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Reciprocal interactions between the stroma and epithelium are considered to be intimately associated with the development of breast cancer. In studies of whole breast tissues, a keen interest exists in the occurrence of the mutagenic DNA lesions 8-hydroxy-2'-deoxyguanosine and 8-hydroxy-2'-deoxyadenosine. However, there is an apparent lack of information on the presence of these lesions in the DNA of the stroma, epithelium, and myoepithelium, despite the fact that these oxidation products may significantly influence reciprocal interactions between these cell types implicated in carcinogenesis. We report age-related increases in concentrations of both lesions in the stromal DNA, which occur roughly commensurate with the known rise in breast cancer incidence between 30 and 40 years of age. However, no further increases in these concentrations occurred in the older women. Plots of lesion concentrations revealed an uneven distribution, with some younger women having relatively high concentrations and some older women having relatively low concentrations. This finding implies that while increased age is a probable factor in lesion accumulations, other factors may also be influential [e.g., cellular concentrations of reactive oxygen species (ROS)]. Distinct differences were found between the base and backbone structures of the stromal DNA from younger women (ages 17 - 30), compared to older women (ages 50 - 62). In addition, comparisons of matched stromal, epithelial, and myoepithelial DNA (from the same individual) showed no differences in DNA damage, suggesting a random attack by the hydroxyl radical on all three groups. Collectively, the findings imply that the structural changes in DNA described may potentially disrupt normal reciprocal interactions between the cell types, thus increasing breast cancer risk.

(5'S)-8,5'-Cyclo-2'-deoxyadenosine (S-cdA), which arises from the reaction of the hydroxyl radical (*OH) with 2'-deoxyadenosine in DNA, is a lesion comprising a base-sugar linkage that distorts the DNA backbone. This structure impedes transcription and blocks polymerase action. Further, a single S-cdA lesion in the TATA box reduces gene expression. Considering the ability of S-cdA to disrupt DNA structure, which is likely associated with increased cancer risk, we determined S-cdA concentrations in the DNA of stroma, epithelium, and myoepithelium from normal breast tissues using liquid chromatography/mass spectrometry (LC/MS). We also identified differences in the base and backbone structures using Fourier transform-infrared (FT-IR) spectroscopy. LC/MS revealed that the lowest concentration of S-cdA in the stroma (0.04 +/- 0.02 lesions/10(6) bases) occurred in women ages 17 to 30. The highest concentration (0.13 +/- 0.07 lesions/10(6) bases) was found in women 33 to 46. FT-IR spectroscopy showed significant base and backbone differences in the stromal DNA between the women under 30 and those over 50. These findings imply that distortions in the geometry of the helix increase with age, reaching significant proportions in older women. No differences were found in the S-cdA concentrations between the three cell types, suggesting that the *OH attack on the base structure may be essentially random. Initial insight is provided on changes in DNA structure that potentially affect gene expression and increase breast cancer risk.

Using the carcinogen 3-methylcholanthrene (MCA), we demonstrate with Fourier transform-infrared spectroscopy that a cancer DNA phenotype is produced well in advance of palpable tumors. We further demonstrate that the administration of cyclophosphamide markedly inhibits the development of the cancer phenotype and concomitantly delays tumor formation. MCA, injected into the hind legs of mice, produced a variety of significant structural changes in the nucleotide bases and phosphodiester-deoxyribose backbone, as reflected in a substantial (34%) difference between the mean DNA spectra of the control and the MCA-injected mice. Strikingly, 57 days before the mean appearance of tumors, we could not distinguish the DNA structure of the histologically normal tissues of the MCA-injected mice from the DNA structure of the tumor tissues. This finding indicates the early development of a cancer phenotype. Confirmatory evidence was obtained when tissues from a group of mice injected with both MCA and cyclophosphamide did not manifest the cancer phenotype, and their mean DNA structure closely resembled that of the control mice. Accordingly, we propose that the cancer DNA phenotype, as evinced by Fourier transform-infrared spectroscopy, is a promising early indicator of tumor formation, and we postulate that agents capable of inhibiting this phenotype may delay or prevent carcinogenesis.

We have used statistical models based on Fourier transform-infrared spectra to differentiate between the DNA structure of normal granulocytes and those obtained from patients with myelodysplastic syndrome (MDS). The substantial degree of discrimination achieved between the two DNA groups is attributed to differences in the nucleotide base and backbone structures. These structural differences allowed for the development of a discriminant analysis model that predicted, with high sensitivity and specificity, which DNA came from normal granulocytes vs. granulocytes from MDS patients. The findings are a promising basis for developing a blood test to diagnose and predict the occurrence of MDS, for which there is currently a paucity of molecular markers.

Structural differences were identified in gill DNA from two groups of English sole collected from Puget Sound, Washington, in October 2000. One group was from the industrialized Duwamish River (DR) in Seattle and the other from relatively clean Quartermaster Harbor (QMH). Chemical markers of sediment contamination [e.g., polynuclear aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)] established that the DR was substantially more contaminated than QMH. The levels of these chemicals in the sediments of both sites were consistent with levels of cytochrome P450 1A (CYP1A) expression in the gills of English sole from the same sites. Structural differences in gill DNA between the groups were evinced via statistical models of Fourier transform-infrared (FT-IR) spectra. Marked structural damage was found in the gill DNA of the DR fish as reflected in differences in base functional groups (e.g., C-O and NH2) and conformational properties (e.g., arising from perturbations in vertical base stacking interactions). These DNA differences were used to discriminate between the two fish groups through principal components analysis of mean FT-IR spectra. In addition, logistic regression analysis allowed for the development of a "DNA damage index" to assess the effects of contaminants on the gill. The evidence implies that environmental chemicals contribute to the DNA changes in the gill. The damaged DNA is a promising marker for identifying, through gill biopsies, contaminant effects on fish.

Using statistical analyses of Fourier transform-IR spectra, we show that DNA of the histologically normal prostates of men 16-80 years old undergoes structural changes in the bases and backbone with increasing age. Of the older men (ages 55-80), 42% exhibited a DNA phenotype mimicking that of primary prostate tumors from a comparable age group. This cancer-like phenotype, which was not found in the younger men (ages 16-36), appears to arise from progressive age-related damage to DNA. The mean concentrations of 8-hydroxypurine lesions (e.g., 8-hydroxyguanine) were substantially higher for the older men than for the younger men. This finding suggests that the hydroxyl radical contributed to the structural changes that characterize the cancer-like phenotype. Strikingly, we were additionally able to discriminate between the DNA of primary prostate tumors and the DNA of primary prostate tumors from which distant metastases had been identified. Moreover, logistic regression analysis was able to predict the probability that a tumor had metastasized with approximately 90% sensitivity and specificity. Collectively, these findings are particularly promising for identifying men at risk for developing prostate cancer, as well as for the early determination of whether a primary tumor has progressed to the metastatic state. This is highly important because the prognosis of histologically similar prostate carcinomas varies, thus creating a need to predict which cancers are most likely metastatic.

N-acetylcysteine (NAC), a strong antioxidant, has antigenotoxic and anticarcinogenic properties currently being investigated in clinical trials. NAC detoxifies free radicals (e.g., the hydroxyl radical,.OH) that cause DNA changes implicated in disease (e.g., cancer). The.OH reacts with purines to form mutagenic 8-hydroxypurine (8-OH) and putatively nonmutagenic formamidopyrimidine (Fapy) lesions. Fapy lesions inhibit DNA synthesis likely modulating the mutagenic potential of the 8-OH lesions, which would suggest that the ratio of these oxidized bases is biologically important. However, little is known about how NAC modifies oxidized DNA structure or how such modifications may affect cellular processes, such as replication and transcription. By using gas chromatography-mass spectrometry and Fourier transform-infrared spectroscopy, we found that dietary NAC (5% in the diet for 14 days) affected.OH-induced structural changes in DNA of the hind leg of the BALB/c mouse. For example, mutagenic 8-hydroxyguanine (8-OH-Gua) was reduced approximately 50% (P = 0.02) in mice fed NAC compared with controls. NAC reduced the log(10) (8-OH-Gua/FapyGua) ratio from 0.58 +/- 0.15 to essentially zero, a virtually neutral redox status. DNA from control mice had a remarkably high variance compared with mice fed NAC. Moreover, the DNA from treated and control mice was distinct with respect to base structure and vertical base-stacking interactions. The findings showing that NAC lowered the concentration of 8-OH-Gua, the log ratio, and the variance (previously associated with neoplastic changes) suggest that NAC reduces the mutagenic potential of oxidized DNA. These benefits could be offset by the other structural changes found after NAC exposure, which may affect the fidelity of DNA synthesis.