Brian P. Bradley

Protein expression signatures identified in Mytilus edulis exposed to PCBs, copper and salinity stress.

Applied to environmental toxicology, proteome analysis may be used to isolate chemical-specific protein expression signatures (PES). In this project specific PES were isolated in mussels, Mytilus edulis, from the Baltic Sea subjected in the laboratory to treatment with copper (70 ppb), Aroclor 1248 (1 ppb), and to lowered salinity. Four mussels in each treatment group were acclimated in the laboratory for 24 h before beginning the 7-day exposure. Whole body tissue was homogenized and separated using two-dimensional gel electrophoresis. The protein gels were scanned to TIFF files and compared using MELANIE II 2D gel analysis software (BioRad). Protein expression signatures including proteins induced and repressed by exposure were isolated for each treatment group. The specificity of PES due to environmental changes shows promise in bioindication, toxicity testing and in helping identify possible toxicity mechanisms.

Protein expression signatures: an application of proteomics

The methods of proteomics, the study of the protein complement of the genome, are applicable to environmental testing. Sets of proteins specific to different stressors can be isolated using computer imaging software. Individual proteins can be identified by mass spectrometry. The Protein Expression Signatures (PES) obtained have potential in diagnosing adverse factors in the environment. The challenge is to demonstrate their feasibility in complex environments. We have shown that PES for three endocrine disrupting compounds in trout (Onchorhynchus mykiss), can be detected in mixed sewage effluent. Other studies support these results. As protein databases expand, identification becomes routine, and capture molecules specific to each protein are developed, the possibility of simple field tests for multiple stressors becomes real.

Protein expression signatures and lysosomal stability in Mytilus edulis exposed to graded copper concentrations

Protein expression and lysosomal stability were observed in Mytilus edulis exposed to 0, 20, 40, 60, and 80 ppb copper sulfate for 24 h in a static exposure system. Haemolymph was removed from each mussel and analyzed for lysosomal damage using the Neutral Red retention assay expressed as the ratio of lysosomal size to cell area, the lysosomal destabilization ratio. Gill tissue from the same individuals was dissected, homogenized and the proteins extracted and then separated using two-dimensional gel electrophoresis. Two-dimensional protein maps from each concentration were analyzed using MELANIE II 2D gel analysis software (Biorad). Lysosomal damage increased with each dosage, with LDRs ranging from 0.18 +/- 0.01 at 0 ppb copper sulfate to 0.32 +/- 0.11 at 80 ppb copper sulfate. Orthogonal comparisons showed that the control, 20 and 40 ppb classes together differed significantly (P < 0.05) from the 60 and 80 ppb classes together. Specific protein expression signatures were identified at each dosage.

Heavy metal uptake, physiological response and survival of the blue mussel (Mytilus edulis) from marine and brackish waters in relation to the induction of heat-shock protein 70

Earlier studies demonstrate that blue mussels (Mytilus edulis) from the Baltic population are more sensitive, in terms of physiological response and survival when exposed to toxic substances, compared to mussels from a more marine environment. The question whether this can be explained by environmental factors or genetic differences in the ability to synthesise a common stress-inducible protein (HSP 70) was addressed in two experiments. In the first experiment mussels from the North and Baltic Seas were acclimatised to an intermediate salinity of 15‰ S in the laboratory. The physiological performance was studied when the heavy metal cadmium was added and accumulated by the animals during a one week exposure. Tissue concentration was measured and related to physiological response. The level of expression of HSP 70 was analysed by densitometry on Western blots. North Sea mussels rapidly induced high levels and multiple forms of HSP 70, reached a comparatively low tissue concentration of cadmium, and showed only a minor physiological response and low mortality rates. Baltic Sea mussels had low levels of HSP 70, induced at slow rate, reached higher tissue concentrations of cadmium, and showed a more pronounced physiological response and higher mortality rates. High levels of stress proteins and a rapid induction corresponded well with physiological fitness, and the mussels from the North Sea thus seem to have a more efficient detoxification system, probably including stress-inducible proteins. In the second experiment, juvenile mussels from the Baltic population were acclimatised for a month to North Sea conditions in a field transplant. The mussels were then exposed to low-dose copper in the laboratory and the accumulation, physiological response and synthesis of stress inducible proteins were assessed. The results indicate that the physiological differences described between the populations are to a large extent explained by environmental factors. However, some differences can still he observed between the populations, e.g., a lower rate of induction of a major stress protein in Baltic than in North Sea mussels. It can thus be suggested that a reduced ability to stress protein induction, in their natural low saline habitat, might be a contributing factor to the higher pollution sensitivity earlier demonstrated for Baltic blue mussels.

