Enhanced sensitivity of 32P-postlabeling analysis of aromatic carcinogen:DNA adducts.
نویسنده
چکیده
We have previously described a 32P assay for the detection and quantitation of aromatic carcinogen:DNA adducts (R. C. Gupta et al., Carcinogenesis (Lond.), 3: 1081-1092, 1982). The method entails enzymatic digestion of DNA to deoxynucleoside 3'-monophosphates which are then converted to deoxynucleoside 3',5'-[5'-32P]diphosphates by T4 polynucleotide kinase-catalyzed 32P transfer from adenosine [gamma-32P]triphosphate. Labeled adducts are purified and resolved by four-directional thin-layer chromatography. This procedure can detect one adduct in 10(7)-10(8) nucleotides but quantitation of adduct concentrations of one adduct in greater than 5 X 10(6) nucleotides becomes exceedingly difficult. I have now found that isolation of DNA adducts by extraction with 1-butanol in the presence of the phase-transfer agent tetrabutylammonium chloride prior to the labeling allows one to use excess carrier-free adenosine [gamma-32P]triphosphate (100-200 microCi), thus enabling quantitative analysis of a single adduct in 10(9)-10(10) nucleotides when 1-10 micrograms of the DNA are used. Further increase in the sensitivity of the assay requires higher amount of DNA. The four-directional thin-layer chromatography system has been modified so as to analyze simultaneously as many as 35-40 DNA samples. The new protocol, as applied to a number of carcinogenic aromatic amines and polycyclic aromatic hydrocarbons of diverse structure, is capable of detecting and quantitating adducts at the level of one adduct per 10(10) nucleotides.
منابع مشابه
Detection of multiple polycyclic aromatic hydrocarbon-DNA adducts by a high-performance liquid chromatography-32P-postlabeling method.
A 32P-postlabeling procedure for identifying and quantifying hydrophobic DNA adducts was developed (by modifying the method of Randerath and co-workers) in which labeled adducts are separated by high-performance liquid chromatography (HPLC) and quantified by liquid scintillation counting. This method was first developed for fluoranthene-DNA adducts, and methods for optimal detection and quantif...
متن کاملAnalysis of cigarette-smoke-induced DNA adducts by butanol extraction and nuclease P1-enhanced 32P-postlabeling in human lymphocytes and granulocytes.
In an earlier study, we analyzed the aromatic DNA adducts separated from lymphocytes and granulocytes of smokers and nonsmokers using the nuclease P1-enhanced 32P-postlabeling assay. Here we compare the butanol extraction and nuclease P1-enhanced procedure on the same kind of samples. The DNA adducts of 42 per 10(8) nucleotides from smokers' lymphocytes were statistically higher (p < 0.05) than...
متن کامل32P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas.
Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to norma...
متن کامل32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.
Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A 32P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10(7) to 10(8) nucleotides has been described previously. In the standard procedure, DNA is en...
متن کاملDNA adducts in peripheral blood lymphocytes from aluminum production plant workers determined by 32P-postlabeling and enzyme-linked immunosorbent assay.
32P-Postlabeling analysis and enzyme-linked immunosorbent assay (ELISA) have been used to detect DNA adducts in peripheral blood lymphocytes from primary aluminum production plant workers who were exposed occupationally to a mixture of polycyclic aromatic hydrocarbons (PAHs). Preliminary results reported here are from a comparative study being performed in two aluminum plants. The levels of aro...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- Cancer research
دوره 45 11 Pt 2 شماره
صفحات -
تاریخ انتشار 1985