phosphorus-radioisotopes and fluoranthene

A 32P-postlabeling procedure for identifying and quantifying fluoranthene (FA)-DNA adducts has been developed through modifications of the method of Randerath and collaborators. In this modified procedure, labeled adducts are separated chromatographically by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation counting. FA-modified DNA is digested to nucleotide 3'-monophosphates and nucleotide 3'-monophosphate adducts; unmodified nucleotides are then separated from adducts using a disposable C18 cartridge. Residual unmodified nucleotides, which reduce the efficiency of 32P-postlabeling of FA adducts, are removed by brief digestion with nuclease P1. This treatment selectively dephosphorylates unmodified nucleotide 3'-monophosphates, while FA adducts are minimally affected. FA adducts are then 5'-phosphorylated with polynucleotide kinase and [gamma-32P]ATP. Prolonged treatment with nuclease P1 then is employed to remove the unlabeled 3'-phosphate from adducted diphosphate nucleotides, following which adducts are separated by HPLC and quantified by liquid scintillation counting. Postlabeled microsomally-activated FA-modified DNA contained adducts derived from anti- and syn-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydrofluoranthene. The identity of the major adduct in DNA-bound microsomally-activated FA was confirmed by this HPLC-32P-postlabeling method as an anti-2,3-dihydroxy-1,10b-epoxy-fluoranthene nucleotide adduct. Each step in the procedure was optimized with respect to experimental conditions, and the recovery of adducts was determined by analysis of DNA modified with [3H]FA. In repeated analyses of 2-50 micrograms DNA containing 1.8 adducts per 10(8) nucleotides, 10-15% of total DNA-bound [3H]FA was recovered as the major adduct; recovery was greater from DNA containing higher levels of adducts. The reproducibility of multiple analyses of the same sample was approximately 5%, and multiple analyses at different times were reproducible within experimental error. The limit of detection of the method was approximately 0.1 fmol adduct, representing a binding level of approximately 3 adducts per 10(8) nucleotides in 1 microgram DNA or approximately 1 adduct per 10(10) nucleotides in 500 micrograms DNA. Because the method is not limited with respect to the amount of DNA that can be subjected to analysis, the inherent sensitivity for adduct detection can be greatly enhanced by analysis of larger quantities of DNA.

Topics: Animals; Biotransformation; Chromatography, High Pressure Liquid; DNA; Fluorenes; Liver; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rats; Tritium

The dose-dependence of hemoglobin binding as well as distribution of fluoranthene and fluoranthene-DNA adducts in various tissues was characterized in male rats 24 h after a single i.p. injection of [3H]fluoranthene. Formation and distribution of DNA adducts after chronic administration of fluoranthene in the diet was also studied. Fluoranthene-derived radioactivity was widely distributed throughout the animal after a single dose, and excreta contained the greatest amounts of radioactivity at all dose levels. Fluoranthene binding to globin was proportional to dose over the range of 2 nmol/kg to 177 mumol/kg, and the adducted protein was cleared at the same rate as unmodified hemoglobin, indicating that the adducts are stable in vivo. In contrast, fluoranthene-DNA adducts were not present at detectable levels in liver or kidney 24 h after one dose; low levels of adducts were found only in the lung at the highest dose level. Chronic administration of fluoranthene in the diet, however, resulted in DNA adduct formation in most tissues examined, including liver, kidney, lung, small intestine, heart, spleen and lymphocytes; adducts were not detectable in testes DNA. The major fluoranthene-DNA adduct found in rat tissues was identified by its chromatographic similarity to the major fluoranthene adduct formed in vitro using microsomally-activated fluoranthene and calf thymus DNA, previously identified as a reaction product of anti-2,3-dihydroxy-1,10b-epoxy-1,2,3-trihydro-fluoranthene with N2-deoxyguanosine. The unusual stability of this diol epoxide at physiological pH may allow transport of this ultimate DNA-binding metabolite to virtually all tissues. These results demonstrate the applicability of the HPLC-32P-postlabelling procedure to detect and quantify fluoranthene-DNA adducts formed in vivo, and suggest that analysis of these adducts in accessible tissues such as lymphocytes may be a means of assessing chronic, high level exposure to fluoranthene. Our results also indicate that hemoglobin adducts of fluoranthene could be useful dosimeters for detecting short-term or chronic exposure to this compound if a suitable method for their detection were developed.

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; DNA; Dose-Response Relationship, Drug; Fluorenes; Hemoglobins; Male; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rats; Rats, Inbred Strains; Tissue Distribution; Tritium