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Santhanam Swaminathan - One of the best experts on this subject based on the ideXlab platform.
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Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by 32P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-Aminobiphenyl
Environmental and molecular mutagenesis, 2002Co-Authors: James F. Hatcher, Santhanam SwaminathanAbstract:DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-Aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP), were analyzed by the 32P-postlabeling method. Two adducts detected by 32P-postlabeling were previously identified as the 3′,5′-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-Aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955–961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295–301). In contrast to the dG-C8-ABP adduct, which was 3′-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-Aminobiphenyl with deoxyguanosine-3′-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3′-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N2-yl)-4-azobiphenyl (dG-NN-ABP). 32P-Postlabeling analysis of the nuclease P1–enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-NN-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA. Environ. Mol. Mutagen. 39:314–322, 2002. © 2002 Wiley-Liss, Inc.
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Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-Aminobiphenyl using 32P-postlabeling method
Chemico-biological interactions, 2002Co-Authors: Santhanam Swaminathan, James F. HatcherAbstract:The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-Aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl and N-(deoxyadenosin-8-yl)-4-Aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-Aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-Aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-Aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.
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Detection of deoxyadenosine-4-Aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-Aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis.
Carcinogenesis, 1995Co-Authors: James F. Hatcher, Santhanam SwaminathanAbstract:To characterize the DNA adducts in human uroepithelial cells (HUC) exposed to 4-Aminobiphenyl and its proximate N-hydroxy metabolites, we used 32 P-postlabeling analyses following butanol extraction of the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-Aminobiphenyl (pdGp-ABP) as a major adduct and N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-Aminobiphenyl (pdAp-ABP) as a minor adduct in an immortalized non-tumorigenic cell line of HUC following exposure to N-hydroxy-4-Aminobiphenyl (N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeled the synthetic N-(deoxyadenosin-3'-phospho-8-yl)-4-Aminobiphenyl (dAp-ABP) adduct to generate pdAp-ABP and determined its chromatographic (TLC and HPLC) properties and sensitivity to nuclease P1 digestion. In contrast to pdGp-ABP, which was cleaved to the corresponding 5'-monophosphate by nuclease P1, the pdAp-ABP adduct was unaffected when incubated with nuclease P1 under similar conditions. To test whether nuclease P1 digestion could be adopted for enrichment of the dAp-ABP adduct in HUC samples, postlabeling analyses were carried out after butanol extraction following nuclease P1 digestion of the DNA hydrolysate. Under these conditions, the pdAp-ABP adduct was detected in DNA from HUC E7 cells treated with N-OH-ABP and in calf thymus DNA reacted with N-OH-ABP under acidic (pH 5.0) conditions. These data indicate that pdGp-ABP and pdAp-ABP adducts are generated in HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichment may provide a method for qualitative and quantitative analyses of the pdAp-ABP adduct in DNA
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Mutagenic activation of 4-Aminobiphenyl and its N-hydroxy derivatives by microsomes from cultured human uroepithelial cells
Mutagenesis, 1993Co-Authors: James F. Hatcher, K. Prabhakar Rao, Santhanam SwaminathanAbstract:Activation of the human bladder carcinogen 4-Aminobiphenyl (ABP) and its N-hydroxy derivatives was investigated using lysates and subcellular enzyme preparations from cultured human uroepithelial cells (HUC). Mutagenic activation was determined using Salmonella typhimurium strains TA98; TA98/1,8-DNP6, a derivative deficient in acetyl coenzyme A:N-hydroxyarylamine O-acetyltransferase (OAT); and YG1024, a derivative of TA98 with elevated OAT activity and enhanced sensitivity to mutation by N-hydroxyarylamines. Mutagenicity of ABP catalyzed by HUC microsomes was detected in YG1024 but not in the parent strain TA98. HUC microsomes also catalyzed the mutagenic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and the relative sensitivity of the tester strains was YG1024 > TA98 > TA98/1,8-DNP6, indicating