VI.6 Detection of DNA adducts in the human endometrium: a lack of evidence

Carmichael, Paul L.; Sardar, S.; Neven, Patrick; Hoof, I. Van; Ugwumadu, Austin; Bourne, Tom; Tomás, Eija; Hellberg, Pår; Hewer, Alan; Phillips, David H.

doi:10.1016/s0959-8049(98)00124-5

Cited by 4 publications

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“…This idea gains support from our observation that even the adding of an external activating system did not appreciably influence DNA adduction in the human endometrial carcinoma cells (Figure 6). Our overall findings confirm the previously determined tamoxifen-DNA adducts in the genomic DNA of the liver (24) and the nondetectability or equivocal detectability of these adducts in the genomic DNA of the endometrium (16,17,(25)(26)(27) from tamoxifen-treated patients.…”

Section: Discussionsupporting

confidence: 89%

“…The widely utilized 32 P-postlabelingbased assays have used dissimilar digestion of DNA and variable adduct enrichment and chromatographic procedures in different studies. Additionally, incomparable phosphorylation efficiencies as well as the use (versus not using) of authentic DNA adduct standards have contributed to this uncertainty (16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Regardless of these discrepancies, a common feature of all applied assays is the detection of tamoxifen-DNA adducts in the overall genome but not at the level of nucleotide resolution.…”

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confidence: 99%

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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

Besaratinia

Pfeifer

2005

Biochemistry

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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 < 0.01), leaving a unique mutation spectrum (p < 0.0001) and a signature mutation of G:C --> 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.

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Section: Discussionsupporting

confidence: 89%

mentioning

confidence: 99%

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

Besaratinia

Pfeifer

2005

Biochemistry

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“…Furthermore, binding of [ 14 C]tamoxifen to endometrial DNA has been demonstrated by accelerator mass spectrometry, following treatment of women with a single 14 C-labeled therapeutic dose (10), which highlights the need to understand the consequences of tamoxifen DNA adduct formation in human cells. This is however a contentious issue because other investigators have failed to find evidence of tamoxifen DNA adduct formation in endometrial tissue of treated patients, using both 32 P-postlabeling (11,12) and electrospray mass spectrometry analysis (13). Reported differences in the level of tamoxifen adducts in uterine samples from women receiving tamoxifen, particularly an absence of detectable adducts in half of the patients examined in one study (8), have been suggested as being due to individual variability in the extent of metabolic activation or DNA repair capacity.…”

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confidence: 99%

Mutation Spectra Induced by α-Acetoxytamoxifen−DNA Adducts in Human DNA Repair Proficient and Deficient (Xeroderma Pigmentosum Complementation Group A) Cells

et al. 2005

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Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.

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“…Among the diastereoisomers, a trans form (fr-2) of dG-N 2 -TAM and dG-N 2 -N-desTAM were detected as a major TAM-DNA adduct in rodents (11)(12)(13) and monkeys (14,15). TAM-DNA adducts were also detected in the endometrium of women treated with TAM (16)(17)(18), although there is some contradiction regarding the detection of TAM-DNA adducts in human tissues (19)(20)(21)(22). A high frequency of K-ras mutations was recently observed in the endometrium of women treated with TAM (23); the mutational specificity was consistent with that occurring at the dG-N 2 -TAM adduct (24).…”

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confidence: 98%

Translesion Synthesis Past Tamoxifen-Derived DNA Adducts by Human DNA Polymerases η and κ

et al. 2006

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The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.

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VI.6 Detection of DNA adducts in the human endometrium: a lack of evidence

Cited by 4 publications

References 0 publications

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

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

Mutation Spectra Induced by α-Acetoxytamoxifen−DNA Adducts in Human DNA Repair Proficient and Deficient (Xeroderma Pigmentosum Complementation Group A) Cells

Translesion Synthesis Past Tamoxifen-Derived DNA Adducts by Human DNA Polymerases η and κ

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