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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.

Fish living in contaminated environments accumulate toxic chemicals in their tissues. Biomarkers are needed to identify the resulting health effects, particularly focusing on early changes at a subcellular level. We used a suite of complementary biomarkers to signal contaminant-induced changes in the DNA structure and cellular physiology of the livers and gills of English sole (Parophrys vetulus) . These sediment-dwelling fish were obtained from the industrialized lower Duwamish River (DR) in Seattle, Washington, and from Quartermaster Harbor (QMH) , a relatively clean reference site in south Puget Sound. Fourier transform-infrared (FT-IR) spectroscopy, liquid chromatography/mass spectrometry (LC/MS) , and gas chromatography/mass spectrometry (GC/MS) identified potentially deleterious alterations in the DNA structure of the DR fish livers and gills, compared with the QMH fish. Expression of CYP1A (a member of the cytochrome P450 multigene family of enzymes) signaled changes in the liver associated with the oxidation of organic xenobiotics, as previously found with the gill. The FT-IR models demonstrated that the liver DNA of the DR fish had a unique structure likely arising from exposure to environmental chemicals. Analysis by LC/MS and GC/MS showed higher concentrations of DNA base lesions in the liver DNA of the DR fish, suggesting that these base modifications contributed to this discrete DNA structure. A comparable analysis by LC/MS and GC/MS of base modifications provided similar results with the gill. The biomarkers described are highly promising for identifying contaminant-induced stresses in fish populations from polluted and reference sites and, in addition, for monitoring the progress of remedial actions.

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.

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 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.

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.

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.

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.

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.

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.

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.

High concentrations of toxic chemicals in sediment and white croaker (Genyonemus lineatus), as well as liver diseases (e.g., carcinomas) in this species, were found in the Los Angeles area. The highest concentrations of aromatic hydrocarbons (AHs) in the sediment were in San Pedro Bay, and the highest concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) derivatives were in sediment from near the White Point sewer outfall. Concentrations of AHs, polychlorobiphenyls (PCBs), and DDT derivatives were generally higher in food organisms from the croaker's stomach than in sediment. Moreover, croaker from San Pedro Bay and White Point were substantially contaminated with DDT derivatives and metabolites of aromatic compounds, compared to croaker from the Hyperion outfall and Dana Point. The evidence suggest that the observed pathological conditions of the liver were associated with exposure of the croaker to toxic chemicals, which occurred, at least in part, through the ingestion of contaminated food organisms.

Hydroxyl radical-induced DNA lesions were found in hepatic tumors of English sole exposed to environmental chemicals (Malins et al., Carcinogenesis, 11, 1045–1047, 1990; Biochem. Biophys. Res. Commun., 173, 614–619, 1990): they were 8-hydroxyguanine. 2.6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 8-hydroxyadenine and 4,6-diamino-5-formamidopyrimidine (FapyAde). Histologically normal livers from a tumor-bearing population have now been studied. With the exception of FapyAde, the DNA lesion concentrations in the livers were intermediate between those of the initially studied tumor-bearing fish and healthy fish from non-contaminated sites. Thus, the DNA lesions can occur prior to tumor formation, which is consistent with the concept that they are causally linked to tumorigenesis.

Epizootiological analyses of field data on prevalances of hepatic neoplasms and other hepatic lesions in English sole (Parophrys vetulus) from Puget Sound suggest a link between these lesions and sediment-associated xenobiotics, particularly aromatic hydrocarbons (AHs). To further investigate this relationship, English sole from a minimally contaminated site were parenterally exposed at monthly intervals for 1 yr to an organic solvent extract of a contaminated urban sediment containing high levels of AHs (30 or 75 mg/kg body weight) or to the model carcinogen benzo[a]pyrene (BaP, 12 mg/kg body weight). Control groups included English sole exposed to an extract of a relatively uncontaminated reference sediment, sole exposed to the carrier only, and untreated sole. Eighteen months after the initial injections, examination of livers revealed a significantly different (increased, [Formula: see text]) incidence of a spectrum of hepatic lesions, including presumptive preneoplastic foci of cellular alteration, in sole exposed to the urban sediment extract or BaP. Sole in control groups did not develop any of these hepatic lesions. These results represent the first direct demonstration of the hepatotoxicity of sediment-associated chemical contaminants in English sole and strengthen the case for a cause-and-effect relationship between exposure to AHs and epizootic neoplastic hepatic lesions in this species.

