Age-related radical-induced DNA damage is linked to prostate cancer.

We measured concentrations and ratios of mutagenic (8-OH) lesions to putatively nonmutagenic formamidopyrimidine (Fapy) lesions of adenine (Ade) and guanine (Gua) to elucidate radical (.OH)-induced changes in DNA of normal, normal from cancer, and cancer tissues of the prostate. The relationship between the lesions was expressed using the mathematical model log(10)[(8-OH-Ade + 8-OH-Gua)/(FapyAde + FapyGua)]. Logistic regression analysis of the log ratios for DNA of normal and cancer tissues discriminated between the two tissue groups with high sensitivity and specificity. Correlation analysis of log ratios for normal prostates revealed a highly significant increase in the proportion of mutagenic base lesions with age. Data from correlation analysis of the log ratios for normal tissues from cancer were consistent with an age-dependent, dose-response relationship. The slopes for both correlations intersected at approximately 61 years, an age when prostate cancer incidence is known to rise sharply. The age-related increase in the proportion of.OH-induced mutagenic base lesions is likely a significant factor in prostate cancer development.

Single 8-oxo-guanine and 8-oxo-adenine lesions induce marked changes in the backbone structure of a 25-base DNA strand.

Structural changes in a 25-base DNA strand, induced by single 8-oxo-guanine or 8-oxo-adenine substitutions, were shown by using Fourier transform-infrared spectroscopy with multivariate statistics. Pronounced differences were demonstrated between the parent and derivatives with respect to base interactions and changes in the phospho-deoxyribose backbone. The greatest degree of change in the backbone likely occurred immediately adjacent to the 8-oxo group, potentially altering the stereochemistry at a distance. The 8-oxo lesions, formed from reactive oxygen species (e.g., hydroxyl radicals), may appreciably alter the conformational properties of strands at the replication fork, thus affecting the selectivity of polymerases, the proofreading capability of repair enzymes, and the fidelity of the transcriptional machinery.

Factors critical to the design and execution of epidemiologic studies and description of an innovative technology to follow the progression from normal to cancer tissue.

The results obtained from experimental studies of estrogen carcinogenesis need validation in epidemiologic studies. Such studies present additional challenges, however, because variations in human populations are much greater than those in experimental systems and in animal models. Because epidemiologic studies are often used to evaluate modest differences in risk factors, it is essential to minimize sources of errors and to maximize sensitivity, reproducibility, and specificity. In the first part of this chapter, critical factors in designing and executing epidemiologic studies, as well as the influence of sample collection, processing, and storage on data reliability, are discussed. One of the most important requirements is attaining sufficient statistical power to assess small genetic effects and to evaluate interactions between genetic and environmental factors. The second part of this chapter describes innovative technology, namely, Fourier transform-infrared (FT-IR) spectra of DNA that reveal major structural differences at various stages of the progression from normal to cancer tissue. The structural differences become evident from wavenumber-by-wavenumber statistical comparisons of the mean FT-IR spectra of DNA from normal to cancer tissues. This analysis has allowed distinguishing benign tissues from cancer and metastatic tissues in human breast, prostate, and ovarian cancers. This analysis, which requires less than 1 microg of DNA, is predicted to be used for detecting early cancer-related changes at the level of DNA, rather than at the cellular level.

A unified theory of carcinogenesis based on order-disorder transitions in DNA structure as studied in the human ovary and breast.