N-hydroxy-4-Aminobiphenyl (N-OH-ABP) as the mutagenic intermediate. In contrast, the mutagenic activity of N-acetoxy-4-acetylaminobiphenyl incubated with HUC microsomes was approximately equal in TA98 and YG1024, and may involve N-acetoxy-4-Aminobiphenyl (N-OAc-ABP) as the intermediate. High pressure liquid chromatography (HPLC) of the DNA hydrolysate obtained after incubation of [3H]N-OH-ABP with YG1024, showed N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-ABP) as the primary adduct, based on mobility of the radioactivity in comparison with the synthetic standard. Additionally, HUC microsomes catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of 4-acetylaminobiphenyl (AABP), N-OH-AABP and acetyl coenzyme A as acetyl donors, and this binding was blocked by paraoxon. The hydrolysate obtained from incubation of DNA with [3H]N-OH-ABP and HUC microsomes, with AABP as acetyl donor, revealed the formation of dG-ABP adduct.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acetyl transferase-mediated metabolic activation of N-hydroxy-4-Aminobiphenyl by human uroepithelial cells
Carcinogenesis, 1992Co-Authors: Susan M. Frederickson, James F. Hatcher, Catherine A. Reznikoff, Santhanam SwaminathanAbstract:Metabolism and nucleic acid binding of N-hydroxy-4-Aminobiphenyl (N-OH-ABP), a proximate carcinogenic metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP), was investigated using cultured normal human uroepithelial cells (HUC). HPLC and TLC of the ethyl acetate extract of the media from cultured HUC after 4 h exposure to N-OH-ABP revealed the formation of two major metabolites, ABP and 4-acetylaminobiphenyl (AABP), suggesting the presence of N-acetyl transferase(s) in HUC. This was further confirmed by the formation of AABP, during the incubation of ABP with acetyl coenzyme A (AcCoA) and HUC cytosol. To test whether these enzymes also catalyze the AcCoA-dependent O-acetylation, we examined the metabolic activation of N-OH-ABP using cytosolic preparations. Cytosol from HUC catalyzed AcCoA-dependent binding of [3H]N-OH-ABP to RNA; the amount of binding was 757 pmol/mg RNA/mg protein. Binding with DNA was quantitatively similar to RNA. HPLC and TLC analyses of the enzymatic hydrolysate of [3H]N-OH-ABP-bound DNA revealed the major adduct to be N-(deoxyguanosine-8-yl)-4-Aminobiphenyl, based on mobility of the radioactivity in comparison with the authentic synthetic standard. 32P-Post-labeling analysis of the DNA from the cytosol-mediated binding of N-OH-ABP revealed four radioactive spots. In contrast, post-labeling analysis of the DNA from intact HUC exposed to N-OH-ABP showed five adducts, including two of the adducts observed with HUC cytosols, suggesting the possible involvement of additional activation pathway(s) in intact HUC. These results suggest that bioactivation of N-OH-ABP could occur within the HUC, the target organ for ABP, and that cytosolic acetyl transferase(s) may play a critical role in susceptibility to arylamine-induced bladder carcinogenesis.
James F. Hatcher - One of the best experts on this subject based on the ideXlab platform.
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Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by 32P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-Aminobiphenyl
Environmental and molecular mutagenesis, 2002Co-Authors: James F. Hatcher, Santhanam SwaminathanAbstract:DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-Aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP), were analyzed by the 32P-postlabeling method. Two adducts detected by 32P-postlabeling were previously identified as the 3′,5′-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-Aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955–961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295–301). In contrast to the dG-C8-ABP adduct, which was 3′-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-Aminobiphenyl with deoxyguanosine-3′-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3′-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N2-yl)-4-azobiphenyl (dG-NN-ABP). 32P-Postlabeling analysis of the nuclease P1–enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-NN-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA. Environ. Mol. Mutagen. 39:314–322, 2002. © 2002 Wiley-Liss, Inc.
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Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-Aminobiphenyl using 32P-postlabeling method
Chemico-biological interactions, 2002Co-Authors: Santhanam Swaminathan, James F. HatcherAbstract:The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-Aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl and N-(deoxyadenosin-8-yl)-4-Aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-Aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-Aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-Aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.
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Detection of deoxyadenosine-4-Aminobiphenyl adduct in DNA of human uroepithelial cells treated with N-hydroxy-4-Aminobiphenyl following nuclease P1 enrichment and 32P-postlabeling analysis.
Carcinogenesis, 1995Co-Authors: James F. Hatcher, Santhanam SwaminathanAbstract:To characterize the DNA adducts in human uroepithelial cells (HUC) exposed to 4-Aminobiphenyl and its proximate N-hydroxy metabolites, we used 32 P-postlabeling analyses following butanol extraction of the DNA hydrolysates. Using this method, we identified N-(deoxyguanosin-3',5'-bisphospho-8-yl)-4-Aminobiphenyl (pdGp-ABP) as a major adduct and N-(deoxyadenosin-3',5'-bisphospho-8-yl)-4-Aminobiphenyl (pdAp-ABP) as a minor adduct in an immortalized non-tumorigenic cell line of HUC following exposure to N-hydroxy-4-Aminobiphenyl (N-OH-ABP). Towards characterization of pdAp-ABP, we postlabeled the synthetic N-(deoxyadenosin-3'-phospho-8-yl)-4-Aminobiphenyl (dAp-ABP) adduct to generate pdAp-ABP and determined its chromatographic (TLC and HPLC) properties and sensitivity to nuclease P1 digestion. In contrast to pdGp-ABP, which was cleaved to the corresponding 5'-monophosphate by nuclease P1, the pdAp-ABP adduct was unaffected when incubated with nuclease P1 under similar conditions. To test whether nuclease P1 digestion could be adopted for enrichment of the dAp-ABP adduct in HUC samples, postlabeling analyses were carried out after butanol extraction following nuclease P1 digestion of the DNA hydrolysate. Under these conditions, the pdAp-ABP adduct was detected in DNA from HUC E7 cells treated with N-OH-ABP and in calf thymus DNA reacted with N-OH-ABP under acidic (pH 5.0) conditions. These data indicate that pdGp-ABP and pdAp-ABP adducts are generated in HUC E7 on treatment with N-OH-ABP and that nuclease P1 enrichment may provide a method for qualitative and quantitative analyses of the pdAp-ABP adduct in DNA
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Mutagenic activation of 4-Aminobiphenyl and its N-hydroxy derivatives by microsomes from cultured human uroepithelial cells
Mutagenesis, 1993Co-Authors: James F. Hatcher, K. Prabhakar Rao, Santhanam SwaminathanAbstract:Activation of the human bladder carcinogen 4-Aminobiphenyl (ABP) and its N-hydroxy derivatives was investigated using lysates and subcellular enzyme preparations from cultured human uroepithelial cells (HUC). Mutagenic activation was determined using Salmonella typhimurium strains TA98; TA98/1,8-DNP6, a derivative deficient in acetyl coenzyme A:N-hydroxyarylamine O-acetyltransferase (OAT); and YG1024, a derivative of TA98 with elevated OAT activity and enhanced sensitivity to mutation by N-hydroxyarylamines. Mutagenicity of ABP catalyzed by HUC microsomes was detected in YG1024 but not in the parent strain TA98. HUC microsomes also catalyzed the mutagenic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and the relative sensitivity of the tester strains was YG1024 > TA98 > TA98/1,8-DNP6, indicating N-hydroxy-4-Aminobiphenyl (N-OH-ABP) as the mutagenic intermediate. In contrast, the mutagenic activity of N-acetoxy-4-acetylaminobiphenyl incubated with HUC microsomes was approximately equal in TA98 and YG1024, and may involve N-acetoxy-4-Aminobiphenyl (N-OAc-ABP) as the intermediate. High pressure liquid chromatography (HPLC) of the DNA hydrolysate obtained after incubation of [3H]N-OH-ABP with YG1024, showed N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-ABP) as the primary adduct, based on mobility of the radioactivity in comparison with the synthetic standard. Additionally, HUC microsomes catalyzed the binding of [3H]N-OH-ABP to RNA in the presence of 4-acetylaminobiphenyl (AABP), N-OH-AABP and acetyl coenzyme A as acetyl donors, and this binding was blocked by paraoxon. The hydrolysate obtained from incubation of DNA with [3H]N-OH-ABP and HUC microsomes, with AABP as acetyl donor, revealed the formation of dG-ABP adduct.(ABSTRACT TRUNCATED AT 250 WORDS)