A cancer DNA phenotype, identical to the DNA structure of tumors, has been identified in the prostate glands of certain healthy men over 55 years of age. We now show that the same DNA signature exists in normal tissues adjacent to tumors. This finding implies that the phenotype is maintained in normal prostate cells from its inception through tumor development. The presence of the phenotype in tumors, adjacent normal cells, and in the normal prostate cells of certain older men suggests that it is a potentially critical factor in tumor development and may serve as an early biomarker for cancer risk assessment. Intervention to inhibit the development of the phenotype in healthy men, or to eliminate it once formed, may suppress or even prevent tumor formation.

Fourier transform-infrared statistical models have the proven ability to identify subtle structural changes in DNA at various stages of tumor development. Using these models, we show evidence for a metastatic cancer DNA phenotype in histologically normal prostate tissues surrounding metastasizing tumors. Strikingly, the DNA base and backbone structures of the metastatic phenotype are indistinguishable from those of the metastasizing prostate tumors but distinctly different from the structure recently reported for the primary cancer DNA phenotype. These findings suggest that the DNA structure linked to the development of metastasis is preordained in progenitor cells relatively early in multistep tumorigenesis. The substantial structural differences found between the primary and metastatic cancer DNA phenotypes suggest that each evolves through a separate pathway. The metastatic phenotype is potentially an early predictor of metastatic disease. Interventions that inhibit its formation would be expected to also inhibit the development of metastatic tumors.

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.

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 ≈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 ≈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.

We report significant differences in ultraviolet resonance Raman (UVRR) spectra of DNA samples from normal and cancerous tissues. The four bases of DNA, adenosine, thymine, guanosine and cytidine, can be selectively enhanced in UVRR spectra, and their intensities are very sensitive to base stacking and DNA H-bonding. 14 DNA samples from patients at different stages of ovarian cancer, 5 from normal, 2 from primary, 3 from metastasis primary and 4 from distant metastasis tumor tissues, were characterized by 257, 238, 229, 220 and 210 nm -excited UVRR spectra. Raman spectral difference between normal and tumor DNA could be readily detected.

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.

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 ≈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.

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.

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 → 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 × 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.

We have recently discovered, using statistical models of Fourier transform-infrared spectra, two distinct cancer DNA phenotypes in histologically normal tissues surrounding non-metastasizing primary tumors and tumors with evidence of distant metastases. Structural comparisons of the DNA from these tumor types imply that each evolves via a separate pathway from DNA phenotypes originating in progenitor cells. These findings challenge the widely-held concept of metastasis.

Factors that elicit mutagenic damage to the DNA have been intensely studied for over 50 years. These reactions include the introduction of bulky substituents on the DNA bases which are products of two-electron oxidations (1). Recent studies on these DNA changes suggest that they are more prone to blocking DNA replication and transcription than creating mutagenic lesions (2). A second means of creating potentially mutagenic changes to the DNA is through one-electron, radical-induced oxidative modifications of the nucleotide bases (3). One such free radical is the hydroxyl radical, eOH. The .011 is likely generated in vivo from 11202 in a Fe++-catalyzed Fenton reaction (4, 5). The source of 11202 may be through cytochrome P-450 oxidoreductase cycling after exposure of the tissue to various types of endogenous and exogenous chemicals (6). In fact, those chemicals capable of forming bulky adducts with DNA through two-electron oxidations also form one or more moles of 11202 per mole of bulky adduct. Thus, both processes occur simultaneously. Historically, it has been routinely possible to identify and measure the incidence of the bulky adducts by means of techniques such as 32P-postlabeling analysis (7). pg 4. JMD

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 study was conducted to assess the potential for uptake of toxic chemicals by down-stream migrant salmon in an urban estuary. Juvenile chinook salmon (Oncorhynchus tshawytscha) were collected from the Duwamish Waterway (located in Seattle, Washington) and from the Nisqually River (a reference site). The mean concentrations of aromatic hydrocarbons and polychlorinated biphenyls (PCBs) in the stomach contents (food organisms) of salmon from the Duwamish Waterway were approximately 650 times and 4 times, respectively, higher than those in salmon from the Nisqually River. Similarly, the mean concentration of bile metabolites of aromatic compounds which fluoresce at benzo[a]pyrene wavelengths was 24 times higher in the urban salmon compared to the reference salmon, whereas the mean concentration of PCBs in liver of urban salmon was 3 times higher than that in reference salmon. The study clearly demonstrated that, during their residency in this urban estuary, juvenile chinook salmon bioaccumulate substantial levels of toxic chemicals. The possible effects of these chemical exposures on the health and survival of this species are not presently known.

