Use of the 32P-postlabelling assay to study transplacental carcinogens and transplacental carcinogenesis.
The formation of DNA adducts represents a key step in the postnatal initiation of the carcinogenic process. Little is known as yet about the role of prenatally induced adducts in transplacental carcinogenesis in offspring. Measurement of transplacental DNA damage in fetal organs of experimental animals has been difficult in the past because of the small amounts of DNA available and low adduct levels. In principle, these difficulties have been overcome by the recent development of a highly sensitive 32P-postlabelling assay which can be applied to a large number of DNA adducts of diverse structure and requires only microgram amounts of DNA for analysis. In this assay, tissue DNA is degraded to mononucleotides; these are enzymatically 32P-labelled via T4 polynucleotide kinase-catalysed [32P]phosphate transfer from [gamma--32P]ATP, to form 5'--32P-labelled 3',5'-bisphosphate derivatives; the labelled products are separated into normal and adducted [32P]nucleotides and quantified by thin-layer chromatography, autoradiography and scintillation (Cerenkov) counting. This technique allows the detection and quantitation of one adduct in 10(8)-10(10) DNA nucleotides (approximately 1-100 adducts/mammalian genome) using a 10-micrograms DNA sample and has been applied in studies of adduct formation from transplacental carcinogens in fetal and adult rodent tissues. In this paper, we review application of 32P-postlabelling to DNA adducts formed with transplacental or suspected transplacental carcinogens in fetal and maternal tissues. The carcinogens studied include diethylstilboestrol (DES), benzo[a]pyrene, safrole, 4-aminobiphenyl and 4-nitroquinoline-1-oxide, as well as cigarette smoke condensate. In DNA of DES-exposed hamsters, one major and several minor adduct spots were observed, which were absent from vehicle controls. A characteristic adduct, which resembled the major hamster adduct chromatographically, was detected in all exposed mouse tissue, except fetal kidney. Chronic administration of low doses of DES to male Syrian hamsters led to an entirely different pattern of adducts in kidney DNA, the target organ of carcinogenesis. These adducts did not contain covalently bound oestrogen moieties and appeared to be formed indirectly: oestrogen appeared to induce or enhance the synthesis of an endogenous electrophilic metabolite reacting with DNA. Thus, multiple mechanisms exist by which DES can damage DNA. Additional work using 32P-postlabelling has shown that non-hormonal genotoxicants (e.g., benzo[a]pyrene, safrole, 4-aminobiphenyl, 4-nitroquinoline-1-oxide) and cigarette smoke condensate given to pregnant mice can induce specific DNA adduct profiles in fetal tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Randerath
K
K
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030.
Liehr
J G
JG
Gladek
A
A
Randerath
E
E
D015112:4-Nitroquinoline-1-oxide / Q000378:metabolism; D000611:Aminobiphenyl Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001564:Benzo(a)pyrene / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D004247:DNA / Q000378:metabolism*; D004054:Diethylstilbestrol / Q000378:metabolism; D005260:Female; D008431:Maternal-Fetal Exchange; D010761:Phosphorus Radioisotopes; D010947:Plants, Toxic; D011247:Pregnancy; D012451:Safrole / Q000378:metabolism; D012906:Smoke; D014026:Nicotiana
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D010761:Phosphorus Radioisotopes; D012906:Smoke; C006757:4-biphenylamine; D001564:Benzo(a)pyrene; D015112:4-Nitroquinoline-1-oxide; D004054:Diethylstilbestrol; D004247:DNA; D012451:Safrole