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Karam Elbayoumy - One of the best experts on this subject based on the ideXlab platform.

  • abstract 4454 genome wide analysis of dna methylation induced by Environmental Carcinogen dibenzo def p chrysene in ovarian tissues of mice
    Cancer Research, 2016
    Co-Authors: Yuanwan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy, Yuka Imamura Kawasawa, Anna C Salzberg, Kun Ming Chen
    Abstract:

    We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical Carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n = 3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the Carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465. Citation Format: Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. Genome-wide analysis of DNA methylation induced by Environmental Carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4454.

  • abstract 2955 the effects of the Environmental Carcinogen dibenzo a l pyrene on genome wide methylation and the impact of dietary black raspberry in mouse oral tissues
    Cancer Research, 2015
    Co-Authors: Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Yuka Imamura Kawasawa, Anna C Salzberg, Gary D Stoner, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We had previously reported the Carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to Carcinogenesis induced by certain Carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous Carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the Environmental Carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955

  • abstract 5470 genotoxic effect and epigenetic alterations induced by the Environmental Carcinogen dibenzo a l pyrene in oral tissues of mice
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable Carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this Environmental Carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral Carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5470. doi:1538-7445.AM2012-5470

  • abstract 5469 detection of deoxyguanosine adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by lc ms ms
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral Carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469

  • identification and quantification of dna adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by hplc ms ms
    Chemical Research in Toxicology, 2011
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Shantu Amin, Jyh Ming Lin, Arun Sharma, Karam Elbayoumy
    Abstract:

    Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an Environmental pollutant and a tobacco smoke constituent, is the most Carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [15N5]-(±)-anti-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two ad...

Kun Ming Chen - One of the best experts on this subject based on the ideXlab platform.

  • abstract 4454 genome wide analysis of dna methylation induced by Environmental Carcinogen dibenzo def p chrysene in ovarian tissues of mice
    Cancer Research, 2016
    Co-Authors: Yuanwan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy, Yuka Imamura Kawasawa, Anna C Salzberg, Kun Ming Chen
    Abstract:

    We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical Carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n = 3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the Carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465. Citation Format: Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. Genome-wide analysis of DNA methylation induced by Environmental Carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4454.

  • abstract 2955 the effects of the Environmental Carcinogen dibenzo a l pyrene on genome wide methylation and the impact of dietary black raspberry in mouse oral tissues
    Cancer Research, 2015
    Co-Authors: Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Yuka Imamura Kawasawa, Anna C Salzberg, Gary D Stoner, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We had previously reported the Carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to Carcinogenesis induced by certain Carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous Carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the Environmental Carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955

  • abstract 5470 genotoxic effect and epigenetic alterations induced by the Environmental Carcinogen dibenzo a l pyrene in oral tissues of mice
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable Carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this Environmental Carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral Carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5470. doi:1538-7445.AM2012-5470

  • abstract 5469 detection of deoxyguanosine adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by lc ms ms
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral Carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469

  • identification and quantification of dna adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by hplc ms ms
    Chemical Research in Toxicology, 2011
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Shantu Amin, Jyh Ming Lin, Arun Sharma, Karam Elbayoumy
    Abstract:

    Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an Environmental pollutant and a tobacco smoke constituent, is the most Carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [15N5]-(±)-anti-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two ad...

Volker M Arlt - One of the best experts on this subject based on the ideXlab platform.

  • the impact of chemotherapeutic drugs on the cyp1a1 catalysed metabolism of the Environmental Carcinogen benzo a pyrene effects in human colorectal hct116 tp53 tp53 and tp53 cells
    Toxicology, 2018
    Co-Authors: Alexandra J Willis, Radek Indra, David H Phillips, Laura E Wohak, Osman Sozeri, Kerstin Feser, Iveta Mrizova, Marie Stiborova, Volker M Arlt
    Abstract:

    Abstract Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/–) or TP53(–/–) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60–80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(–/–) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(–/–) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.