Juvenile starry flounder (Platichthys stellatus) were force-fed naphthalene, p-cresol, or a mixture of naphthalene and p-cresol in daily doses of 0.3–0.4 mg/kg body weight, for 6 consecutive days. On the 8th day, each fish was force-fed a dose of 12–15 mg/kg of 2,6-dimethyl[14C]naphthalene (DMN). Twenty-four hours later, the fish were killed and 14C-labelled metabolites in the bile were isolated and identified by thin-layer chromatography. Most of the biliary metabolites were recovered as conjugates, principally as glurosides and glucuronides. Analyses of the nonconjugated metabolites and metabolites resulting from enzymatic hydrolysis of the conjugates provided identification of four metabolites of DMN: 2,6-dimethyl-3-naphthol; 2,6-dimethyl-3,4-naphthoquinone; trans-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene (dihydrodiol); and b-methyl-2-naphthalenemethanol (alcohol). Enzymatic hydrolysis of the glucuronides yielded two metabolites: the alcohol, representing metabolism at a methyl substituent, and the dihydrodiol, representing oxidation of an aromatic ring. Exposure to naphthalene and/or p-cresol led to a significant reduction (P

A series of field studies was conducted between 1979 and 1985 in Puget Sound, Washington State, to investigate etiological relationships between prevalences of hepatic neoplasms in bottom-dwelling marine fish species, with emphasis on English sole (Parophrys vetulus), and concentrations of toxic chemicals in sediments and affected fish. Statistically significant (p less than or equal to 0.05) correlations have been found between the prevalences of hepatic neoplasms in English sole and the following parameters: sediment concentrations of aromatic hydrocarbons, and concentrations of the metabolites of aromatic compounds in the bile of affected sole. A significant difference (p less than 0.001) was also found between the relative concentrations of aromatic free radicals in the liver microsomes of English sole with liver lesions compared to sole without liver lesions. Laboratory studies designed to evaluate the etiology of the liver neoplasms in English sole have also yielded evidence that is consistent with the view that high molecular weight aromatic hydrocarbons, e.g., benzo[a]pyrene (BaP), are hepatocarcinogens in English sole. The current status of a series of long-term (up to 18 months) exposures of English sole and rainbow trout (Salmo gairdneri) to selected fractions of Puget Sound sediment extracts, enriched with aromatic hydrocarbons and nitrogen-containing aromatic compounds, and to individual carcinogens (e.g., BaP) is discussed. Images FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12.

Application of a new microtechnique is described for the derivation of acoustic properties (ultrasonic velocity, adiabatic compressibility and acoustic impedance) of lipids. The technique utilizes very small volumes (4 × 10−3 −4 × 10−6 ml) of lipids and thus overcomes the requirement for large volumes inherent in the use of conventional velocimeters. The use of the microtechnique allows acoustic properties to be included among the variety of other physical properties routinely measured in the characterization of lipids, including those isolated in small amounts from complex biological mixtures.

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.

Recently, we have focused attention on organic free radicals in marine organisms exposed to pollutants in the field 1 and laboratory. 2 For example, in Puget Sound, N-oxyl derivatives of heterocycles have been identified in the liver of English sole ( Parophrys vetulus ) containing hepatic neoplasms and other hepatic lesions. 1,2 We now report the occurrence of free radicals in sediments and bottom-dwelling fish from Eagle Harbor, a creosote-polluted area of Puget Sound. The free radicals were analyzed by electron paramagnetic resonance (EPR) spectroscopy. Sediments from Eagle Harbor contained high concentrations of aromatic hydrocarbons (e.g. benzo[ a ]pyrene) and nitrogen heterocycles (e.g. carbazoles). 3 Both types of xenobiotics contain compounds that are noted for their facile conversion to free radicals. 4

