phosphorus-radioisotopes and 1-nitropyrene

This study is part of an ongoing investigation of biomarkers in iron foundry workers exposed to polycyclic aromatic compounds. Foundry workers with the highest exposures had elevated levels of DNA adducts in their white blood cells in previous studies. The purpose of this study was to characterize the nature of DNA reactive chemicals in foundry air samples through incubating the foundry filter extract with DNA and activation enzymes. Calf thymus DNA was incubated with foundry filter extract and activated by either rat liver activation mixture (S9 mix) or xanthine oxidase. A complex pattern of adducts was observed on thin-layer chromatography (TLC) by the 32P-postlabeling assay. Two selected polycyclic aromatic hydrocarbons (PAHs)--1-NP-and anti(+/-)benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide [anti(+/-) BPDE]-DNA adducts--were used as marker compounds in characterizing the postlabeled DNA adducts by TLC combined with high-performance liquid chromatography (HPLC). After an initial separation of DNA adducts by TLC, individual spots were isolated and separated further on HPLC. HPLC analysis and spiking with anti(+/-)BPDE-DNA standard confirmed the co-migration of the anti(+/-)BPDE-DNA standard with one PAH adduct formed by the S9 mix-activated DCM extract in calf thymus DNA.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Air Pollution, Indoor; Animals; Benzo(a)pyrene; Biomarkers; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA Adducts; Humans; In Vitro Techniques; Iron; Leukocytes; Metallurgy; Occupational Exposure; Phosphorus Radioisotopes; Polycyclic Aromatic Hydrocarbons; Pyrenes; Thymus Gland

On the basis of 32P-postlabeling analysis, treatment of rats with 1-nitropyrene (1-NP) resulted in the formation of multiple DNA adducts in the liver, mammary glands, and peripheral lymphocytes. The one adduct resulting from nitroreduction, N-(deoxyguanosin-8-yl)-1-aminopyrene, constitutes only a minor component among the adducts. In the present study, incubation of calf thymus DNA with mutagenic ring-oxidized metabolites of 1-NP in vitro in the presence and absence of xanthine oxidase also resulted in the formation of multiple adducts. On the basis of their chromatographic behavior, it appears that DNA adducts derived from such metabolites may have been formed in vivo; however, this needs to be confirmed. [3H]1-NP was given to male and female F344 rats and Sprague-Dawley rats by gavage at five dose levels in the range of 0.1 to 1000 micrograms/kg bw. This led to stable hemoglobin adducts accounting for 0.08 +/- 0.05% of the dose (n = 3 rats). The radioactivity associated with hemoglobin following administration of [3H]1-NP was cleared with a half-life of about 14 days, which is faster than that of unmodified erythrocytes in the rat (t1/2 = 30 days). Treatment of the hemoglobin with 1% HCl in acetone, to precipitate the globin, released the radioactivity; it was all bound to the heme moiety. The structures of the heme adducts have not been elucidated; yet, because of their stability, they may be useful as dosimeters for human exposure to 1-NP.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Animals; Dose-Response Relationship, Drug; Female; Hemoglobins; Male; Monitoring, Physiologic; Mutagens; Phosphorus Radioisotopes; Pyrenes; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; Tissue Distribution

