The impact of environmental parameters on microcystin production in dialysis bag experiments.

It is important to understand what environmental parameters may regulate microcystin (MC) production and congener type. To determine if environmental conditions in two hydraulically connected lakes can influence MC production and congener ratios, we incubated dialysis bags containing phytoplankton from mesotrophic/eutrophic Muskegon Lake into hypereutrophic Bear Lake (Michigan, USA) and vice versa. Strong cyanobacteria growth was observed in all dialysis bags with Bear Lake phytoplankton in July and August. Phytoplankton communities were dominated by Aphanizomenon aphanizomenoides, Microcystis wesenbergii, Limnothrix redekei. MC concentrations were correlated with M. wesenbergii and A. aphanizomenoides biovolume. MC concentrations in bags incubated in the Muskegon Lake with Bear Lake water were significantly higher than the other bags. The higher light intensity and total nitrogen concentration may have caused the increase of MC production. The MC-LR/MC-RR ratios varied with sample origin but not with lake of incubation, indicating that physical environmental factors (water temperature and turbidity) were not the reasons for different toxin production ratios. Differences in total phosphorus concentrations might be one reason for the dissimilarity of the MC-LR/MC-RR ratio between the two lakes. The higher light intensity and NO3-N concentration in Muskegon Lake are two factors contributing to an increase of MC production.

Sex differences in contaminant concentrations of fish: a synthesis.

A comparison of whole-fish polychlorinated biphenyl (PCB) and total mercury (Hg) concentrations in mature males with those in mature females may provide insights into sex differences in behavior, metabolism, and other physiological processes. In eight species of fish, we observed that males exceeded females in whole-fish PCB concentration by 17 to 43 %. Based on results from hypothesis testing, we concluded that these sex differences were most likely primarily driven by a higher rate of energy expenditure, stemming from higher resting metabolic rate (or standard metabolic rate (SMR)) and higher swimming activity, in males compared with females. A higher rate of energy expenditure led to a higher rate of food consumption, which, in turn, resulted in a higher rate of PCB accumulation. For two fish species, the growth dilution effect also made a substantial contribution to the sex difference in PCB concentrations, although the higher energy expenditure rate for males was still the primary driver. Hg concentration data were available for five of the eight species. For four of these five species, the ratio of PCB concentration in males to PCB concentration in females was substantially greater than the ratio of Hg concentration in males to Hg concentration in females. In sea lamprey (Petromyzon marinus), a very primitive fish, the two ratios were nearly identical. The most plausible explanation for this pattern was that certain androgens, such as testosterone and 11-ketotestosterone, enhanced Hg-elimination rate in males. In contrast, long-term elimination of PCBs is negligible for both sexes. According to this explanation, males not only ingest Hg at a higher rate than females but also eliminate Hg at a higher rate than females, in fish species other than sea lamprey. Male sea lamprey do not possess either of the above-specified androgens. These apparent sex differences in SMRs, activities, and Hg-elimination rates in teleost fishes may also apply, to some degree, to higher vertebrates including humans. Our synthesis findings will be useful in (1) developing sex-specific bioenergetics models for fish, (2) developing sex-specific risk assessment models for exposure of humans and wildlife to contaminants, and (3) refining Hg mass balance models for fish and higher vertebrates.

Differences in Energy Expenditures and Growth Dilution Explain Higher PCB Concentrations in Male Summer Flounder.

Comparison of polychlorinated biphenyl (PCB) concentrations between the sexes of mature fish may reveal important behavioral and physiological differences between the sexes. We determined whole-fish PCB concentrations in 23 female summer flounder Paralichthys dentatus and 27 male summer flounder from New Jersey coastal waters. To investigate the potential for differences in diet or habitat utilization between the sexes, carbon and nitrogen stable isotope ratios were also determined. In 5 of the 23 female summer flounder, PCB concentrations in the somatic tissue and ovaries were determined. In addition, we used bioenergetics modeling to assess the contribution of the growth dilution effect to the observed difference in PCB concentrations between the sexes. Whole-fish PCB concentrations for females and males averaged 87 and 124 ng/g, respectively; thus males were 43% higher in PCB concentration compared with females. Carbon and nitrogen stable isotope ratios did not significantly differ between the sexes, suggesting that diet composition and habitat utilization did not vary between the sexes. Based on PCB determinations in the somatic tissue and ovaries, we predicted that PCB concentration of females would increase by 0.6%, on average, immediately after spawning due to release of eggs. Thus, the change in PCB concentration due to release of eggs did not explain the higher PCB concentrations observed in males. Bioenergetics modeling results indicated that the growth dilution effect could account for males being 19% higher in PCB concentration compared with females. Thus, the bulk of the observed difference in PCB concentrations between the sexes was not explained by growth dilution. We concluded that a higher rate of energy expenditure in males, stemming from greater activity and a greater resting metabolic rate, was most likely the primary driver for the observed difference in PCB concentrations between the sexes.