  • linking Environmental Carcinogen exposure to tp53 mutations in human tumours using the human tp53 knock in hupki mouse model
    FEBS Journal, 2010
    Co-Authors: Jill E Kucab, David H Phillips, Volker M Arlt
    Abstract:

    TP53 is one of the most commonly mutated genes in human tumours. Variations in the types and frequencies of mutations at different tumour sites suggest that they may provide clues to the identity of the causative mutagenic agent. A useful model for studying human TP53 mutagenesis is the partial human TP53 knock-in (Hupki) mouse containing exons 4-9 of human TP53 in place of the corresponding mouse exons. For an in vitro assay, embryo fibroblasts from the Hupki mouse can be examined for the generation and selection of TP53 mutations because mouse cells can be immortalized by mutation of Tp53 alone. Thus far, four Environmental Carcinogens have been examined using the Hupki embryo fibroblast immortalization assay: (a) UV light, which is linked to human skin cancer; (b) benzo[a]pyrene, which is associated with tobacco smoke-induced lung cancer; (c) 3-nitrobenzanthrone, a suspected human lung Carcinogen linked to diesel exposure; and (d) aristolochic acid, which is linked to Balkan endemic nephropathy-associated urothelial cancer. In each case, a unique TP53 mutation pattern was generated that corresponded to the pattern found in human tumours where exposure to these agents has been documented. Therefore, the Hupki embryo fibroblast immortalization assay has sufficient specificity to make it applicable to other Environmental mutagens that putatively play a role in cancer aetiology. Despite the utility of the current Hupki embryo fibroblast immortalization assay, it has several limitations that could be addressed by future developments, in order to improve its sensitivity and selectivity.

Shantu Amin - One of the best experts on this subject based on the ideXlab platform.

  • abstract 4454 genome wide analysis of dna methylation induced by Environmental Carcinogen dibenzo def p chrysene in ovarian tissues of mice
    Cancer Research, 2016
    Co-Authors: Yuanwan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy, Yuka Imamura Kawasawa, Anna C Salzberg, Kun Ming Chen
    Abstract:

    We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical Carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n = 3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the Carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465. Citation Format: Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. Genome-wide analysis of DNA methylation induced by Environmental Carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4454.

  • abstract 2955 the effects of the Environmental Carcinogen dibenzo a l pyrene on genome wide methylation and the impact of dietary black raspberry in mouse oral tissues
    Cancer Research, 2015
    Co-Authors: Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Yuka Imamura Kawasawa, Anna C Salzberg, Gary D Stoner, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We had previously reported the Carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to Carcinogenesis induced by certain Carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous Carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the Environmental Carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955

  • abstract 5470 genotoxic effect and epigenetic alterations induced by the Environmental Carcinogen dibenzo a l pyrene in oral tissues of mice
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable Carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this Environmental Carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral Carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5470. doi:1538-7445.AM2012-5470

  • abstract 5469 detection of deoxyguanosine adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by lc ms ms
    Cancer Research, 2012
    Co-Authors: Shangmin Zhang, Kun Ming Chen, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Karam Elbayoumy
    Abstract:

    Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent Carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral Carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469

  • identification and quantification of dna adducts in the oral tissues of mice treated with the Environmental Carcinogen dibenzo a l pyrene by hplc ms ms
    Chemical Research in Toxicology, 2011
    Co-Authors: Shangmin Zhang, Yuanwan Sun, Kun Ming Chen, Cesar Aliaga, Shantu Amin, Jyh Ming Lin, Arun Sharma, Karam Elbayoumy
    Abstract:

    Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an Environmental pollutant and a tobacco smoke constituent, is the most Carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [15N5]-(±)-anti-DB[a,l]PDE-N6-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two ad...

Suse Broyde - One of the best experts on this subject based on the ideXlab platform.

  • following an Environmental Carcinogen n2 dg adduct through replication elucidating blockage and bypass in a high fidelity dna polymerase
    Nucleic Acids Research, 2007
    Co-Authors: Lida Oum, Lorena S Beese, Nicholas E Geacintov, Suse Broyde
    Abstract:

    We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.

  • toward understanding the mutagenicity of an Environmental Carcinogen structural insights into nucleotide incorporation preferences
    Journal of Molecular Biology, 2002
    Co-Authors: Rebecca A Perlow, Suse Broyde
    Abstract:

    Abstract Bulky Carcinogen–DNA adducts, including (+)- trans - anti -[BP]- N 2 -dG derived from the reaction of (+)- anti -benzo[ a ]pyrene diol epoxide with guanine, often block the progression of DNA polymerases. However, when rare bypass of the lesions does occur, they may be misreplicated. Experimental results have shown that nucleotides are inserted opposite the (+)- trans - anti -[BP]- N 2 -dG adduct by bacteriophage T7 DNA polymerase with the order of preference A>T≥G>C. To gain structural insights into the effects of the bulky adduct on nucleotide incorporation within the polymerase active site, molecular modeling and molecular dynamics simulations were carried out using T7 DNA polymerase to permit the relation of function to structure. We modeled the (+)- trans - anti -[BP]- N 2 -dG adduct opposite incoming dGTP, dTTP and dCTP nucleotides, as well as unmodified guanine opposite its normal partner dCTP as a control, to compare with our previous simulation with dATP opposite the adduct. The modeling required that the (+)- trans - anti -[BP]- N 2 -dG adduct adopt the syn conformation in each case to avoid deranging essential protein–DNA interactions. While the dATP: (+)- trans - anti -[BP]- N 2 -dG pair was well accommodated within the active site of T7 DNA polymerase, dCTP fit poorly opposite the adduct, adopting an orientation perpendicular to the plane of the syn modified guanine during the simulation. Rotation about the glycosidic bond of the dCTP residue to this abnormal position was allowed because only one hydrogen bond between dCTP and the (+)- trans - anti -[BP]- N 2 -dG residue evolved during the simulation, and this hydrogen bond was directly across from the dCTP glycosidic bond. The dTTP and dGTP nucleotides, incorporated with an intermediate preference opposite (+)- trans - anti -[BP]- N 2 -dG, were accommodated reasonably well, but not as stably as the dATP nucleotide, due to a skewed primer-template alignment and more exposed BP moiety, respectively. In addition, the extent of stabilizing interactions between the nascent base-pair in each simulation was correlated positively with the incorporation preference of that particular nucleotide. The dATP nucleotide is accommodated most stably opposite the adduct, with protein–DNA hydrogen bonding interactions and an active-site pocket size that do not deviate significantly from those of the control simulation. The simulations of dTTP and dGTP opposite (+)- trans - anti -[BP]- N 2 -dG exhibited more instability in interactions between the protein and the nascent base-pair than the dATP system. However, the active-site pocket size of the dTTP and dGTP simulations remained stable. The dCTP: (+)- trans - anti -[BP]- N 2 -dG system had the least number of stabilizing interactions, and the active-site pocket of this system increased in size significantly compared to the control and other dNTPs opposite the adduct. These simulations elucidated why A is inserted opposite (+)- trans - anti -[BP]- N 2 -dG most frequently, while T and G are inserted opposite the adduct to an extent intermediate between A and C, and C is most rarely incorporated. Structural rationalization of the incorporation preference opposite (+)- trans - anti -[BP]- N 2 -dG by T7 DNA polymerase contributes to providing a molecular explanation for mutations caused by this Carcinogen–DNA adduct in a model system.

  • evading the proofreading machinery of a replicative dna polymerase induction of a mutation by an Environmental Carcinogen
    Journal of Molecular Biology, 2001
    Co-Authors: Rebecca A Perlow, Suse Broyde
    Abstract:

    DNA replication fidelity is dictated by DNA polymerase enzymes and associated proteins. When the template DNA is damaged by a Carcinogen, the fidelity of DNA replication is sometimes compromized, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical Carcinogenesis by metabolically activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interactions between the Carcinogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are Environmental Carcinogens that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as Carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and fish, and as a contaminant in other foods. BP is metabolically activated into several highly reactive intermediates, including the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide (BPDE). The primary product of the reaction of (+)-anti-BPDE with DNA, the (+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-dG ((+)-ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucidate the structural characteristics and interactions between the DNA polymerase and Carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (+)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP, the nucleotide most commonly mismatched with the (+)-ta-[BP]G adduct during replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (+)-ta-[BP]G:A mismatch can be accommodated stably in the active site so that the fidelity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic base. In this structure, the modified guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. This structure is stabilized by a hydrogen bond between the modified guanine base and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, a hydrogen bond between the modified guanine base and the polymerase, and several hydrogen bonds between the BP moiety and polymerase side-chains. Moreover, the G:A mismatch in this system closely resembles the size and shape of a normal Watson-Crick pair. These features reveal how the polymerase proofreading machinery may be evaded in the presence of a mutagenic Carcinogen-damaged DNA, so that a mismatch can be accommodated readily, allowing bypass of the adduct by the replicative T7 DNA polymerase.