Showing papers by "Ahmad Besaratinia published in 2005"

Mutations induced by ultraviolet light

Abstract: The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA bases. We also discuss the role of DNA damage-tolerant DNA polymerases in UV lesion bypass and mutagenesis.

DNA lesions induced by UV A1 and B radiation in human cells: Comparative analyses in the overall genome and in the p53 tumor suppressor gene

Abstract: The UV components of sunlight (UVA and UVB) are implicated in the etiology of human skin cancer. The underlying mechanism of action for UVB carcinogenicity is well defined; however, the mechanistic involvement of UVA in carcinogenesis is not fully delineated. We investigated the genotoxicity of UVA1 versus UVB in the overall genome and in the p53 tumor suppressor gene in normal human skin fibroblasts. Immuno-dot blot analysis identified the cis-syn cyclobutane pyrimidine-dimer (CPD) as a distinctive UVB-induced lesion and confirmed its formation in the genomic DNA of UVA1-irradiated cells dependent on radiation dose. HPLC/tandem MS analysis showed an induction of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the genomic DNA of UVA1-irradiated cells only. Mapping of DNA damages by terminal transferase-dependent PCR revealed preferential, but not identical, formation of polymerase-blocking lesions and/or strand breaks along exons 5-8 of the p53 gene in UVB- and UVA1-irradiated cells. The UVB-induced lesions detected by terminal transferase-PCR were almost exclusively mapped to pyrimidine-rich sequences; however, the UVA1-induced lesions were mapped to purine- and pyrimidine-containing sequences along the p53 gene. Cleavage assays with lesion-specific DNA repair enzymes coupled to ligation-mediated PCR showed preferential, but not identical, formation of CPDs along the p53 gene in UVB- and UVA1-irradiated cells. Additionally, dose-dependent formation of oxidized and ring-opened purines and abasic sites was established in the p53 gene in only UVA1-irradiated cells. We conclude that UVA1 induces promutagenic CPDs and oxidative DNA damage at both the genomic and nucleotide resolution level in normal human skin fibroblasts.

DNA adduction and mutagenic properties of acrylamide.

Abstract: This review article summarizes our current knowledge on DNA damaging and mutagenic properties of acrylamide. Direct and indirect modes of interaction of acrylamide with DNA are discussed, and the resulting alkylating DNA adducts are highlighted. Emphasis is placed on glycidamide-DNA adducts generated via epoxidation of acrylamide presumably by cytochrome P4502E1. Dosimetry and mapping of acrylamide-induced DNA adducts in vitro and/or in vivo are described. Mutagenic potency and specificity of acrylamide in relation to its respective DNA adducts are discussed. Prospective views are provided on the potential applications of acrylamide-induced DNA adduct dosimetry/mapping and mutation frequency/spectrometry for biomonitoring purposes.

Investigating human cancer etiology by DNA lesion footprinting and mutagenicity analysis

Abstract: Many genotoxic carcinogens are known to leave unique signatures on cancer-related genes. The signature of carcinogens is manifested by the induction of characteristic mutations at distinctive nucleotide positions along oncogenes and/or tumor suppressor genes. Often, the nucleotide positions, wherein mutations occur, co-localize with the sites of initial DNA damage induced by the respective carcinogens. Thus, DNA damage-targeted mutation can be a predictor of carcinogenicity of genotoxins. Today, genomic sequencing technologies for investigating human cancer etiology are based on DNA-lesion footprinting in conjunction with mutagenicity analysis of genotoxic carcinogens. In this review article, we discuss the ligation-mediated PCR and terminal transferase-dependent-PCR, two versatile DNA-lesion footprinting techniques. We highlight the in vitro shuttle vector-based mutation systems for investigating site-specific mutagenicity of carcinogens and the in vivo transgenic rodent mutation systems for exploring DNA damaging and mutagenic properties of carcinogens. We present examples of application of each of these methodologies to human cancer etiology, and provide prospective views on investigations using these technologies for carcinogenicity testing.

Investigating DNA adduct-targeted mutagenicity of tamoxifen: preferential formation of tamoxifen-DNA adducts in the human p53 gene in SV40 immortalized hepatocytes but not endometrial carcinoma cells.

Abstract: Tamoxifen is a widely used drug for chemotherapy and chemoprevention of breast cancer worldwide. Tamoxifen therapy is, however, associated with an increased incidence of endometrial cancer. The carcinogenicity of tamoxifen is ascribed to its genotoxic and estrogen agonist effects. We investigated DNA adduct-targeted mutagenicity of tamoxifen as a function of its genotoxicity in the cII transgene in Big Blue mouse embryonic fibroblasts and mapped the formation of tamoxifen-induced DNA adducts in the p53 tumor suppressor gene in SV40 immortalized human hepatocytes and human endometrial carcinoma cells. We used the terminal transferase-dependent polymerase chain reaction for mapping of DNA adducts in the cII and p53 genes. We utilized a lambda phage-based assay and DNA sequencing for determining cII mutant frequency and mutation spectrum, respectively. Tamoxifen treatment yielded polymerase-blocking DNA adducts at multiple nucleotide positions along the cII transgene. The treatment significantly and dose-dependently increased the cII mutant frequency (p T:A transversions (p < 0.03), relative to the control. Tamoxifen treatment of the immortalized human hepatocytes but not endometrial carcinoma cells, even in the presence of an external activation system, i.e., rat liver S9 mix, induced DNA adducts at specific codons along exons 6 and 8 of the p53 gene. These data suggest a proficient metabolic activation of tamoxifen in human liver and an inefficient activation and/or efficient detoxification of tamoxifen in human endometrium. Because the liver is essentially a mitotically quiescent organ, tamoxifen-DNA adduction in the liver may, at least partially, prevent its reactants from reaching highly proliferative organs via, e.g., circulating blood. Thus, tamoxifen-DNA adduction in the liver may not have as significant biological consequences as it might have in highly proliferative organs. Our findings favor an involvement of a nongenotoxic mechanism in tamoxifen-associated human endometrial cancer.

Investigating human cancer etiology by DNA lesion footprinting and

Abstract: Many genotoxic carcinogens are known to leave unique signatures on cancer-related genes. The signature of carcinogens is manifested by the induction of characteristic mutations at distinctive nucleotide positions along oncogenes and/or tumor suppressor genes. Often, the nucleotide positions, wherein mutations occur, co-localize with the sites of initial DNA damage induced by the respective carcinogens. Thus, DNA damage-targeted mutation can be a predictor of carcinogenicity of genotoxins. Today, genomic sequencing technologies for investigating human cancer etiology are based on DNA-lesion footprinting in conjunction with mutagenicity analysis of genotoxic carcinogens. In this review article, we discuss the ligation-mediated PCR and terminal transferase-dependent-PCR, two versatile DNA-lesion footprinting techniques. We highlight the in vitro shuttle vector-based mutation systems for investigating site-specific mutagenicity of carcinogens and the in vivo transgenic rodent mutation systems for exploring DNA damaging and mutagenic properties of carcinogens. We present examples of application of each of these methodologies to human cancer etiology, and provide prospective views on investigations using these technologies for carcinogenicity testing.