Detection of Aflatoxin B, -DNA Adducts in Human Placenta and Cord Blood1

Human placenta and cord blood are readily available specimens that respond to maternal environmental insult and are being used to investi gate metabolism, bioactivation, and transplacental transfer of procarcinogens. Enzyme-linked immunosorbent assay was used to quantitate 120 placentas and 56 cord bloods from term, uncomplicated pregnancies at Taipei Chang Gung Memorial Hospital, Taiwan, for the presence of the imidazole ring-opened form of aflatoxin B,-DNA (AFB,-DNA) adducts. Of the 120 samples of placentas, 69 (57.5%) contained AFB,-DNA adducts in levels from 0.6 to 6.3 umol/mol DNA. Of the 56 samples of cord bloods, 5 (8.9%) contained AFB,-DNA adducts in levels from 1.4 to 2.7 umol/mol DNA. A higher positive rate was found in samples collected in the summer than in the winter. These results indicate that a significant number of individuals in an area of high liver cancer risk have been exposed to A KH,, and it is possible to transfer A KB, and its metabolites to the progeny through the transplacental unit Thus, monitoring adduct levels in human specimens may provide information not only on carcinogen exposure but also on the relationship among infection with hepatitis B/C virus, dietary exposure to AHI,, and liver cancer. INTRODUCTION There is a high mortality (18.5 cases/100,000/year) of PHC3 in the Chinese population in Taiwan. PHC mortality is significantly higher among the mountainous aborigines and residents living in the Penghu islets, while hepatitis B surface antigen carrier rates are only slightly higher in these areas than for the general population ( 1, 2). Epidemiological studies in Taiwan have found a significant association be tween the development of PHC and hepatitis B virus infection, familial history of liver diseases, alcohol consumption, arsenic con centration in drinking water, and fermented bean product consumption as a source of AFB, (3). These risk factors were more significantly associated with PHC in the younger age groups than in the elderly; this suggests that they may shorten the induction period and accelerate the onset of PHC in the young. Moreover, PHC mortality for children (under age 14) is 0.4/100,000 in Taiwan, which is among the highest in the world. These studies suggest that exposure to environmental risk factors such as aflatoxin at an early age may play an important role in the development of liver cancer. Epidemiological studies have demonstrated a strong association between exposure to AFB, in conjunction with hepatitis virus infec tion and an increased incidence of human hepatocellular carcinoma (4). To better understand the role of AFB, exposure with respect to human PHC incidence, immunoassays for the biological quantitation of free AFB,, its metabolites, and its adduct macromolecules have been developed (5-8). There is considerable evidence that the initi ating event in chemical carcinogenesis is the covalent binding of the carcinogen to cellular DNA (9), so that monitoring carcinogen-DNA adducts may be relevant to the development of cancer. The major Received 10/19/92; accepted 1/6/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by NSC Grant NSC79-0412-B182-40 and Chang Gung Medical Research Grant CMRP296. 2 To whom requests for reprints should be addressed, at Department of Public Health, Chang Gung Medical College, 259 Wen-Hwa 1 Road, Kwei-San, Tao-Yuan, Taiwan, Republic of China. 3 The abbreviations used are: PHC, primary hepatocellular carcinoma; AFB,, aflatoxin B,; ELISA, enzyme-linked immunosorbent assay. DNA adduct of AFB, results from binding of its 8,9-oxide to the N-7 of guanine. This adduct, which is unstable, is either lost from DNA, creating an apurinic site, or converted to the imidazole ring-opened AFB,-FAPy adduct (10, 11). It is possible that AFB,-FAPy, the stable form of AFB!-guanine, may play an important role in the development of PHC. We have previously developed monoclonal antibodies rec ognizing this stable form of the adduct and used these antibodies in a competitive ELISA to quantitate DNA adducts in liver tissues of animals treated with AFB, and in human liver tissue specimens from PHC patients in Taiwan (12). A quantitative indirect immunofluorescence method using monoclonal antibody 6A10 has also been used to measure AFB,-DNA adducts in liver tissues (13). Exposure to genotoxic chemicals is widespread in human popula tions, and it is believed that the quantitation of carcinogen-associated DNA adduct is a valuable parameter for molecular epidemiological studies. The placenta is an important source of material for such studies because it is readily available and responsive to maternal exposures to environmental pollutants. Previous studies have shown that human and animal placentas contain enzymes which can bioactivate genotoxic carcinogens to form covalent carcinogen-DNA ad ducts (14-16). AFB, and its metabolites have been found in human cord sera, which suggests that transplacental transfer of AFB, may play a biological role in the initiation of PHC in progeny (17). This study was designed to quantitate the levels of the imidazole ring-opened form of AFB,-DNA adducts, as a marker of AFB, expo sure in human placenta and cord blood from term, uncomplicated pregnancies. MATERIALS AND METHODS Chemicals. [3H1AFB, (25 Ci/mmol) was obtained from Moravek Biochemicals (City of Industry, CA). Dichloromethane and m-chloroperoxybenzoic acid were purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). AFB i, calf thymus DNA, goat anti-mouse IgG-alkaline phosphatase conjugates, and p-nitrophenyl phosphate (Sigma 104) were purchased from Sigma Chemical Co. (St. Louis, MO). RNase A and proteinase K were pur chased from Boehringer Mannheim GmbH (Mannheim, Germany). The imi dazole ring-opened form of AFB,-DNA was prepared as described previously (12). Human Samples. Sixty placenta samples were collected from term, un complicated pregnancies at Taipei Chang Gung Memorial Hospital during August 1990 and in January 1991, respectively. Because many women do not deliver naturally, only 27 and 29 cord blood samples could be collected in parallel in August 1990 and January 1991, respectively. Placenta! cotyledons containing the chorionic villus (placenta! lobules) were dissected into small pieces to avoid nonparenchymal tissue contamination. These were thoroughly washed with 0.15 MNaCl and 0.015 Msodium citrate containing 1 mM ZnCl2 at 4°C,frozen in liquid nitrogen, and stored at -70°C. Thirty-rive ml of cord blood were collected per subject into a 50-ml conical tube containing 0.5 ml of 1000 units heparin sodium salt in 0.9% saline; serum, buffy coat, and RBC were separated by centrifugation and stored at -70°C. Preparation of DNA Samples. Placenta! DNA nuclei were separated into the following two fractions using the technique described by Resendez-Perez et al. (18): nuclear fraction IV containing knotted nuclei from syncytiotrophoblasts and nuclear fraction III containing free nuclei from syncytiotrophoblasts plus nuclei from other placenta! cell types as well as from contaminating maternal and fetal leukocytes. The nuclear fractions were treated with protein ase K (200 Mg/ml, 2 h, 37°C)in 10 mm Tris buffer (pH 8.0) containing 1 mM EDTA and 0.4 MNaCl and subjected to phenol extraction. Nucleic acids were recovered by ethanol (95%) precipitation, dissolved in Tris buffer, and treated