the material which had been bound to the column, revealed the presence of a number of discrete adduct spots in autoradiograms of the more heavily adducted samples. Sample DNA not retained by the columns was also analysed; the chromatographic pattern obtained was a dense zone of radioactive material migrating from the origin. This evidence suggests that the composition of PAH-DNA adducts found in human lung samples exhibits wide inter-individual variation.
King
M M
MM
Jack Birch Unit for Environmental Carcinogenesis, Department of Biology, University of York, Heslington, UK.
Cuzick
J
J
Jenkins
D
D
Routledge
M N
MN
Garner
R C
RC
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D000906:Antibodies; D002846:Chromatography, Affinity; D004247:DNA; D018736:DNA Adducts; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008168:Lung / Q000737:chemistry* / Q000187:drug effects; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds / Q000032:analysis*
D000906:Antibodies; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA