Biomarkers signal contaminant effects on the organs of English sole (Parophrys vetulus) from Puget Sound.

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.

Oxidative changes in the DNA of stroma and epithelium from the female breast: potential implications for breast cancer.

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.

Structural alterations in breast stromal and epithelial DNA: the influence of 8,5'-cyclo-2'-deoxyadenosine.

(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.

A cancer DNA phenotype in healthy prostates, conserved in tumors and adjacent normal cells, implies a relationship to carcinogenesis.

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.

Development of a cancer DNA phenotype prior to tumor formation.

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.

Models of granulocyte DNA structure are highly predictive of myelodysplastic syndrome.

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.

Metastatic cancer DNA phenotype identified in normal tissues surrounding metastasizing prostate carcinomas.

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.