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Acetyl transferase-mediated metabolic activation of N-hydroxy-4-Aminobiphenyl by human uroepithelial cells
Carcinogenesis, 1992Co-Authors: Susan M. Frederickson, James F. Hatcher, Catherine A. Reznikoff, Santhanam SwaminathanAbstract:Metabolism and nucleic acid binding of N-hydroxy-4-Aminobiphenyl (N-OH-ABP), a proximate carcinogenic metabolite of the human bladder carcinogen 4-Aminobiphenyl (ABP), was investigated using cultured normal human uroepithelial cells (HUC). HPLC and TLC of the ethyl acetate extract of the media from cultured HUC after 4 h exposure to N-OH-ABP revealed the formation of two major metabolites, ABP and 4-acetylaminobiphenyl (AABP), suggesting the presence of N-acetyl transferase(s) in HUC. This was further confirmed by the formation of AABP, during the incubation of ABP with acetyl coenzyme A (AcCoA) and HUC cytosol. To test whether these enzymes also catalyze the AcCoA-dependent O-acetylation, we examined the metabolic activation of N-OH-ABP using cytosolic preparations. Cytosol from HUC catalyzed AcCoA-dependent binding of [3H]N-OH-ABP to RNA; the amount of binding was 757 pmol/mg RNA/mg protein. Binding with DNA was quantitatively similar to RNA. HPLC and TLC analyses of the enzymatic hydrolysate of [3H]N-OH-ABP-bound DNA revealed the major adduct to be N-(deoxyguanosine-8-yl)-4-Aminobiphenyl, based on mobility of the radioactivity in comparison with the authentic synthetic standard. 32P-Post-labeling analysis of the DNA from the cytosol-mediated binding of N-OH-ABP revealed four radioactive spots. In contrast, post-labeling analysis of the DNA from intact HUC exposed to N-OH-ABP showed five adducts, including two of the adducts observed with HUC cytosols, suggesting the possible involvement of additional activation pathway(s) in intact HUC. These results suggest that bioactivation of N-OH-ABP could occur within the HUC, the target organ for ABP, and that cytosolic acetyl transferase(s) may play a critical role in susceptibility to arylamine-induced bladder carcinogenesis.
Paul Vouros - One of the best experts on this subject based on the ideXlab platform.
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Recent technical and biological development in the analysis of biomarker N-deoxyguanosine-C8-4-Aminobiphenyl.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2018Co-Authors: Zhidan Chen, Yuesheng Zhang, Paul VourosAbstract:4-Aminobiphenyl (4-ABP) which is primarily formed during tobacco combustion and overheated meat is a major carcinogen responsible for various cancers. Its adducted form, N-deoxyguanosine-C8-4-Aminobiphenyl (dG-C8-4-ABP), has long been employed as a biomarker for assessment of the risk for cancer. In this review, the metabolism and carcinogenisity of 4-ABP will be discussed, followed by a discussion of the current common approaches of analyzing dG-C8-4-ABP. The major part of this review will be on the history and recent development of key methods for detection and quantitation of dG-C8-4-ABP in complex biological samples and their biological applications, from the traditional 2P-postlabelling and immunoassay methods to modern liquid chromatography-mass spectrometry (LC-MS) with the latter as the focus. Many vital biological discoveries based on dG-C8-4-ABP have been published by using the nanoLC-MS with column switching platform in our laboratory, which has also been adopted and further improved by many other researchers. We hope this review can provide a perspective of the challenges that had to be addressed in reaching our present goals and possibly bring new ideas for those who are still working on the frontline of DNA adducts area.
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Quantification of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl adducts in human lymphoblastoid TK6 cells dosed with N-hydroxy-4-acetylaminobiphenyl and their relationship to mutation, toxicity, and gene expression profiling.
Analytical chemistry, 2006Co-Authors: Elaine M. Ricicki, Wen Luo, Wenhong Fan, Lue Ping Zhao, Helmut Zarbl, Paul VourosAbstract:Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-Aminobiphenyl, a mutagenic metabolite of 4-Aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-Aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-Aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.
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Detection and quantification of N-(deoxyguanosin-8-yl)-4-Aminobiphenyl adducts in human pancreas tissue using capillary liquid chromatography-microelectrospray mass spectrometry.
Chemical research in toxicology, 2005Co-Authors: Elaine M. Ricicki, John R. Soglia, Candee H. Teitel, Robert Kane, And Fred Kadlubar, Paul VourosAbstract:Cigarette smoking has been associated with various cancers including bladder and pancreas. 4-Aminobiphenyl has been isolated as a constituent of cigarette smoke and has been established as a carcinogen in various animal models and humans. In rodents and humans, 4-Aminobiphenyl is N-hydroxylated and forms adducts to DNA, the predominant one being N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-C8-ABP). In this study, we report a micro-electrospray mass spectrometric (μESI-MS) isotope dilution method for the detection and quantification of dG-C8-ABP in human pancreatic tissue. A reverse phase capillary column (320 μm ID) was connected to a triple quadrupole mass spectrometer via a commercially available micro-ESI source. The system was operated in the selected reaction monitoring mode transmitting the [M + H]+ → [M + H − 116]+ transitions for both the analyte and the isotopically labeled internal standard. Twelve human pancreas samples were analyzed, where six were current smokers (three male and three female) a...
Steven R. Tannenbaum - One of the best experts on this subject based on the ideXlab platform.
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INVESTIGATION OF THE MECHANISM OF MUTAGENESIS BY THE DNA ADDUCTS OF THE
2016Co-Authors: Steven R. Tannenbaum, John M. Essigmann, Susan Bina, Malaikal Verghis, Human Bladder CarcinogenAbstract:in Toxicology This study investigated the mutagenicity of the major DNA adduct formed by the human bladder carcinogen 4-Aminobiphenyl (ABP), N-(deoxyguanosin-8-yl)-4-Aminobiphenyl (dG-C8-ABP), in the bacteriophage M13mpl9 single-stranded (ss) genome replicated in Escherichia coli. To determine if DNA adducts formed by ABP could induce mutations in the M13 ss genome, two complementary mutational analyses in E. coli were undertaken. In the first study, mutagenesis assays in the lacZ gene of globally ABP-modified M13mplO ss and duplex (ds or RF) DNAs were conducted to determine which of the known DNA adducts of ABP were likely to be mutagenic in the M13mplO ss genome and to compare the ABP mutational spectrum in ss DNA to that established in the lacZ gene of ABP-modified RF DNA. M13 globally ABP-modified ss and RF DNAs were replicated in excision-repair proficient (uvr*) and excision-repair deficient (uvr~) E. coli strains that wer
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Monoclonal antibodies and rabbit antisera recognizing 4-Aminobiphenyl—DNA adducts and application to immunoaffinity chromatography