Congener Patterns of Persistent Organic Pollutants Establish the Extent of Contaminant Biotransport by Pacific Salmon in the Great Lakes.

In the Great Lakes, introduced Pacific salmon (Oncorhynchus spp.) can transport persistent organic pollutants (POPs), such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs), to new environments during their spawning migrations. To explore the nature and extent of POP biotransport by salmon, we compared 58 PCB and 6 PBDE congeners found in spawning salmon directly to those in resident stream fish. We hypothesized that stream fish exposed to salmon spawners would have congener patterns similar to those of salmon, the presumed contaminant source. Using permutational multivariate analysis of variance (PERMANOVA) and nonmetric multidimensional scaling (NMDS), we found that POP congener patterns of Pacific salmon varied among regions in the Great Lakes basin (i.e., Lake Huron, Lake Michigan, or Lake Superior), tissue type (whole fish or eggs), and contaminant type (PCB or PBDE). For stream-resident fish, POP congener pattern was influenced by the presence of salmon, location (i.e., Great Lakes Basin), and species identity (i.e., brook trout [Salvelinus fontinalis] or mottled sculpin [Cottus bairdii]). Similarity in congener patterns indicated that salmon are a source of POPs to brook trout in stream reaches receiving salmon spawners from Lake Michigan and Lake Huron but not from Lake Superior. Congener patterns of mottled sculpin differed from those of brook trout and salmon, suggesting that brook trout and mottled sculpin either use salmon tissue to differing degrees, acquire POPs from different dietary sources, or bioaccumulate or metabolize POPs differently. Overall, our analyses identified the important role of salmon in contaminant biotransport but also demonstrated that the extent of salmon-mediated POP transfer and uptake in Great Lakes tributaries is location- and species-specific.

Laboratory estimation of net trophic transfer efficiencies of PCB Congeners to lake trout (Salvelinus namaycush) from its prey.

A technique for laboratory estimation of net trophic transfer efficiency (γ) of polychlorinated biphenyl (PCB) congeners to piscivorous fish from their prey is described herein. During a 135-day laboratory experiment, we fed bloater (Coregonus hoyi) that had been caught in Lake Michigan to lake trout (Salvelinus namaycush) kept in eight laboratory tanks. Bloater is a natural prey for lake trout. In four of the tanks, a relatively high flow rate was used to ensure relatively high activity by the lake trout, whereas a low flow rate was used in the other four tanks, allowing for low lake trout activity. On a tank-by-tank basis, the amount of food eaten by the lake trout on each day of the experiment was recorded. Each lake trout was weighed at the start and end of the experiment. Four to nine lake trout from each of the eight tanks were sacrificed at the start of the experiment, and all 10 lake trout remaining in each of the tanks were euthanized at the end of the experiment. We determined concentrations of 75 PCB congeners in the lake trout at the start of the experiment, in the lake trout at the end of the experiment, and in bloaters fed to the lake trout during the experiment. Based on these measurements, γ was calculated for each of 75 PCB congeners in each of the eight tanks. Mean γ was calculated for each of the 75 PCB congeners for both active and inactive lake trout. Because the experiment was replicated in eight tanks, the standard error about mean γ could be estimated. Results from this type of experiment are useful in risk assessment models to predict future risk to humans and wildlife eating contaminated fish under various scenarios of environmental contamination.

Polychlorinated biphenyl congener distributions in burbot: evidence for a latitude effect.

The authors compared the distributions of the congeners of polychlorinated biphenyls (PCBs) detected in whole-body samples of burbot (Lota lota) from Great Slave Lake and Lake Erie. Total PCB concentrations in Great Slave Lake burbot were about one-sixtieth of the concentrations in Lake Erie burbot. Burbot from Great Slave Lake contained a higher proportion of lower-chlorinated (2-6 chlorines) congeners than did burbot from Lake Erie; the reverse occurred for more highly chlorinated (7-9 chlorines) congeners. Hexachloro congeners, followed by pentachloro congeners, dominated the proportions of total PCBs in burbot from both lakes. There were no differences between sexes in whole-body samples or between gonad and somatic tissues in the proportions of the 39 congeners and 3 sets of coeluters detected in burbot from Great Slave Lake. In contrast, there were distinct sex differences in congener distributions for older burbot from Lake Erie. The results generally supported a prediction of higher proportions of lower-chlorinated PCB homologs in organisms in remote polar areas. However, the latitudinal effect on PCB congener distribution may be more complex than that portrayed in previous studies.

Sexual difference in PCB congener distributions of burbot (Lota lota) from Lake Erie.

Concentrations of 86 congeners of polychlorinated biphenyls (PCBs) were determined for 25 adult female and 25 adult male burbot (Lota lota) from Lake Erie. Significant differences in mean proportions of total PCB concentration between males and females ages 14-17 were found for 26 congeners. Males generally had higher mean proportions of hexachloro congeners, whereas females had higher mean proportions of more highly chlorinated congeners. In contrast, only four congeners exhibited differences between males and females ages 6-13 in mean proportions of total PCB. Therefore, the sexual difference in PCB congener distribution widened for older burbot. Males ages 14-17 also had higher proportions of certain hexachloro congeners than the other three demographic groups (males ages 6-13, females ages 6-13, and females ages 14-17) we examined. The reverse was true for more highly chlorinated congeners. The results supported a previous hypothesis that older male burbot spent a substantial amount of time feeding in the vicinity of mouths of rivers with sediment contaminated with PCBs. However, additional studies are needed to clarify and corroborate this apparent "hot spot" effect, such as seasonal movements, spatial distributions, and diet of burbot; and PCB congener distributions of various species of prey fishes captured at various locations throughout the eastern basin of Lake Erie and at different locations in the contaminated rivers.

Net trophic transfer efficiencies of polychlorinated biphenyl congeners to lake trout (Salvelinus namaycush) from its prey.

Lake trout (Salvelinus namaycush) were fed bloater (Coregonus hoyi) in eight laboratory tanks over a 135-d experiment. At the start of the experiment, four to nine fish in each tank were sacrificed, and the concentrations of 75 polychlorinated biphenyl (PCB) congeners within these fish were determined. Polychlorinated biphenyl congener concentrations were also determined in the 10 lake trout remaining in each of the eight tanks at the end of the experiment as well as in the bloater fed to the lake trout. Each lake trout was weighed at the start and the end of the experiment, and the amount of food eaten by the lake trout was recorded. Using these measurements, net trophic transfer efficiency (γ) from the bloater to the lake trout in each of the eight tanks was calculated for each of the 75 congeners. Results showed that γ did not vary significantly with the degree of chlorination of the PCB congeners, and γ averaged 0.66 across all congeners. However, γ did show a slight, but significant, decrease as log K(OW) increased from 6.0 to 8.2. Activity level of the lake trout did not have a significant effect on γ.

Resident fishes display elevated organic pollutants in salmon spawning streams of the great lakes.

Pacific salmon (Oncorhynchus spp.) can transport bioaccumulated organic pollutants to stream ecosystems where they spawn and die. We quantified PCBs, DDE, and PBDEs in resident fishes from 13 Great Lakes tributaries to assess biotransport of pollutants associated with introduced Pacific salmon. Resident fishes sampled from salmon spawning reaches had higher mean pollutant concentrations than those from upstream reaches lacking salmon (93.5 and 4.1 µg x kg(-1) [PCB], 24.0 and 3.1 µg x kg(-1) [DDE], 8.5 and 1.0 µg x kg(-1) [PBDE], respectively), but differences varied substantially among lake basins. In Lake Michigan tributaries, PCB concentrations in resident fishes from salmon reaches were over four times higher than those from salmon reaches in Lake Huron and over 30 times higher than those from Lake Superior. Moreover, resident fish pollutant concentrations were better explained by pollutant inputs from salmon (µg x m(-2); R(2) = 0.76 [PCB], 0.64 [DDE], 0.64 [PBDE]) than by land development/agriculture, watershed area, resident fish species, body length, or lipid content. These results suggest that pollutant dispersal to stream ecosystems via biotransport is an often overlooked consequence of salmon stocking and historical food web contamination in the Great Lakes. Our findings have implications for Great Lakes management, including dam removal and wildlife conservation.

Glucose deprivation inhibits multiple key gene expression events and effector functions in CD8+ T cells.

