Immunohistochemical detection of anti-(+/-)-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)p yrene-bound adduct in nuclei of cultured HeLa cells and mouse lung tissue.
Rabbit IgG against anti-BP-DE-modified calf thymus DNA was characterized and utilized for detection of adducts formed in cultured HeLa cells treated with anti-BP-DE or in lung tissues from mice given BP. Four weekly treatments with 1 mg of the modified (1.2% of total nucleotides) DNA were used to raise the rabbit antiserum, which even at 10(6)-fold dilution clearly recognized adducts in an ELISA. The detection limit was 25 fmol of anti-BP-DE adduct with 10(3)-fold diluted IgG fraction. The IgG did not cross-react with BP, BP tetraol, guanine, or 7-methyl-guanine, and only slightly with the syn form of BP-DE, (+/-)-4,5-dihydrobenzo(a)-pyrene-4,5-epoxide, aflatoxin B1, and N-methyl-N-nitrosourea-modified DNA. Although syn-BP-DE-modified DNA inhibited the reaction between IgG and anti-BP-DE adduct, the inhibition rate was low, not correlating with the numbers of modified bases. Essentially similar values for level of bound adducts in HeLa cells treated with anti-BP-DE were generated by both ELISA and associated radioactivity derived from anti-3H-BP-DE. Immunohistochemical detection of adduct formation was dependent on the amounts of anti-BP-DE added to the culture medium of HeLa cells. Similar positive binding was obtained in mouse lung alveolar cell nuclei after intragastric administration of BP. These observations indicate that the prepared IgG is highly specific for anti-BP-DE-modified DNA and that should prove useful for detection of adducts formed in tissue samples exposed to anti-BP-DE or BP.
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis*; D000818:Animals; D002467:Cell Nucleus / Q000032:analysis*; D002478:Cells, Cultured; D004101:Dihydroxydihydrobenzopyrenes / Q000032:analysis*; D006367:HeLa Cells / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D008168:Lung / Q000648:ultrastructure*; D051379:Mice
D004101:Dihydroxydihydrobenzopyrenes; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide