phosphorus-radioisotopes and 2-amino-3-methylimidazo(4-5-f)quinoline

Capillary liquid chromatography/microelectrospray mass spectrometry has been applied to the detection of deoxyribonucleoside adducts of the food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) from in vitro and in vivo sources. Constant neutral loss (CNL) and selective reaction monitoring (SRM) techniques with a triple-quadrupole mass spectrometer enabled sensitive and specific detection of IQ adducts in vitro and in animals. Detection of 1 adduct in 10(4) unmodified bases is achieved using CNL scanning detection, while the lower detection limits using SRM approach 1 adduct in 10(7) unmodified bases using 300 microg of DNA. The DNA adducts N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4, 5-f]quinoline (dG-C8-IQ) and 5-(deoxyguanosin-N(2)-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N(2)-IQ) were detected in kidney tissues of chronically treated cynomolgus monkeys at levels and in proportions consistent with previously published (32)P-postlabeling data [Turesky, R. J., et al. (1996) Chem. Res. Toxicol. 9, 403-408]. Thus, capillary tandem LC/MS is a highly sensitive technique, which can be used to screen for DNA adducts in vivo.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Liquid; DNA; DNA Adducts; Isotope Labeling; Kidney; Macaca fascicularis; Mass Spectrometry; Mutagens; Phosphorus Radioisotopes; Quinolines

DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) have been measured in the liver, kidney, and colo-rectum of male Fischer-344 rats given a single oral dose of IQ (20 mg/kg). The pattern and distribution of DNA adducts examined by 32P-postlabeling was similar in all tissues. N-(Deoxyguanosin-8-yl)-2-amino-3-methylimidazo-[4,5-f]quinoline (dG-C8-IQ) was the principal adduct identified and it accounted for approximately 50-70% of the observed radioactivity, followed by (deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-N2-IQ) which accounted for 15-20% of the radioactivity. Twenty-four hours after IQ treatment, DNA modification was greatest in the liver at a level of 7.64 +/- 1.08 adducts per 10(7) bases, followed by kidney at 2.04 +/- 0.32 adducts per 10(7) bases, and colorectum at 1.08+/-0.22 adducts per 10(7) bases. Liver and colo-rectum are target tissues of tumorigenesis in the rat during chronic feeding studies with IQ; however, tumors are not formed in the kidney. Therefore, factors in addition to IQ-guanine adduct formation, such as adduct persistence, error-prone repair, and tumor promotion must contribute to organ susceptibility of IQ-induced carcinogenesis.

Topics: Animals; Carcinogens; Colon; DNA Adducts; Intestinal Mucosa; Intestines; Kidney; Liver; Male; Phosphorus Radioisotopes; Quinolines; Rats; Rats, Inbred F344; Rectum

The DNA adducts of the food mutagens/carcinogens IQ and PhIP were studied by means of 32P-postlabelling techniques. Adducts were generated in vitro and in vivo by three techniques: by photolysis of azido-IQ and azido-PhIP in the presence of dGp, calf-thymus DNA or Salmonella; by administration of IQ and PhIP to rats; and by incubation of cultured COS-1 cells with IQ. These cells expressed the human cytochromes P450 1A1 or P450 1A2 and/or the human N-acetyltransferases NAT1 or NAT2. The data demonstrate that, in the photolytic, rat and human systems, a common IQ metabolite and a common PhIP metabolite are formed together with common sets of IQ adducts and PhIP adducts. The data obtained in the human system show that N-hydroxy-IQ, formed by cytochrome P450, binds poorly to DNA, whereas more efficient binding occurs in the presence of NAT1 and most efficient binding in presence of NAT2. This indicates an O-acetyltransferase activity of human NAT1 and NAT2 and formation of N-acetoxy-IQ as an intermediate and immediate precursor of the ultimate arylnitrenium ion. The effect of the polymorphic NAT2 suggests a critical role for the human acetylation polymorphism in the DNA-binding of IQ in humans and in its genotoxic implications.

Topics: Animals; Arylamine N-Acetyltransferase; Biotransformation; Carcinogens; Cell Line; DNA; DNA Damage; Humans; Imidazoles; In Vitro Techniques; Male; Mutagens; Phosphorus Radioisotopes; Quinolines; Rats; Rats, Inbred F344; Ultraviolet Rays

The potent heterocyclic food mutagen IQ is carcinogenic in the CDF1 mouse, affecting the liver, lung and forestomach. Using 32P-postlabelling methods we have isolated up to five IQ-DNA adducts from both target and non-target organs after oral administration of IQ. Up to four additional non-specific adducts, found when the 32P-postlabelling assay was run under intensification conditions, could be attributed to the use of a certain brand of microcentrifuge tube in the assay. CLA is a mixture of heat-generated derivatives of linoleic acid, each with a conjugated double bond system, that has chemopreventive properties in rodents. To examine the effect of CLA on the formation of IQ-DNA adducts in CDF1 mice, we administered CLA by gavage every other day for 45 days, followed by a single oral dose (50 mg/kg) of IQ. Tissues collected 24 h later were analysed for IQ-DNA adducts by 32P-postlabelling. Compared to controls, CLA treatment caused inhibition of adduct formation in the liver, lung, large intestine and kidney. In the kidneys of females, CLA treatment inhibited IQ-DNA adduct formation almost completely (95.2%). Male F344 rats were fed a control diet or an isocaloric diet containing 20% menhaden oil (MO), a rich source of omega-3 fatty acids, for six weeks, then given a single oral dose of IQ. Analysis for IQ-DNA adducts by 32P-postlabelling one or six days later revealed that on day 1 the MO diet caused a 6-7 fold decrease in adduct formation in the liver and an up to 2-fold decrease in both the small and large intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Dietary Fats, Unsaturated; DNA; DNA Damage; DNA Repair; Fatty Acids; Female; Male; Mice; Mutagens; Phosphorus Radioisotopes; Quinolines; Rats; Rats, Inbred F344; Tissue Distribution

Relatively few reported attempts have been made to substitute HPLC for the thin-layer ion-exchange chromatography (TLC) conventionally used in the 32P-postlabelling assay. Using a reverse-phase phenyl-modified silica gel column and a gradient of methanol in 0.5 M sodium phosphate buffer (pH 2.0), we were able to improve the resolution of very similar adducts. Combined with on-line detection of Cerenkov radiation, this method allows separation of sub-femtomole quantities of 32P-labelled nucleoside 3',5'-bisphosphates modified by bulky carcinogens. Using this method, we were able to separate nine of the ten major adducts formed by reaction of the diol-epoxides of ten polycyclic aromatic hydrocarbons with DNA, and resolve different adducts formed by a single carcinogen. The major adducts formed by benzo[b]fluoranthene (BbF) or dibenz[a,h]anthracene in mouse skin in vivo have been shown to be distinct from the adducts formed directly by the bay-region diol-epoxides. The heterocyclic amines IQ and MeIQ have each been shown to form one major DNA adduct in several in vitro and in vivo systems; using HPLC we were able to resolve the two adducts formed by these food mutagens. HPLC is especially useful for the identification of adducts by means of chromatographic comparisons and in the analysis of the multiple adducts formed by complex mixtures of environmental carcinogens. The major adducts formed by benzo[a]pyrene (BaP) and BbF in mouse skin in vivo that were not resolved on TLC were well separated by HPLC and thus a major DNA adduct formed in the skin of mice treated topically with coal tar was found to be derived from BaP rather than BbF.