Petroleum is composed of a complex mixture of hydrocarbons that readily undergo chemical and biological conversions on entering aquatic environments. These conversions lead to the formation of a host of oxygenated products, some of which are potentially toxic to marine life and to the consumer of fishery products. State-of-the-art analytical methods, as employed in our laboratories, utilize glass-capillary gas chromatography in conjunction with mass spectrometry to analyze environmental samples containing trace amounts of aliphatic and aromatic petroleum hydrocarbons. These procedures are applied on a routine basis to the analysis of seawater, sediments and tissues of marine organisms. Despite this analytical proficiency, a need exists for analyzing oxygenated and other polar petroleum products in environmental samples. For example, techniques such as high-performance liquid chromatography (HPLC), in conjunction with on-line fluorometric assay techniques and mass spectrometry, make possible the analysis of polar oxygenated compounds resulting from both chemical and biological conversions. These methodologies are first steps toward the development of routine assay procedures for environmental samples. Current techniques for hydrocarbon analyses and new methods for analyzing polar aromatic compounds are discussed.

(1-/sup 14/C)Naphthalene and the (1-/sup 14/C)naphthalene complexed with BSA at 8 to 12 ppb in flowing sea water produced 100 percent mortality in 24 to 36 hr in newly hatched Dungeness crab (Cancer magister) zoea and stage I and V spot shrimp (Pandalus platyceros). Maximum accumulation of (1-/sup 14/C)naphthalene in the stage V spot shrimp exposed to (1-/sup 14/C)naphthalene was nearly four times greater than in shrimp exposed to the (1-/sup 14/C)naphthalene-BSA complex (820 and 220 ppb, respectively). These values represent a magnification of 25 to 100 times the exposure levels. Metabolic products of (1-/sup 14/C)naphthalene in larval tissues exposed to the complexed and noncomplexed naphthalene reached a maximum of 21 and 9 percent, respectively, of the total radioactive compounds (based on the molecular weight of naphthol). (1-/sup 14/C)Naphthalene was almost entirely depurated from the tissues in 24 to 36 hr, whereas metabolic products were strongly resistant to depuration.

Creosote oil and organic extracts of marine sediments were subjected to silica/alumina column chromatography to obtain fractions greatly enriched in nitrogen-containing aromatic compounds (NCAC), which were then characterized by gas chromatography (GC) with nitrogen-specific detection and GC/mass spectrometry. A large number of NCAC were identified in sediments from creosote-contaminated Eagle Harbor, Puget Sound, WA, as well as in the sample of commercially available creosote oil. No NCAC were detected in sediments from a pristine reference area (detection limit 10 ng/g). The total NCAC concentration in the Eagle Harbor sediments ranged from about 200 to 1200 ..mu..g/g of sediment (dry weight). Because many NCAC are known mutagens/carcinogens/teratogens, their presence in high concentrations in sediments may pose various health risks for marine biota.

Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine from the melon of the porpoise Tursiops gilli were shown to contain 7.9,7.2, and 0.8 moles % of long-chain iso acids (C14-C16), respectively. The occurrence of the long-chain iso acids is an anomalous finding in the phospholipids of higher animals. Isovaleric acid was not detected in the melon phospholipids, despite the presence of large amounts of neutral lipids rich in this short-chain, branched acid. Both isovaleric and the longchain iso acids were virtually absent from the phospholipids of the post-cranial blubber. The positional distribution of the acids in phosphatidylcholine indicated that the iso acids show a strong preference for position 2. This finding is consistent with the general rule that structural modifications in the acyl chains of phospholipids are usually accommodated at position 2, rather than at position 1. The long-chain triacylglycerols (C10-C18 acyl chains) contained 31.6 moles % of iso acids which were confined to chain lengths of C12-C16. These iso acids may replace C16-C20 monoenoic acids commonly present in the lipids of other mammalian systems. Positional analyses of these glycerides revealed that, with the exception of isopentadecanoic acid, no distinct tendency existed to deposit the iso acids at position 2 in preference to positions 1 and 3. Moreover, no preference was shown for the deposition of the polyenoic acids at position 2. Such a lack of positional specificity is shown by the polyenoic acids in triacylglycerols of some marine mammals

Statistical comparisons were made between the relative mean concentrations of metabolites of aromatic compounds in bile (as estimated by a high-performance liquid chromatography/fluorescence detection method) and idiopathic liver lesions in English sole (Parophrys vetulus) from eleven Puget Sound sites. A significant positive correlation was found between prevalences of neoplasms, foci of cellular alteration, megalocytic hepatosis and total hepatic lesions and the concentrations of metabolites of aromatic compounds. These findings provide further evidence of the putative relationship between aromatic compounds and serious idiopathic liver diseases in bottom-dwelling fish.