DNA damage, histologial changes and DNA repair in larval Japanese medaka (Oryzias latipes) exposed to ultraviolet-B radiation

Cyclobutane dimer formation, photorepair capability and histological damage were compared among four differently pigmented strains of larval Japanese medaka (Oryzias latipes) to determine whether pigmentation modifies the level of UV-B radiation (290-320 nm) inducible damage in these fish. One-day post-hatch medaka were exposed to one of several UV-B fluence rates with or without photoreactivating light for 5 days for 7 h per day. Their DNA was extracted for analysis by ELISA for cyclobutane pyrimidine dimers or the larvae were processed for histological examination. At the higher UV-B fluence rates tested, wild-type melanophore-containing medaka formed significantly more dimers than at least one of the other strains tested. Wild-type medaka also showed significantly less photorepair capability than the white melanophore-lacking medaka. The wild-type larvae had significantly more necrosis than the orange-red melanophore-lacking larvae at the lower UV-B fluence rate tested and at the higher fluence rate used, the wild-type medaka also exhibited significantly more necrosis than the white melanophore-lacking larvae. Of the 19 medaka observed with cellular hyperplasia, six were wild-type. These six individual larvae showed the greatest degree of cellular hyperplasia. Cellular hyperplasia appeared to be greatest at the lowest UV-B fluence rate used. The presence of melanophores in the wild-type medaka may have contributed to an increased level of tissue damage in this strain when compared to the other strains.

Salinity stress proteins in Eurytemora affinis

Seasonal densities of Eurytemora affinis, a calanoid copepod in the Chesapeake Bay, seem to be controlled by temperature and salinity. To examine the role of osmotic stress we analyzed protein synthesis under various conditions of temperature and osmotic stress. Adult females were exposed in groups for 5 hours to different temperature and salinity regimes in the presence of isotope-labelled amino acid. Newly synthesized (stress) proteins could be separated and identified using polyacrylamide gel electrophoresis and autoradiography. The protein profiles occurring in copepods experiencing osmotic shock alone were different from those of control animals. Copepods transferred to lower (2 and 5‰) and higher (15 and 20‰) salinities showed differences in the up- and down-regulation of specific proteins. Concurrent heat stress changed these protein patterns. Animals experiencing osmotic and heat shock at the same time exhibited enhanced expression of another set of proteins. Variation in induced proteins occurred among individuals.

Zinc concentration effect at the organismal, cellular and subcellular levels in the eastern oyster

The purpose of this research was to demonstrate a concentration effect of zinc exposure at organismal, cellular and sub-cellular levels in the eastern oyster and to find associated protein expression signatures (PES) for each concentration of zinc. Oysters were exposed to six concentrations of zinc for 48 h in a controlled environment. At the organismal level, fecal material was observed as a measure of physiological health during metal exposures. At the cellular level, lysosomal destabilization was measured using hemolymph. This cellular response was significant only at the highest concentration, when the fecal index was lowest. Protein responses were monitored in the oyster following exposure to zinc. Gill tissue was excised and homogenized, and then analyzed using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and digital image analysis. Protein expression signatures were found to be specific to each concentration. The protein responses were linked to the other biological parameters measured, each of which followed a concentration gradient of zinc.

Models for Physiological and Genetic Adaptation to Variable Environments

The challenge of a temporally or spatially varying environment to a population can be met at the individual level, at the population level, or at both. Individual organisms may adjust physiologically to the entire range of conditions. Populations may systematically change in gene frequencies (where the environmental variation is sufficiently predictable). Likewise, both physiological and genetic changes may occur in space and time.

HSP70 levels in physiologically stressed Baltic Sea mussels

Abstract Mussels, Mytilus edulis L., from the Baltic Sea were exposed to stressful cadmium and temperature environments. The expression of the 70 000 kD heat shock protein family (HSP70) and of several organismal responses was observed in two phases. Mussels exposed to 20 °C for one day had higher levels of HSP70 than controls at 4 °C, with 20 μg litre−1 cadmium or without cadmium. The higher temperature increased respiration and ammonia excretion and decreased clearance rate and scope for growth. In the second phase, mussels pretreated at 20 °C for 5 days and reacclimated to 4 °C for 5 days still showed significantly elevated HSP70, with 20 μg litre−1 cadmium and without cadmium. The organismal responses in the second phase were much less affected by treatment, although scope for growth declined in pretreated animals exposed to cadmium. HSP70 seems to be a suitable indicator of both present and recent stress. A functional relationship between HSP70 levels and adaptation to heat and cadmium is suggested but not established.