Topics: Animals; Benz(a)Anthracenes; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Coal Tar; DNA; DNA Damage; Evaluation Studies as Topic; Female; Fluorenes; Humans; In Vitro Techniques; Male; Mice; Phosphorus Radioisotopes; Quinolines; Rats

The 32P-postlabeling method was used to examine the adducts in DNA, polynucleotides, and mononucleotides reacted in vitro with the N-hydroxy and N-acetoxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Adduct profiles were compared to those found in vivo in liver of cynomolgus monkeys fed IQ, MeIQx or PhIP. The N-acetoxy derivatives of IQ, MeIQx and PhIP (generated in situ from the corresponding N-hydroxylamine in the presence of acetic anhydride) each formed three principal adducts in DNA. Adduct 1 of IQ, MeIQx and PhIP was chromatographically identical to the 32P-labeled bis(phosphate) derivative of N-(deoxyguanosin-8-yl)-IQ, N-(deoxyguanosin-8-yl)-MeIQx, and N-(deoxyguanosin-8-yl)-PhIP respectively, and this adduct comprised approximately 65% of total adduct levels found in DNA in vitro. The C8-guanine adduct and the two minor adducts were also found in poly(dG-dC). poly(dG-dC), suggesting that the two minor adducts of IQ, MeIQx and PhIP are also formed on the guanine base. The N-acetoxy derivatives of IQ, MeIQx, and to a much lesser extent PhIP, also formed adducts with adenine-containing polynucleotides including poly(dA), poly(dA).poly(dT) and poly(dA-dT).poly(dA-dT), but these adenine adducts were chromatographically different from those found in DNA. The three guanine adducts of N-acetoxy-IQ, -MeIQx and -PhIP found in vitro in DNA and in guanine-containing polynucleotides were also found in the liver of monkeys fed IQ, MeIQx or PhIP respectively, indicating that metabolic activation via N-hydroxylation and esterification occurred in vivo in monkeys. With each compound, the C8-guanine adduct was the predominant adduct found in vivo. The results indicate similarities among IQ, MeIQx and PhIP in the DNA adducts formed in vitro and in vivo and substantiate the use of the 32P-postlabeling method for comparative adduct studies.

Topics: Animals; Autoradiography; Chromatography, High Pressure Liquid; DNA; DNA Damage; Haplorhini; Imidazoles; Liver; Mutagens; Phosphorus Radioisotopes; Polynucleotides; Quinolines; Quinoxalines

Male and female CDF1 mice were administered a single oral dose of 3 mumol of the food mutagens 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) or 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and killed 24 h later. DNA was isolated from the livers, lungs, kidneys, colon and forestomach and analysed by 32P-postlabelling for the presence of IQ and MeIQ adducts. Several adduct-enrichment procedures were investigated, including ATP-deficient labelling conditions, butanol extraction and nuclease P1 digestion, and only the ATP-deficient procedure was found to produce the same adduct pattern on polyethyleneimine--cellulose TLC as the standard procedure. Up to nine adduct spots were detected in liver DNA from IQ-treated mice, two of which were not detected in other tissues. The levels of binding in both male and female mice were in the order liver greater than kidney greater than colon greater than forestomach greater than lung. Analysis of DNA from MeIQ-treated mice revealed the presence of up to seven adducts, one of which was detected in liver but not in other tissues. The relative order of DNA binding was kidney greater than liver greater than or equal to colon greater than forestomach greater than lung. As dietary feeding of IQ induces liver, lung and forestomach tumours, and MeIQ induces liver and forestomach tumours in this mouse strain, these binding levels do not correlate with the susceptibility of the organs to carcinogenesis induced by these compounds; the results may indicate the importance of additional factors in determining organ specificity of carcinogenicity.

Topics: Animals; Autoradiography; Carcinogens; Chromatography, Thin Layer; DNA; Female; Male; Mice; Mice, Inbred Strains; Phosphorus Radioisotopes; Quinolines; Tissue Distribution

Eight DNA adducts of 2-amino-3-methylimidazolo[4,5-f]-quinoline (IQ) were found by the standard 32P-postlabeling method in livers from male Cynomolgus monkeys fed IQ (5 days/week, 3 weeks, 20 mg/kg, nasal-gastric intubation). The IQ-DNA adduct fingerprints observed in monkeys were identical to those observed in rats that received IQ (0.03%) in the diet for 2 weeks. The C8-guanine-IQ adduct was identified by comigration with the synthetic 3',5'-bisphosphate derivative of N(-deoxyguanosin-8-yl)-IQ. DNA modified in vitro with N-hydroxy-IQ showed seven adducts, including the C8-guanine-IQ adduct, that were identical to those found in monkeys and rats. Thus it appears that N-hydroxy-IQ, the reactive metabolite of IQ, was responsible for all adducts found in vivo, except one. In order to detect adducts in other organs that were present at lower levels, the intensification (ATP-deficient) method for 32P-postlabeling was used. Five of the adducts detected under standard conditions, including the C8-guanine-IQ adduct, were also detected under intensification conditions. The total level of DNA-IQ adducts was highest in the liver, followed by the kidney, colon and stomach, and bladder. The adduct patterns were identical in all organs examined. The results indicate that IQ is potentially genotoxic in primates and therefore a likely human carcinogen.

Topics: Animals; Autoradiography; Deoxyguanosine; DNA; Macaca fascicularis; Male; Mutagens; Phosphorus Radioisotopes; Quinolines; Rats; Rats, Inbred F344; Tissue Distribution

The nonmutagenic carcinogen methapyrilene, together with several noncarcinogenic analogues, was administered to rats p.o. for as long as 4 wk at concentrations of 0.1%. DNA was isolated from the liver and other organs and hydrolyzed, and the identification of covalent adducts was made using the 32P postlabeling method of Randerath. Some modified procedures were also used to deal with the possibility of very mobile adducts being formed from these hydrophilic amines. Although the rats had received as much as 2 g of amine per kg of body weight, no evidence of formation of DNA adducts in liver or other organs was seen; the level of detection was between 1 in 10(8) and 1 in 10(9) nucleotides. Adduct formation from much lower doses of the mutagenic food pyrolysis product 2-amino-3-methylimidaz(4, 5f)quinoline was detectable at a level of 1 in 10(6) nucleotides in parallel analyses. These results add to the evidence that carcinogenesis by methapyrilene is through an indirect or nonmutagenic mechanism.

Topics: Aminopyridines; Animals; DNA; Liver; Male; Methapyrilene; Mutation; Phosphorus Radioisotopes; Quinolines; Rats; Rats, Inbred F344

The formation of DNA adducts by the fried meat mutagen and carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was studied by means of 32P-postlabeling of DNA digests and four-directional t.l.c. Three major and five minor adducts were detected in assays of DNA digests obtained from Salmonella typhimurium TA98 cells after treatment with IQ and rat liver postmitochondrial supernatant (S9). A qualitatively identical adduct pattern was obtained with nitro-IQ (3-methyl-2-nitroimidazo[4,5-f]quinoline), a new analogue of IQ with a nitro instead of the amino group. These two compounds, therefore, form the same ultimate metabolite. The same adduct pattern was also found after TA98/1,8-DNP6 (acetyltransferase-deficient) cells were treated with nitro-IQ; this is probably due to a residual acetyltransferase activity in this strain. Upon treatment of TA98 cells with 1 mM IQ for 3 h one adduct was detected in 4.7 x 10(5) total bases; a considerably higher adduct frequency, one in 4.2 x 10(3), was induced by nitro-IQ (70 microM, 30 min). The IQ isomer 2-amino-1-methylimidazo[4,5-f]quinoline (isoIQ) and its nitro-analogue nitro-isoIQ (1-methyl-2-nitroimidazo[4,5-f]quinoline) also produced identical adducts. Their common adduct pattern was very similar to the IQ adduct pattern but was located in a position different from that of the IQ adduct pattern. DNA from Syrian hamster embryo (SHE) cells treated with IQ and S9 exhibited adducts apparently identical with those of Salmonella DNA.

Topics: Acetyltransferases; Animals; Cricetinae; DNA; Mutagens; Phosphorus Radioisotopes; Quinolines; Salmonella