Correlated responses for DNA damage, phagocytosis activity and lysosomal function revealed in a comparison between field and laboratory studies: Fathead minnow exposed to tritium.

Tritium entering the aquatic environment can confer a whole body internal radiological dose to aquatic organisms. Multiple stressors inherent in natural environments, however, confound estimates for observable radiation specific responses. To disentangle differences between field and laboratory outcomes to tritium exposures, a multivariate analysis comparing biomarkers for radiation exposure at the cellular level with changes in biological processes within tissues is described for fathead minnows (Pimephales promelas). Over tritium activity concentrations up to 180,000 Bq/L, DNA damage in the field were lower than DNA damage in the laboratory. This finding does not support an increase in morbidity of biota in field exposures. Energy deposited by tritium decay produces oxidised free radicals, yet the biological responses in brain, muscle and liver to oxidative stress differed between the studies and were not related to the tritium. For both studies, DNA damage in gonad and blood increased with increased tritium as did the fluorescence associated with lysosomal function in spleen. The studies differed in spleen phagocytosis activity were, in the laboratory but not the field, activity increased with increased tritium-and was correlatd with lysosomal function (Spearman coefficient of 0.98 (p = 0.001). The higher phagocytosis activity in the field reflects exposures to unmeasured factors that were not present within the laboratory. In the laboratory, DNA damage and lysosomal function were correlated: Spearman coefficients of 0.9 (Comet, p = 0.03) and 0.9 (micronuclei, p = 0.08). In the field, DNA damage by the Comet assay, but not by micronucleus frequency, correlated with lysosomal function: Spearman coefficients of 0.91 (Comet, p < 0.001) and 0.47 (micronuclei, p = 0.21). These observations highlight a need for better physiologic understanding of linkages between radiation-induced damage within cells and responses at higher levels of biological organization.

Spatial Autocorrelation, Source Water and the Distribution of Total and Viable Microbial Abundances within a Crystalline Formation to a Depth of 800 m.

Proposed radioactive waste repositories require long residence times within deep geological settings for which we have little knowledge of local or regional subsurface dynamics that could affect the transport of hazardous species over the period of radioactive decay. Given the role of microbial processes on element speciation and transport, knowledge and understanding of local microbial ecology within geological formations being considered as host formations can aid predictions for long term safety. In this relatively unexplored environment, sampling opportunities are few and opportunistic. We combined the data collected for geochemistry and microbial abundances from multiple sampling opportunities from within a proposed host formation and performed multivariate mixing and mass balance (M3) modeling, spatial analysis and generalized linear modeling to address whether recharge can explain how subsurface communities assemble within fracture water obtained from multiple saturated fractures accessed by boreholes drilled into the crystalline formation underlying the Chalk River Laboratories site (Deep River, ON, Canada). We found that three possible source waters, each of meteoric origin, explained 97% of the samples, these are: modern recharge, recharge from the period of the Laurentide ice sheet retreat (ca. ∼12000 years before present) and a putative saline source assigned as Champlain Sea (also ca. 12000 years before present). The distributed microbial abundances and geochemistry provide a conceptual model of two distinct regions within the subsurface associated with bicarbonate - used as a proxy for modern recharge - and manganese; these regions occur at depths relevant to a proposed repository within the formation. At the scale of sampling, the associated spatial autocorrelation means that abundances linked with geochemistry were not unambiguously discerned, although fine scale Moran's eigenvector map (MEM) coefficients were correlated with the abundance data and suggest the action of localized processes possibly associated with the manganese and sulfate content of the fracture water.

Local and Regional Diversity Reveals Dispersal Limitation and Drift as Drivers for Groundwater Bacterial Communities from a Fractured Granite Formation.

Microorganisms found in terrestrial subsurface environments make up a large proportion of the Earth's biomass. Biogeochemical cycles catalyzed by subsurface microbes have the potential to influence the speciation and transport of radionuclides managed in geological repositories. To gain insight on factors that constrain microbial processes within a formation with restricted groundwater flow we performed a meta-community analysis on groundwater collected from multiple discrete fractures underlying the Chalk River Laboratories site (located in Ontario, Canada). Bacterial taxa were numerically dominant in the groundwater. Although these were mainly uncultured, the closest cultivated representatives were from the phenotypically diverse Betaproteobacteria, Deltaproteobacteria, Bacteroidetes, Actinobacteria, Nitrospirae, and Firmicutes. Hundreds of taxa were identified but only a few were found in abundance (>1%) across all assemblages. The remainder of the taxa were low abundance. Within an ecological framework of selection, dispersal and drift, the local and regional diversity revealed fewer taxa within each assemblage relative to the meta-community, but the taxa that were present were more related than predicted by chance. The combination of dispersion at one phylogenetic depth and clustering at another phylogenetic depth suggest both niche (dispersion) and filtering (clustering) as drivers of local assembly. Distance decay of similarity reveals apparent biogeography of 1.5 km. Beta diversity revealed greater influence of selection at shallow sampling locations while the influences of dispersal limitation and randomness were greater at deeper sampling locations. Although selection has shaped each assemblage, the spatial scale of groundwater sampling favored detection of neutral processes over selective processes. Dispersal limitation between assemblages combined with local selection means the meta-community is subject to drift, and therefore, likely reflects the differential historical events that have influenced the current bacterial composition. Categorizing the study site into smaller regions of interest of more closely spaced fractures, or of potentially hydraulically connected fractures, might improve the resolution of an analysis to reveal environmental influences that have shaped these bacterial communities.

A novel method for detection of nitric oxide binding sites by using a chemiluminescence-headspace gas technique.

Nitric oxide (NO) is important in many physiological, pharmacological, and pathological processes. According to current concepts, guanylyl cyclase is considered to be a receptor for NO in vascular and nonvascular smooth muscle and other tissues. Since there are no suitable radioisotopes of oxygen and nitrogen available for conventional radioligand-receptor binding studies for NO, a novel method was developed to identify NO binding site(s). A chemiluminescence-headspace gas assay was utilized to measure the sequestration of NO in biological systems, and this was used as an index of NO binding. In the present report, myoglobin (a hemoprotein, Mb) was used as a prototype macromolecule to develop the binding assay for subsequent application to studies of putative NO receptors. Solutions containing various concentrations of Mb were incubated with NO in sealed micro-Fernbach flasks at 37 degrees C in an argon atmosphere for 30 min; NO remaining in the headspace gas was analyzed by means of the chemiluminescence assay. The magnitude of NO sequestration was dependent on Mb concentration, and 5 nM Mb was the lowest Mb concentration for which NO sequestration was measurable. Application of the method to the measurement of NO sequestration by bovine serum albumin (BSA) and pulmonary artery medial layer homogenate (BPA-M) revealed that the lowest BSA concentration at which NO sequestration was measurable was 1.6 microM, which was 320 times greater than that for Mb. Applicability of the method to address the question of putative NO receptors was indicated by significant NO sequestration after incubation with 20% (w/v) homogenate of BPA-M, which is responsive to NO and putative NO prodrugs.

Selective sequestration of nitric oxide by subcellular components of vascular smooth muscle and platelets: relationship to nitric oxide stimulation of the soluble guanylyl cyclase.

Sequestration of nitric oxide (NO) by subcellular fractions isolated from bovine pulmonary arterial medial layer (BPA) and rabbit platelets (RP) was studied utilizing a novel chemiluminescence--headspace gas technique. Sequestration in all fractions was similarly rapid (5 min) and remained constant for at least 30 min. When incubated with 108 pmol of NO, the BPA mitochondrial, microsomal, and nuclear fractions sequestered 22.8 +/- 1.9, 20.5 +/- 2.2 and 15.2 +/- 3.6% of the NO, respectively (n = 14). However, significantly more of the 108 pmol of NO, 36.8 +/- 2.8 and 32.9 +/- 3.6%, respectively, was sequestered by the BPA homogenate (about 2 mg protein/mL) and BPA cytosolic fraction (about 1 mg protein/mL) (n = 19). Also, RP cytosolic fraction (about 3 mg protein/mL) sequestered a greater amount of NO than any BPA fraction when incubated with 108 pmol of NO (83.0 +/- 1.0%; n = 3). Analysis of the binding data obtained for the BPA homogenate and cytosolic fraction was consistent with the existence of two binding sites, one site with a Kd of approximately 100 nM and another with a Kd of approximately 1 microM. Both the BPA homogenate fraction and the cytosolic fraction as well as the RP cytosolic fraction were shown to have soluble guanylyl cyclase activity. The nitrovasodilator sodium nitroprusside (SNP) caused a concentration-dependent increase in the activity of this enzyme in all these fractions. Maximum stimulations caused by 1 mM SNP in BPA homogenate fraction, BPA cytosolic fraction, and RP cytosolic fraction were equivalent to 2-, 4- and 3-fold increases in catalytic activity, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)