Carcinogenesis, 1992Co-Authors: John D. Groopman, Fred F. Kadlubar, Paul L. Skipper, Paul R. Donahue, Laura J. Trudel, Michael Wildschutte, Steven R. TannenbaumAbstract:Monoclonal antibodies and rabbit antisera were produced that recognized 4-Aminobiphenyl, its major DNA adducts and other metabolites. The antigens used to raise these antibodies were synthesized by coupling the aromatic amine to protein through a diazotization reaction. The goal of this immunization strategy was to induce antibodies that also cross-reacted with most 4-Aminobiphenyl-derived metabolites. A total of 20 mice and four rabbits were immunized and every animal produced a strong immune response for 4-Aminobiphenyl and its derivatives. Two IgG1 monoclonal antibodies, 3D6 and 2E11, were isolated from two different mouse spleen cell fusions. One of the monoclonal antibodies, 3D6, had a high recognition for the three major 4-Aminobiphenyl-DNA adducts: N-(deoxyguanosine-8-yl)-4-Aminobiphenyl, N-(deoxyadenosin-8-yl)-4-Aminobiphenyl and N-(deoxyguanosine-N2-yl)-4-Aminobiphenyl, with affinity constants between 2 and 4 x 10(9) l/mol. In addition, one of the rabbit anti-sera had an affinity constant for the DNA adducts of 2.1 x 10(9) l/mole. Thus, the strategy to use a diazotization coupling reaction was successful at producing high-affinity aminobiphenyl-DNA adduct-specific antibodies. Preparative immunoaffinity resins were made for each monoclonal antibody. These resins quantitatively bound 500 ng each [3H]N-acetyl-aminobiphenyl, [3H]N-aminobiphenyl and [3H]N-(deoxyguanosine-8-yl)-4-Aminobiphenyl. Preliminary experiments were performed to test the applicability of the preparative monoclonal antibody immunoaffinity column to isolate [3H]4-Aminobiphenyl-derived metabolites in dosed rat and dog urine. About 70% of the radioactivity in rat or dog urine could be bound to the immunoaffinity columns. The combined immunoaffinity column/HPLC analysis of the dog urine led to the identification of a novel urinary metabolite, N-formyl-aminobiphenyl. HPLC analysis of a rat urine sample tentatively found 4-Aminobiphenyl, N-acetyl-4-Aminobiphenyl and N-formyl-4-Aminobiphenyl by co-chromatography, and these compounds accounted for 20, 6.8 and 6.5% of the total radioactivity in the chromatogram respectively. Taken together, these data show that these 4-Aminobiphenyl-specific monoclonal antibodies can be used in immunoaffinity columns to isolate metabolites and DNA adducts from biological samples.
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Detection of carcinogen-DNA adducts in exfoliated urothelial cells of cigarette smokers: association with smoking, hemoglobin adducts, and urinary mutagenicity.
Cancer Epidemiology Biomarkers & Prevention, 1991Co-Authors: Glenn Talaska, Helmut Bartsch, Marlene Schamer, Leonard Unruh, Fred F. Kadlubar, Christian Malaveille, Paul L. Skipper, Neil E Caporaso, Steven R. Tannenbaum, Paolo VineisAbstract:The presence of covalent modifications in DNA obtained from exfoliated urothelial cells of smokers and nonsmokers was determined using 32P postlabeling methods. Urine and blood samples were procured from 73 persons. Cells were removed from the urine by filtration. DNA was isolated using an enzyme-solvent extraction method and then coprecipitated with glycogen. Sufficient DNA to detect 1 carcinogen-DNA adduct/10(9) normal nucleotides was obtained from 40 of the 73 samples. DNA was hydrolyzed to 3'-phosphodeoxynucleotides and then 32P postlabeled under conditions of excess [32P]ATP. Carcinogen-DNA adducts were resolved using anion-exchange thin-layer chromatography and visualized by autoradiography; film exposures lasted as long as 7 days. Twelve different carcinogen-DNA adducts and a diagonal zone of radioactivity could be found, but no sample contained all adducts. At least four adducts appeared to be cigarette smoking related. These adducts were from 2 to 20 times higher in the smokers than the nonsmokers. Two carcinogen-DNA adducts were qualitatively very similar to adducts described earlier in a study of human bladder biopsies. One of these corresponded to N-(deoxyguanosin-8-yl)-4-Aminobiphenyl. Adducts were correlated significantly with the levels of 4-Aminobiphenyl hemoglobin adducts and number of cigarettes smoked. In addition, levels of the putative N-(deoxyguanosin-8-yl)-4-Aminobiphenyl adduct and a measure of total adducts were correlated with the mutagenic activity of the individual's urine. These data suggest that noninvasive, biological monitoring techniques can be applied to the study of carcinogen-DNA adducts in humans at high risk for bladder cancer.