Nitropolynuclear aromatic hydrocarbons (nitro-PAHs) are widely distributed in the environment. For several chemicals in this class of compounds, mutagenic activity in bacterial and mammalian systems and tumorigenic activity in laboratory animals have been clearly documented. Procedures for assessing the risk to humans from exposure to nitro-PAHs have not been clearly defined, despite the wide-spread occurrence of such agents in the environment and their possible involvement in the etiology of some human cancers. Several methods are available for determining exposure, uptake, and metabolic activation of genotoxic carcinogens in humans. DNA adducts currently are regarded as the most direct markers of genotoxicity. However, several proteins are equally capable of forming adducts with electrophiles derived from xenobiotics. We focused on developing methods to detect and quantify adducts of 1-nitropyrene and 1,6-dinitropyrene with proteins and with DNA. 1-Nitropyrene is the most abundant nitro-PAH in emissions from combustion sources such as diesel engines. Although 1,6-dinitropyrene is far more mutagenic and more tumorigenic than 1-nitropyrene, it is present in the environment at lower levels. Seeking a highly sensitive method, we have utilized the 32P-postlabeling technique to establish the pattern of the DNA adducts formed in rat tissues, as well as in peripheral blood lymphocytes, following administration of both 1-nitropyrene and 1,6-dinitropyrene. We also present results on hemoglobin and albumin adducts formed after administration of these nitro-PAHs. [3H]1-Nitropyrene was given to male or female Fischer-344 or Sprague-Dawley rats by gavage at five dose levels ranging from 0.1 to 1,000 micrograms/kg of body weight. This led to stable hemoglobin adducts, which accounted for 0.08% +/- 0.05% of the dose. The radioactivity associated with hemoglobin following administration of [3H]1-nitropyrene was cleared with a half-life of 13.6 days. This is faster than the clearance of unmodified erythrocytes in the rat (half-life of 30 days). Treating the hemoglobin with 1% hydrochloric acid in acetone, to precipitate the globin, released the radioactivity so that none remained bound to the globin. Rather, the radioactivity remained bound to the heme moiety. To obtain structural information about the heme adducts, we incubated [3H]1-nitrosopyrene and [3H]4,5-epoxy-4,5-dihydro-1-nitropyrene with rat hemoglobin. In each case, [3H] was bound mainly to globin and, to a les

Topics: Acetylation; Albumins; Animals; Binding Sites; Chromatography, Gas; DNA; Environmental Monitoring; Female; Globins; Heme; Hemoglobins; Humans; Male; Mass Spectrometry; Mutagens; Phosphorus Radioisotopes; Pilot Projects; Pyrenes; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity

Recent reports have indicated that 1-nitropyrene is tumorigenic in laboratory animals. Since it is generally accepted that the covalent binding of carcinogens to DNA is causally related to tumorigenesis, we used 32P-postlabeling to examine the DNA adducts present in target tissues. 1-Nitropyrene (99.85-99.98% 1-nitropyrene, 0.15-0.02% 1,3-, 1,6- and 1,8-dinitropyrene by mass spectral analyses) was administered to Sprague-Dawley rats, CD-1 mice and A/J mice according to three tumorigenesis protocols. In DNA obtained from the injection site of Sprague-Dawley rats, two major adducts were observed. Based upon their chromatographic behavior and sensitivities to treatment with nuclease P1 and hydrazine, these adducts were identified as N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP) and N-(deoxyguanosin-8-yl)-1-amino-3-, 6- and/or 8-nitropyrene (dG-C8-ANP), which are adducts derived from the nitroreduction of 1-nitropyrene and dinitropyrenes respectively. In mammary gland DNA from Sprague-Dawley rats, two adducts were found. One of these had chromatographic characteristics and hydrazine and nuclease P1 sensitivities similar to dG-C8-AP, while the identity of the other adduct is presently unknown. The only DNA adduct detected in the livers of newborn CD-1 mice and the lungs of A/J mice was dG-C8-ANP. The presence of dG-C8-AP in the injection site and mammary gland of the Sprague-Dawley rats indicates that nitroreduction is involved in the metabolic activation of 1-nitropyrene in these tissues. Since an unidentified adduct was also found in the mammary gland, other pathways are important in this tissue. The presence of only dinitropyrene DNA adducts in the livers of CD-1 mice and lungs of A/J mice indicates that dinitropyrenes are activated very efficiently to electrophilic metabolites, to an extent far better than 1-nitropyrene.

Topics: Animals; Autoradiography; Biotransformation; DNA; Female; Male; Mass Spectrometry; Mice; Mice, Inbred A; Mice, Inbred Strains; Phosphorus Radioisotopes; Pyrenes; Rats; Rats, Inbred Strains; Tritium