Fourier transform-infrared/statistics models demonstrate that the malignant transformation of morphologically normal human ovarian and breast tissues involves the creation of a high degree of structural modification (disorder) in DNA, before restoration of order in distant metastases. Order-disorder transitions were revealed by methods including principal components analysis of infrared spectra in which DNA samples were represented by points in two-dimensional space. Differences between the geometric sizes of clusters of points and between their locations revealed the magnitude of the order-disorder transitions. Infrared spectra provided evidence for the types of structural changes involved. Normal ovarian DNAs formed a tight cluster comparable to that of normal human blood leukocytes. The DNAs of ovarian primary carcinomas, including those that had given rise to metastases, had a high degree of disorder, whereas the DNAs of distant metastases from ovarian carcinomas were relatively ordered. However, the spectra of the metastases were more diverse than those of normal ovarian DNAs in regions assigned to base vibrations, implying increased genetic changes. DNAs of normal female breasts were substantially disordered (e.g., compared with the human blood leukocytes) as were those of the primary carcinomas, whether or not they had metastasized. The DNAs of distant breast cancer metastases were relatively ordered. These findings evoke a unified theory of carcinogenesis in which the creation of disorder in the DNA structure is an obligatory process followed by the selection of ordered, mutated DNA forms that ultimately give rise to metastases.

Infrared spectral models demonstrate that exposure to environmental chemicals leads to new forms of DNA.

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 approximately 10(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.

Models of DNA structure achieve almost perfect discrimination between normal prostate, benign prostatic hyperplasia (BPH), and adenocarcinoma and have a high potential for predicting BPH and prostate cancer.

In our previous studies of DNA, wavenumber-absorbance relationships of infrared spectra analyzed by principal components analysis (PCA) were expressed as points in space. Each point represented a highly discriminating measure of structural modifications that altered vibrational and rotational motion, thus changing the spatial orientation of the points. PCA/Fourier transform-infrared technology has now provided a virtually perfect separation of clusters of points representing DNA from normal prostate tissue, BPH, and adenocarcinoma. The findings suggest that the progression of normal prostate tissue to BPH and to prostate cancer involves structural alterations in DNA that are distinctly different. The hydroxyl radical is likely a major contributor to these structural alterations, which is consistent with previous studies of breast cancer. Models based on logistic regression of infrared spectral data were used to calculate the probability of a tissue being BPH or adenocarcinoma. The models had a sensitivity and specificity of 100% for classifying normal vs. cancer and normal vs. BPH, and close to 100% for BPH vs. cancer. Thus, the PCA/Fourier transform-infrared technology was shown to be a powerful means for discriminating between normal prostate tissue, BPH and prostate cancer and has considerable promise for risk prediction and clinical application.

Mutagenic DNA base modifications are correlated with lesions in nonneoplastic hepatic tissue of the English sole carcinogenesis model.

Hydroxyl radical-induced mutagenic base modifications have been linked to neoplasia in a number of biological systems, including English sole from chemically contaminated urban environments. However, virtually no information exists on the relationship between the mutagenic base modifications and preneoplastic and other lesions found in tumor-free tissues prone to cancer. We studied six hepatic lesions in immature, neoplasm-free English sole exposed to an urban and reference environment and established correlations between the lesion incidence and concentrations of the mutagenic base modifications 8-hydroxyguanine and 8-hydroxyadenine. The lesions were putatively preneoplastic basophilic foci, hepatocellular karyomegaly, megalocytic hepatosis, hepatocellular vacuolar change, hyalin droplet formation, and apoptosis. With the exception of hepatocellular vacuolar change, significant positive correlations were found between the lesions and the mutagenic base modifications. The hydroxyl radical may be a common etiological factor in the formation of the base modifications and hepatic lesions.

Tumor progression to the metastatic state involves structural modifications in DNA markedly different from those associated with primary tumor formation.

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 degrees difference in direction between the progression of normal DNA-->primary tumor DNA and the progression of primary tumor DNA-->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 x 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 alter-ations 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.

Progression of human breast cancers to the metastatic state is linked to hydroxyl radical-induced DNA damage.

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 radical-induced 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.

The etiology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals progressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion of normal women.

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 > or = 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.

Fourier-transform infrared spectroscopy and gas chromatography-mass spectrometry reveal a remarkable degree of structural damage in the DNA of wild fish exposed to toxic chemicals.

