Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

Comparative study of nucleotide excision repair defects between XPD-mutated fibroblasts derived from trichothiodystrophy and xeroderma pigmentosum patients.

To get a clue to understand how mutations in the XPD gene result in different skin cancer susceptibilities in patients with xeroderma pigmentosum (XP) or trichothiodystrophy (TTD), a thorough understanding of their nucleotide excision repair (NER) defects is essential. Here, we extensively characterize the possible causes of NER defects in XP-D and in TTD fibroblasts. The 3 XP-D cell strains examined were similarly deficient in repairing UV-induced cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs) from genomic DNA. The severity of NER defects correlated with their UV sensitivities. Possible alterations of TFIIH (which consists of 10 subunits including XPD) were then examined. All XP-D cell strains were normal in their concentrations of TFIIH, and displayed normal abilities to recruit TFIIH to sites of UV-induced DNA damage. However, replication protein A (RPA; single-stranded DNA binding protein) accumulation at DNA damage sites, which probably reflects the in vivo XPD helicase activity of TFIIH, is similarly impaired in all XP-D cell strains. Meanwhile, all 3 TTD cell strains had approximately 50% decreases in cellular TFIIH content. Importantly, 2 of the 3 TTD cell strains, which carry the major XPD mutations found in TTD patients, showed defective recruitment of TFIIH to DNA damage sites. Moreover, RPA accumulation at damage sites was impaired in all TTD cell strains to different degrees, which correlated with the severity of their NER defects. These results demonstrate that XP-D and TTD cells are both deficient in the repair of CPDs and 6-4PPs, but TTD cells have more multiple causes for their NER defects than do XP-D cells. Since TFIIH is a repair/transcription factor, TTD-specific alterations of TFIIH possibly result in transcriptional defects, which might be implication for the lack of increased incidence of skin cancers in TTD patients.

Carotid atherosclerosis is associated with brain atrophy in Japanese elders.

BACKGROUND: The relation between atherosclerosis and brain atrophy remains unclear in patients with risk factors for cardiovascular diseases. OBJECTIVE: This study was performed to clarify the relation between brain atrophy and carotid atherosclerosis. METHODS: A total of 142 patients (78 women and 64 men, mean age 74 years) with no neurologic disturbances were studied. Brain atrophy was evaluated on the basis of the brain atrophy index (BAI, BAI = brain parenchyma/intracranial space A 100%), calculated by means of digitized computed tomographic scans obtained at the level of the basal ganglia. Carotid atherosclerosis was evaluated on the basis of the plaque score (PS), defined as the sum of all plaque heights in both carotid arteries, intima-media thickness (IMT), and vessel diameter (VD) of the common carotid artery as assessed by ultrasonography. RESULTS: Age negatively correlated with BAI in both men (r = -0.587, p < 0.001) and women (r = -0.724, p < 0.001). PS of the carotid artery also negatively correlated with BAI in men (r = -0.502, p < 0.001) as well as women (r = -0.480, p < 0.001). VD and IMT of the right carotid artery negatively correlated with BAI in women (VD; -0.256, p < 0.05, IMT; -0.216, p < 0.05) but not in men. Other characteristics were unrelated to BAI. Multiple regression analysis showed that age and PS were independent predictors of brain atrophy in both sexes. The percentage of variance of BAI values explained by this model in women (51.9%) was much greater than that in men (35.5%). CONCLUSION: Carotid atherosclerosis may be a useful morphological index of brain atrophy.