Quantifying loading, toxic concentrations, and systemic persistence of chloride in a contemporary mixed-land-use watershed using an experimental watershed approach.

A nested-scale experimental watershed study was implemented to quantify loading and persistence of chloride in an urbanizing, mixed-land-use watershed. A Midwest USA (Missouri) watershed was partitioned into five sub-basins with contrasting dominant land use. Streamwater was tested for chloride concentration four days per week from October 2009 through May 2014 at each site. Monitoring sites included co-located gauging and climate stations recording variables at 30-minute intervals. Results indicate significant (p<0.01) differences in chloride concentrations and loading between sites. Loading consistently increased from the forested headwaters (average=507kgday-1) to primarily urban watershed terminus (average=7501kgday-1). Chloride concentrations were highest (average=83.9mgL-1) with the greatest frequency of acutely toxic conditions (i.e. 860mgL-1) mid-watershed. This finding is in-part attributable to the ratio of chloride application to streamflow volume (i.e. increasing flow volume with stream distance resulted in chloride dilution, offsetting increased percent urban land use with stream distance). Results highlight the important, yet often confounding, interactions between pollutant loading and flow dynamics. Chloride peaks occurred during late winter/early spring melting periods, implicating road salt application as the primary contributor to the chloride regime. Floodplain groundwater analysis indicated seasonal sink/source relationships between the stream and floodplain, which could contribute to chronic toxicity and persistent low Cl- concentrations in streamwater year-round. Results hold important implications for resource managers wishing to mitigate water quality and aquatic habitat degradation, and suggest important water quality limitations to stream restoration success in complex urban aquatic ecosystems.

Short-term breast cancer prediction by random periareolar fine-needle aspiration cytology and the Gail risk model.

BACKGROUND: : Biomarkers are needed to refine short-term breast cancer risk estimates from epidemiologic models and to measure response to prevention interventions. The purpose of our study was to determine whether the cytologic appearance of epithelial cells obtained from breast random periareolar fine-needle aspirates or molecular marker expression in these cells was associated with later breast cancer development. METHODS: : Four hundred eighty women who were eligible on the basis of a family history of breast cancer, prior precancerous biopsy, and/or prior invasive cancer were enrolled in a single-institution, prospective trial. Their risk of breast cancer according to the Gail model was calculated, and random periareolar fine-needle aspiration was performed at study entry. Cells were characterized morphologically and analyzed for DNA aneuploidy by image analysis and for the expression of epidermal growth factor receptor, estrogen receptor, p53 protein, and HER2/NEU protein by immunocytochemistry. All statistical tests are two-sided. RESULTS: : At a median follow-up time of 45 months after initial aspiration, 20 women have developed breast cancer (invasive disease in 13 and ductal carcinoma in situ in seven). With the use of multiple logistic regression and Cox proportional hazards analysis, subsequent cancer was predicted by evidence of hyperplasia with atypia in the initial fine-needle aspirate and a 10-year Gail projected probability of developing breast cancer. Although expression of epidermal growth factor receptor, estrogen receptor, p53, and HER2/NEU was statistically significantly associated with hyperplasia with atypia, it did not predict the development of breast cancer in multivariable analysis. CONCLUSION: : Cytomorphology from breast random periareolar fine-needle aspirates can be used with the Gail risk model to identify a cohort of women at very high short-term risk for developing breast cancer. We recommend that cytomorphology be studied for use as a potential surrogate end point in prevention trials.

Breast cytology and biomarkers obtained by random fine needle aspiration: use in risk assessment and early chemoprevention trials.

In a prospective pilot study, we performed breast fine needle aspirations (FNAs) on 224 high-risk and 30 low-risk women and analyzed these aspirates for cytologic changes and biomarker abnormalities of aneuploidy and overexpressed estrogen receptor (ER), epidermal growth factor receptor (EGFR), p53 and HER-2/neu. High-risk women had a first-degree relative with breast cancer (74%), prior biopsy indicating premalignant breast disease (25%), a history of breast cancer (13%), or some multiple of these risk factors (12%). Median ages of the high- and low-risk groups were 44 and 42, respectively. Seventy percent of high-risk and 17% of low-risk women had cytologic evidence of hyperplasia with or without atypia (P < .0001). Aneuploidy and overexpression of EGFR and p53 occurred in 27, 37, and 29% of high-risk subjects but only 0, 3, and 3% of low-risk subjects (P < .0023). Overexpression of ER and HER-2/neu occurred in 7 and 20% of high-risk women but in none of the low-risk subjects. Biomarker abnormalities were more frequent with increasing cytologic abnormality. Restricting the analysis to those 3 biomarkers most frequently overexpressed in the high-risk group (ploidy, EGFR, p53), 13% of high-risk women with normal cytology, 19% of high-risk women with epithelial hyperplasia, and 49% of high-risk women with hyperplasia with atypia had abnormalities of 2 or more of these 3 biomarkers (P = .00004). At a median follow-up of 32 months, four women have been diagnosed with invasive cancer and two with ductal carcinoma in situ (DCIS). Later detection of these neoplastic conditions was associated (P < or = .016) by univariate analysis with prior FNA evidence of hyperplasia with atypia; overexpression of p53 and EGFR; the modified Gail risk of breast cancer development at 10 years; and multiple biomarker abnormalities. By multivariate analysis, later detection of cancer was primarily predicted by the number of biomarker abnormalities in the 3-test battery (P = .0005) and secondarily by the Gail risk at 10 years (P = .0049). In turn, hyperplasia with atypia was associated with multiple biomarker abnormalities, particularly p53 and EGFR overexpression. Thus, hyperplasia with atypia and cytologic markers in breast FNAs have promise as risk predictors and as surrogate endpoint biomarkers for breast cancer chemoprevention trials.

Prevalence of aneuploidy, overexpressed ER, and overexpressed EGFR in random breast aspirates of women at high and low risk for breast cancer.

Breast tissue biomarkers which accurately predict breast cancer development within a 10 year period in high risk women are needed but currently not available. We initiated this study to determine 1) the prevalence of one or more breast tissue abnormalities in a group of women at high risk for breast cancer, and 2) if the prevalence of biomarker abnormalities is greater in high risk than in low risk women. Eligible high risk women were those with a first degree relative with breast cancer, prior breast cancer, or precancerous mastopathy. Low risk women were those without these or other major identifiable risk factors. Ductal cells were obtained via random fine needle aspirations and cytologically classified. Biomarkers included DNA ploidy, estrogen receptor (ER), and epidermal growth factor receptor (EGFR). The prevalence of DNA aneuploidy was 30%, overexpression of ER 10%, and overexpression of EGFR 35%, in the 206 high risk women whose median 10 year Gail risk (projected probability) of developing breast cancer was 4.5%. The prevalence of aneuploidy and overexpressed EGFR was significantly higher in the high risk women than in the 25 low risk controls (p < 0.002), whose median 10 year Gail risk was 0.7%. The difference in the prevalence of ER overexpression between high and low risk groups was not statistically significant (p = 0.095). This may be due to the low prevalence of overexpressed ER and the small number of controls. A significant difference was noted in the prevalence of one or more abnormal biomarkers between the high risk and low risk women (p < 0.001). A large prospective trial is needed to determine if one or more of these biomarkers, is predictive of breast cancer development.

Biomarker and cytologic abnormalities in women at high and low risk for breast cancer.

Fine needle aspirates (FNA) from 106 high-risk women and 25 low-risk women were evaluated for overexpression of estrogen receptor (ER), epidermal growth factor receptor (EGFR), mutant p53, and HER-2/neu by immunocytochemistry, and for aneuploidy by image analysis. Aspirates were also classified cytologically as normal, apocrine metaplasia, epithelial hyperplasia (EH), or dysplasia. High-risk women were those with a first-degree relative with breast cancer (76%), precancerous breast disease (26%), prior cancer of the contralateral breast (9%), or multiple abnormalities (11%). Low-risk women had none of the above risk factors, nor a prior breast biopsy or clinical evidence of fibrocystic disease. The median 10-year Gail risk for the high-risk group was 4%, compared to 0.7% for the low-risk group. There were significant differences (p < 0.01) between high- and low-risk women in the prevalences of hyperplasia (55% versus 12%), dysplasia (19% versus 0%), aneuploidy (32% versus 0%), overexpressed EGFR (32% versus 4%), and overexpressed p53 (29% versus 4%). The prevalence of multiple biomarker abnormalities was also greater in high-risk than in low-risk women (28% versus 0%; p < 0.01). Four percent (4%) of FNAs from high-risk women with normal cytology, 29% of aspirates with hyperplastic cytology, and 60% of those with dysplasia were associated with two or more biomarker abnormalities. The differences in the prevalence of multiple biomarker abnormalities among various cytologic categories were statistically significant (p = 0.02, normal versus EH; p = 0.02, EH versus dysplasia; p < 0.01, normal versus dysplasia).(ABSTRACT TRUNCATED AT 250 WORDS)

Matrix vesicle biogenesis in vitro by rachitic and normal rat chondrocytes.

Calcifying matrix vesicles (MVs) are released from chondrocytes and osteoblasts in monolayer culture. In the present studies, we tested the ability of rachitic versus normal rat growth plate chondrocytes in micromass or monolayer primary cultures to produce MVs. Unlike earlier reports of in vitro MV biogenesis by chicken chondrocytes in which most MVs were released into the medium, we found that most of the released rat matrix vesicles were entrapped in a newly formed cartilaginous matrix enveloping the cells. These matrix-associated MVs could be isolated by mild collagenase treatment and concentrated by differential centrifugation. Vesicle production slowed in the older 2- to 4-week-old cultures and, unlike vesicle release from cultured chicken chondrocytes, active vesicle production did not show a second burst of activity at 3 to 4 weeks. Alkaline phosphatase (ALP) activity diminished with time in culture in cells and matrix vesicles, suggesting a decrease in differentiative expression. Protein profiles on SDS polyacrylamide gels of native matrix vesicles and culture-derived MVs from rachitic and normal cells were quite similar and showed a typical simplified protein pattern as compared to chondrocyte plasma membrane proteins. There were distinctive proteins migrating at 130, 80 to 95, 66, 43, 20, and 14 kd. Culture-derived MVs showed vigorous in vitro calcifying activity that was ALP related. We conclude that 1) rachitic chondrocytes are essentially normal in their matrix vesicle production; 2) matrix entrapment of MVs is a characteristic of rat chondrocyte cultures; and 3) culture-produced MVs are similar to native MVs in protein profile and calcifiability, and thus can be studied as a model for normal MV composition and calcification.

Enzyme cytochemical localization of alkaline phosphatase in cultures of chondrocytes derived from normal and rachitic rats.

Epiphyseal growth plate cartilages were removed from rats which had been maintained on normal laboratory chow or a rachitogenic diet. Chondrocytes were released from the growth plates by collagenase digestion and cultured in tissue chamber slides. After 7, 10 and 12 days of culture, the chondrocytes were removed as intact multilayers and processed for electron microscopical enzyme cytochemical studies. Alkaline phosphatase activity in the cultures was visualized by means of a cerium based capture method. Electron-dense cerium phosphate deposits were localized on the membrane of matrix vesicles and plasma membranes of chondrocytes derived from normal and rachitic animals. The appearance of first crystals within matrix vesicles was characterized by a concomitant decrease in alkaline phosphatase activity in the membrane of these structures. Calcification was initiated at approximately the same time in cultures of chondrocytes derived from normal or rachitic animals. The results suggest that rickets has no serious effects on the capacity of chondrocytes to support matrix calcification in vitro. Additionally, the evidence indicates that alkaline phosphatase-positive matrix vesicles play a significant role in the initiation of this process.

Presence and activity of alkaline phosphatase in two human osteosarcoma cell lines.

The presence and activity of alkaline phosphatase in SAOS-2 and TE-85 human osteosarcoma cells grown in culture were examined at the ultrastructural level. A monoclonal antibody raised against purified human bone osteosarcoma alkaline phosphatase was used to localize the enzyme in cultures of the osteosarcoma cells. Similar cultures were analyzed for alkaline phosphatase activity using an enzyme cytochemical method with cerium as the capture agent. Alkaline phosphatase was immunolocalized at the light microscopic level in an osteogenic sarcoma and ultrastructurally on the SAOS-2 cell membrane and the enclosing membrane of extracellular vesicular structures close to the cells. In contrast, the TE-85 cells were characterized by the absence of all but a few traces of immunolabeling at the cell surface. Enzyme cytochemical studies revealed strong alkaline phosphatase activity on the outer surface of the SAOS-2 cell membrane. Much lower enzyme activity was observed in the TE-85 cells. The results support biochemical data from previous studies and confirm that SAOS-2 cells have a significantly greater concentration of alkaline phosphatase at the plasma membrane.