The use of gas chromatography-mass spectrometry with selected ion monitoring (GC-MS/SIM) and Fourier-transform infrared (FT-IR) spectroscopy revealed a remarkable degree of damage in the hepatic DNA of fish exposed to toxic environmental chemicals, compared with controls. The exposed fish, which were neoplasm-free, were part of a population with a high incidence of liver cancer. GC-MS/SIM showed markedly high concentrations of hydroxyl radical-induced ring-opening products (e.g., 2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-hydroxy adducts of adenine and guanine (e.g., 8-hydroxyguanine) in the DNA. FT-IR spectroscopy revealed substantial changes in spectral areas, such as those assigned to NH vibrations of nucleotide bases and CO vibrations of deoxyribose. This diverse and extensive damage to DNA provides a perspective of premalignant changes resulting from xenobiotic exposure and a promising basis for predicting cancer risk in animals and humans.

Identification of hydroxyl radical-induced lesions in DNA base structure: biomarkers with a putative link to cancer development.

Hydroxyl radical-induced DNA base lesions of guanine and adenine were originally found in neoplastic and microscopically normal livers of fish exposed to environmental carcinogens. They were later identified in a mammalian tissue--the cancerous female breast. This evidence suggested that the base lesions are broadly present in the cancerous and microscopically normal tissues of a variety of eukaryotic organisms. The base lesion concentrations in both neoplastic tissues frequently exceeded 1 modified base in 1000 normal bases. By contrast, the base lesion:normal base ratios in healthy tissues were generally 10-100 times less. A greater variety of base lesions was found subsequently in the cancerous lung, brain, and other human tissues, although information relating to their biological significance is largely confined to the originally found purine derivatives. The biochemistry of the base lesions and the relationship of ring-opening (Fapy) derivatives to OH adducts in the DNA of normal and cancerous tissues is discussed with regard to the etiology of cancer and the potential use of the lesions as biomarkers for cancer risk assessment.

The etiology of breast cancer. Characteristic alteration in hydroxyl radical-induced DNA base lesions during oncogenesis with potential for evaluating incidence risk.

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.

Major alterations in the nucleotide structure of DNA in cancer of the female breast.

DNA of invasive ductal carcinomas from five women was analyzed for structural alterations in the purine nucleotides using gas chromatography-mass spectrometry with selected ion monitoring. The results were compared to those for a normal DNA control. The carcinoma DNA showed dramatically higher concentrations of the base modifications 8-hydroxy-guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 8-hydroxyadenine. For example, the concentration of total identified base modifications represented a more than 9-fold increase over the control value. Base modifications of this type, which arise from radical-induced hydroxylation and cleavage reactions of the purine ring, likely play a major role in initiation and probably contribute to the further transformation of neoplastic cells in cancer of the female breast.

4,6-Diamino-5-formamidopyrimidine, 8-hydroxyguanine and 8-hydroxyadenine in DNA from neoplastic liver of English sole exposed to carcinogens.

Attack of the hydroxyl radical on the 8-carbon of guanine results in the formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). This modified nucleotide was recently found in neoplastic livers of wild English sole exposed to aromatic hydrocarbons (Malins et al., Carcinogenesis, 11, 1045-1047, 1990). Gas-chromatographic-mass spectrometric evidence reported here indicates that three other modified nucleotides are also present in the neoplastic liver: 4,6-diamino-5-formamidopyrimidine, 8-hydroxyguanine and 8-hydroxyadenine. These findings support the hypothesis that the purine nucleotides were modified by the hydroxyl radical and that these changes play a crucial role in tumor formation.

A novel DNA lesion in neoplastic livers of feral fish: 2,6-diamino-4-hydroxy-5-formamidopyrimidine.

DNA from neoplastic hepatic tissues of feral fish environmentally exposed to carcinogens was shown to contain the guanine-derived lesion 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). This modification of DNA appears to arise from the attack of the hydroxyl radical at C-8 of guanine. Concentrations of the lesion ranged from 0.97 to 5.11 nmol/mg DNA. FapyGua was not detected in nonneoplastic tissue and has not been previously reported to occur in the DNA of animal tissues. The present findings support the view that reactive oxygen species damage DNA in living systems and thus may play an important role in the formation of neoplastic tissues.