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Measurement of 4-Aminobiphenyl-hemoglobin adducts in lung cancer cases and controls.
Cancer research, 1991Co-Authors: Ainsley Weston, Paul L. Skipper, Neil E Caporaso, Steven R. Tannenbaum, Koli Taghizadeh, Robert N. Hoover, James H. Resau, Benjamin F. Trump, Curtis C. HarrisAbstract:Abstract Hemoglobin adducts of the activated carcinogenic aromatic amine 4-Aminobiphenyl have been measured in a case-control study of lung cancer. Data obtained for lung cancer cases are compared to those obtained for controls that consisted of patients with either chronic obstructive pulmonary disease or non-pulmonary cancers. Both simple and multivariate analysis found a positive association of 4-Aminobiphenyl-hemoglobin adducts with the quantity of tobacco smoked as determined by either urine cotinine or questionnaire data. No association was found between 4-Aminobiphenyl-hemoglobin adducts and cancer diagnosis, and adduct levels were not related to remote tobacco use, i.e., total pack years of smoking. There was no association between the levels of adducts detected and the ability of an individual to metabolize debrisoquine (debrisoquine metabolic phenotype, CYP2D6). Whereas 4-Aminobiphenyl-hemoglobin adduct levels reflected recent tobacco smoking, they were not correlated with lung cancer risk.
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4-Aminobiphenyl hemoglobin adducts in fetuses exposed to the tobacco smoke carcinogen in utero.
Journal of the National Cancer Institute, 1991Co-Authors: J. Coghlin, Paul L. Skipper, P. H. Gann, S. K. Hammond, Koli Taghizadeh, M. Paul, Steven R. TannenbaumAbstract:Maternal-fetal exchange of a potent tobacco-related human carcinogen, 4-Aminobiphenyl, was studied in smoking (n = 14) and nonsmoking (n = 38) pregnant women. N-Hydroxy-4-Aminobiphenyl, the active metabolite of 4-Aminobiphenyl, forms chemical addition products (adducts) with hemoglobin. Levels of 4-Aminobiphenyl hemoglobin adducts were measured in maternal-fetal paired blood samples obtained from smoking and nonsmoking women during labor and delivery. Carcinogen-hemoglobin adducts were detected in all maternal and fetal blood samples. Levels of such adducts were significantly higher (P less than .001) in maternal and fetal blood samples from smokers: the mean 4-Aminobiphenyl hemoglobin adduct level was 92 +/- 54 pg/g of hemoglobin in blood samples from fetuses of smokers, and 17 +/- 13 pg/g of hemoglobin in blood samples from fetuses of nonsmokers; the mean maternal 4-Aminobiphenyl hemoglobin adduct level was 183 +/- 108 pg/g of hemoglobin in smokers, and 22 +/- 8 pg/g of hemoglobin in nonsmokers. Fetal carcinogen-adduct levels were consistently lower than maternal levels: the mean maternal to fetal ratio was 2.4 +/- 1.1 in smokers and 1.9 +/- .98 in nonsmokers. Fetal 4-Aminobiphenyl hemoglobin adduct levels were strongly associated (correlation coefficient [r2] = .51, P = .002) with maternal 4-Aminobiphenyl hemoglobin adduct levels when paired samples from smoking mothers were analyzed. A measure of third-trimester tobacco smoke exposure based on number of cigarettes smoked per day, amount of each cigarette smoked, and depth of inhalation was associated (r2 = .59, P = .029) with maternal 4-Aminobiphenyl levels but not with fetal 4-Aminobiphenyl levels. This study demonstrates that a potent tobacco-related carcinogen, 4-Aminobiphenyl, or its active metabolite, N-hydroxy-4-Aminobiphenyl, crosses the human placenta and binds to fetal hemoglobin in concentrations that are significantly higher in smokers than in nonsmokers.
David W. Hein - One of the best experts on this subject based on the ideXlab platform.
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N-acetyltransferase 2 acetylator genotype-dependent N-acetylation of 4-Aminobiphenyl in cryopreserved human hepatocytes.
