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Hydroxyl radical damage in metastatic tumor DNA was elucidated in women with breast cancer, and a comparison was made with nonmetastatic tumor DNA. The damage was identified by using statistical models of modified base and Fourier transform-infrared spectral data. The modified base models revealed a greater than 2-fold increase in hydroxyl radical damage in the metastatic tumor DNA compared with the nonmetastatic tumor DNA. The metastatic tumor DNA also exhibited substantially greater base diversity than the nonmetastatic DNA, and a progression of radicalinduced base damage was found to be associated with the growth of metastatic tumors. A three-dimensional plot of principal components from factor analysis, derived from infrared spectral data, also showed that the metastatic tumor DNA was substantially more diverse than the tightly grouped nonmetastatic tumor DNA. These cohesive, independently derived findings suggest that the hydroxyl radical generates DNA phenotypes with various metastatic potentials that likely contribute to the diverse physiological properties and heterogeneity characteristic of metastatic cell populations.

Background. Substantial hydroxyl radical (.OH)‐induced base lesions, recently found in the DNA of invasive ductal carcinoma of the female breast, are likely to be intimately related to oncogenesis. However, virtually no information was available regarding relationships between the different base lesions in the normal and cancerous breast. Such information is essential in understanding initial stages in the development of breast cancer and the potential of the base lesions as early predictors of cancer risk. Methods. The OH‐induced DNA base lesions in normal reduction mammoplasty tissue (RMT) were compared with those from invasive ductal carcinoma (IDC) and nearby microscopically normal tissue (MNT). Comparisons were then undertaken on relationships between the base lesion profiles in the normal and cancerous breast using 22 statistical models. Results. DNA from the RMT was characterized by a high ratio of ring‐opening products (e.g., 4,6‐diamino‐5‐formamidopyrimidine) to hydroxy‐adducts of adenine and guanine. A dramatic shift in this relationship in favor of carcinogenic hydroxy‐adducts (e.g., 8‐hydroxyguanine) was found in the cancerous breast. Statistical models with a high sensitivity (91%) and specificity (97%) provided a consistent means of classifying tissues (e.g., 96% correct). Conclusions. The dramatic shift in the DNA base lesion relationships in oncogenesis is attributed to alterations in the redox potential of the breast favoring oxidative conditions and cancer formation. These findings suggest that base lesion profiles are potential sentinels for cancer risk assessment. Further, intervention in controlling the tissue redox potential may provide benefit in delaying or preventing early oncogenic changes and the ultimate manifestation of cancer.

Background. The authors previously have shown by gas chromatography‐mass spectrometry that the hydroxyl radical (.OH) induces alterations in the DNA base structure of the female breast, which are premalignant markers of breast cancer. Fourier transform‐infrared (FT‐IR) spectroscopy also has a high potential for revealing a broad array of structural changes in DNA that may provide important new insight into breast cancer etiology and prediction. Methods. DNA from normal reduction mammoplasty tissue, invasive ductal carcinoma, and nearby microscopically normal tissue was analyzed by FT‐IR spectroscopy. Statistical models based on DNA spectral properties were developed and compared with a statistical model previously used with base modifications. Results. Substantial differences were found in the spectral properties of DNA from women with normal and cancerous breast tissue, indicating an ability to discriminate cancerous tissue from noncancerous tissue with a sensitivity and specificity of 83%. Most importantly, the normal population was divided into subgroups in which a nonrandom progression was identified and a cancer‐like DNA phenotype that was highly correlated (r ⩾ 0.90) with that of the patients with cancer was exhibited in 59% of the women. The spectral data, which also were highly correlated with the base‐model data, were used to establish a model for predicting the probability of breast cancer. Consistent with the high cancer reoccurrence rate in the ipsilateral breast, 8 of 10 of the microscopically normal tissue specimens remaining after tumor excision were classified as cancerous using this model. Conclusions. Progressive structural changes in the DNA of the normal female breast, leading to a premalignant cancer‐like phenotype in a high proportion of women, are the basis for a new paradigm for understanding the etiology of breast cancer and predicting its occurrence at early stages of oncogenesis. The results also suggest therapeutic strategies for potentially reversing the extent of DNA damage, which may be useful in disease prevention and treatment. Cancer 1995;75:503‐17.

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Wavenumber-absorbance relationships of infrared spectra of DNA analyzed by principal components analysis may be expressed as points in space. Each point represents a highly discriminating measure of DNA structure. Structural modifications of DNA, such as those induced by free radicals, alter vibrational and rotational motion and consequently change the spatial location of the points. Using this technology to analyze breast tumor DNA, we revealed a 94؇ difference in direction between the progression of normal DNA 3 primary tumor DNA and the progression of primary tumor DNA 3 metastatic tumor DNA (P < 0.001). This sharp directional change was accompanied by a substantial increase in the structural diversity of the metastatic tumor DNA (P ‫؍‬ 0.003), which, on the basis of the volume of the core cluster of points, could comprise as many as 11 ؋ 10 9 different phenotypes. This suggests that the heterogeneity and varied physiological properties known to characterize malignant tumor cell populations may at least partially arise from these diverse phenotypes. The evidence suggests that the progression to the metastatic state involves structural modifications in DNA that are markedly different from the modifications associated with the formation of the primary tumor. Overall, the findings of this and earlier studies imply that the observed DNA alterations are a pivotal factor in the etiology of breast cancer and a formidable barrier to overcome in intervention to control the disease. In terms of cancer etiology and prediction, the technology described has potentially wide application to studies in which the structural status of DNA is an important consideration.A significant body of evidence points to the involvement of the hydroxyl radical (⅐OH) in introducing mutagenic structures into DNA of the normal female breast, thus statistically increasing the probability of breast cancer (1-5). In a recent study (5), the transformation of primary breast tumors to the metastatic state was shown to involve a Ͼ2-fold increase in ⅐OH damage in DNA, as indicated by modified nucleotide base models comprising mutagenic 8-hydroxyadenine (6) and the putatively nonmutagenic ring-opened product 4,6-diamino-5-formamidopyrimidine (fapyadenine; refs. 7-11). In addition, plots of the modified nucleotide base model log 10 (fapyadenine͞8-hydroxyadenine) versus the size of metastatic and nonmetastatic breast tumors revealed that the metastatic tumor DNA had significantly greater structural diversity than the nonmetastatic tumor DNA (P ϭ 0.01; ref. 5).Principal components analysis (PCA) of data obtained by Fourier transform-infrared (FT-IR) spectroscopy yielded plots in which individual spectra were represented as points in twoor three-dimensional space. Each point was a highly discriminating representation of an individual DNA structure in that spatially and visually close points had Ͻ3% average spectral difference over the range 1750-700 cm Ϫ1 (5). The core cluster of metastatic tumor DNA points was substantially larger than the core c...

Environmental chemicals are known to induce a high degree of hydroxyl radical-mediated damage in DNA. Accordingly, we tested the hypothesis that this exposure leads to new forms of DNA using principal components analysis of Fourier transform infrared spectra. The hepatic DNA of English sole (controls) from an essentially clean environment was compared with that of sole inhabiting a chemically contaminated environment. All livers studied were cancer-free; however, a high incidence of liver cancer has been found in the exposed population. The exposed sole were sampled twice, 2 years apart, while the sediments in which they live were under remediation. After obtaining infrared spectra, the first three principal components (PC1, PC2, and PC3) were calculated and found to represent 97% of the total spectral variance. When the principal component scores were plotted in 3-dimensional space, clusters of points were obtained that represented the DNA from the control and exposed groups. Each of the points was derived from Ϸ10 6 wavenumber-absorbance correlations. The spatial location of a point was a highly discriminating measure of DNA structure. The clusters of points were completely separated, demonstrating that the three groups could be 100% correctly classified. The points from the control group were tightly clustered whereas those from the exposed groups were highly diverse. The findings demonstrate that exposure to environmental chemicals results in new, structurally diverse forms of DNA that likely play an important role in carcinogenesis.English sole exposed to environmental chemicals in their natural habitat (Duwamish River, Seattle, WA) have been shown to have a remarkably high degree of base damage resulting from the attack of the hydroxyl radical (⅐OH) on DNA (1). The base damage included substantial increases in mutagenic hydroxy derivatives [8-hydroxyadenine (8-OHAde) and 8-hydroxyguanine (8-OH-Gua)] (2, 3) and ringopened products [e.g., 2,6-diamino-4-hydroxy-5-formamidopyrimidine] compared with controls. In another study of English sole from the Duwamish River (4), positive correlations were found between 8-OH-Ade and 8-OH-Gua and incidences of a variety of nonneoplastic hepatic lesions. These included basophilic foci (a putatively preneoplastic lesion) and megalocytic hepatosis (a putatively degenerative or cytotoxic lesion) (5, 6). The ⅐OH was likely a common etiological factor in the formation of the base modifications and the hepatic lesions, which is consistent with its proposed role in tumor formation (7)(8)(9)(10)(11). The sole population from the Duwamish River was shown to have a high incidence of liver cancer; however, this condition rarely occurs in fish from uncontaminated environments (5, 6).The ⅐OH is believed to arise from H 2 O 2 via the Fe 2ϩ -mediated Fenton reaction (12). H 2 O 2 , which readily crosses the nuclear membrane, is likely produced via redox cycling of sediment contaminants, such as polynuclear aromatic hydrocarbons and chlorinated pesticides, to which the fish are ex...

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