Pharmacogenetics and genomics, 2020Co-Authors: Mariam R. Habil, Mark A. Doll, David W. HeinAbstract:Arylamine N-acetyltransferases are xenobiotic-metabolizing enzymes responsible for detoxification of many drugs and carcinogens. Two N-acetyltransferase proteins (NAT1 and NAT2) are expressed in humans and they both N-acetylate aromatic amine carcinogens such as 4-Aminobiphenyl. Arylamines such as 4-Aminobiphenyl represent a large class of chemical carcinogens. Exposure to 4-Aminobiphenyl occurs in the chemical, dye and rubber industries as well as in hair dyes, paints, and cigarette smoke. NAT2 is subject to a genetic polymorphism resulting in rapid, intermediate and slow acetylator phenotypes. We investigated the role of the NAT2 genetic polymorphisms on the N-acetylation of 4-Aminobiphenyl in cryopreserved human hepatocytes in which NAT2 genotype and deduced phenotype were determined. Differences in sulfamethazine (selectively N-acetylated via NAT2) and 4-Aminobiphenyl (N-acetylated by both NAT1 and NAT2) N-acetylation rates among rapid, intermediate, and slow NAT2 acetylator genotypes were tested for significance by one-way analysis of variance. In vitro 4-Aminobiphenyl N-acetyltransferase activities differed significantly between rapid, intermediate and slow acetylators at 10 µM (P = 0.0102) or 100 µM (P = 0.0028). N-acetylation of 4-Aminobiphenyl in situ also differed significantly between human hepatocytes from rapid, intermediate, and slow acetylators at 10 µM (P = 0.0015) and 100 µM (P = 0.0216). A gene dose-response relationship was exhibited as intermediate acetylators catalyzed 4-Aminobiphenyl N-acetylation both in vitro and in situ at rates arithmetically between rapid and slow acetylators. In conclusion, N-acetylation of 4-Aminobiphenyl is NAT2 genotype-dependent in human hepatocytes. These results suggest refinement of the exposure limit and safety for arylamine carcinogens according to NAT2 genotype.
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Acetylator genotype (NAT2)-dependent formation of aberrant crypts in congenic Syrian hamsters administered 3,2'-dimethyl-4-Aminobiphenyl
Cancer research, 1996Co-Authors: Yi Feng, R. J. Wagner, Adrian J. Fretland, William K. Becker, A. M. Cooley, T. P. Pretlow, K. J. Lee, David W. HeinAbstract:Abstract Some but not all human epidemiological studies suggest a higher incidence of colon cancer in rapid acetylator individuals. Aberrant crypts, the earliest morphologically evident preneoplastic lesions in chemical colon carcinogenesis, were measured in rapid and slow acetylator congenic Syrian hamsters administered 3,2′-dimethyl-4-Aminobiphenyl, an aromatic amine colon carcinogen, to investigate the specific role of the acetylator genotype (NAT2) in colon carcinogenesis. Age-matched rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/H-Pats) acetylator female Syrian hamsters congenic at the NAT2 locus received a s.c. injection of 3,2′-dimethyl-4-Aminobiphenyl (100 mg/kg) at the start of weeks 1 and 2. After 10 and 14 weeks, the hamsters were sacrificed, and each whole cecum, colon, and rectum was stained with 0.2% methylene blue, fixed in 4% paraformaldehyde, and examined under a dissecting microscope for the presence of aberrant crypts. Aberrant crypts were identified in the cecums and colons of both rapid and slow acetylator congenic hamsters treated with 3,2′-dimethyl-4-Aminobiphenyl but not in vehicle controls. The size of the aberrant crypt foci was larger in the colon than in the cecum, and the highest frequency of aberrant crypt foci was observed in the cecum. No aberrant crypts were detected in the rectum. The frequency of aberrant crypt foci was significantly higher (2–3-fold) in rapid versus slow acetylator congenic hamsters in both cecum (P = 0.0352) and colon (P = 0.0006). These results support human epidemiological studies that suggest the rapid acetylator genotype is associated with higher risk of colon cancer induced by aromatic amines.