We recently reported that differentiation of CD8(+) T cells from the naïve to the effector state involves the upregulation of glucose-dependent metabolism. Glucose deprivation or inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) selectively inhibited production of IFN-gamma but not of IL-2. To determine a more global role of glucose metabolism on effector T-cell function, we performed gene array analysis on CD8(+) effector T cells stimulated in the presence or absence of 2-DG. We observed that expression of only 10% of genes induced by TCR/CD28 signaling was inhibited by 2-DG. Among these were genes for key cytokines, cell cycle molecules, and cytotoxic granule proteins. Consistent with these results, production of IFN-gamma and GM-CSF, cell cycle progression, upregulation of cyclin D2 protein, cytolytic activity, and upregulation of granzyme B protein and also conjugate formation were exquisitely glucose-dependent. In contrast to glucose, oxygen was little utilized by CD8(+) effector T cells, and relative oxygen deprivation did not inhibit these CTL functional properties. Our results indicate a particularly critical role for glucose in regulating specific effector functions of CD8(+) T cells and have implications for the maintenance of the effector phase of cellular immune responses in target tissue microenvironments such as a solid tumor.

Net trophic transfer efficiencies of polychlorinated biphenyl congeners to lake whitefish (Coregonus clupeaformis) from their food.

Lake whitefish (Coregonus clupeaformis) were fed rainbow smelt (Osmerus mordax) in four laboratory tanks over a 133-d experiment. At the start of the experiment, 10 to 14 of the fish in each tank were sacrificed, and the concentrations of 40 polychlorinated biphenyl (PCB) congeners within these fish were determined. Polychlorinated biphenyl congener concentrations were also determined in the 15 lake whitefish remaining in each of the four tanks at the end of the experiment as well as in the rainbow smelt fed to the lake whitefish. Each lake whitefish was weighed at the start and the end of the experiment, and the amount of food eaten by the lake whitefish during the experiment was tracked. Using these measurements, net trophic transfer efficiency (gamma) from the rainbow smelt to the lake whitefish in each of the four tanks was calculated for each of the 40 PCB congeners. Results showed that gamma decreased exponentially as log K(OW) for the congeners increased from 6 to 8. Further, gamma averaged 0.70 for the tetrachloro congeners but averaged only 0.45 for the higher chlorinated congeners.

Induction of cytotoxic granules in human memory CD8+ T cell subsets requires cell cycle progression.

Memory CD8(+) T cell responses are thought to be more effective as a result of both a higher frequency of Ag-specific clones and more rapid execution of effector functions such as granule-mediated lysis. Murine models have indicated that memory CD8(+) T cells exhibit constitutive expression of perforin and can lyse targets directly ex vivo. However, the regulated expression of cytotoxic granules in human memory CD8(+) T cell subsets has been underexplored. Using intracellular flow cytometry, we observed that only a minor fraction of CD45RA(-)CD8(+) T cells, or of CD8(+) T cells reactive to EBV-HLA2 tetramer, expressed intracellular granzyme B (GrB). Induction of GrB-containing cytotoxic granules in both CD45RA(+) and CD45RA(-) cells was achieved by stimulation with anti-CD3/anti-CD28 mAb-coated beads, required at least 3 days, occurred after several rounds of cell division, and required cell cycle progression. The strongest GrB induction was seen in the CCR7(+) subpopulations, with poorest proliferation being observed in the CD45RA(-)CCR7(-) effector-memory pool. Our results indicate that, as with naive T cells, induction of cytotoxic granules in human Ag-experienced CD8(+) T cells requires time and cell division, arguing that the main numerical advantage of a memory T cell pool is a larger frequency of CTL precursors. The fact that granule induction can be achieved through TCR and CD28 ligation has implications for restoring lytic effector function in the context of antitumor immunity.

Cutting edge: cytotoxic granule polarization and cytolysis can occur without central supramolecular activation cluster formation in CD8+ effector T cells.

The functional implication of molecular segregation within the immunological synapse remains uncertain. We recently reported that effector but not naive TCR transgenic murine CD8+ T cells formed immunological synapses containing a central supramolecular activation cluster (cSMAC), suggesting that execution of effector functions such as cytolytic activity might be facilitated by the cSMAC structure. We have now explored this hypothesis using two approaches. First, by simultaneously imaging cSMAC formation and mobilization of cytotoxic granules to the synapse, we observed no correlation between the presence of a cSMAC and granule reorientation. Second, we took advantage of the observation that CD28 costimulation markedly enhances cSMAC formation. Granule polarization to the contact site was indistinguishable with B7-1+ and B7-1- target cells, and cytolytic activity against B7-1+ or B7-1- targets was similar and granule-dependent. Together, our results indicate that the formation of a cSMAC is not required for cytolytic activity in CD8+ effector T cells.

Formation of a central supramolecular activation cluster is not required for activation of naive CD8+ T cells.

Although both naive and effector T lymphocytes interact with antigen-expressing cells, the functional outcome of these interactions is distinct. Naive CD8(+) T cells are activated to proliferate and differentiate into effector cytolytic T lymphocytes (CTL), whereas CTL interact with specific targets, such as tumor cells, to induce apoptotic death. We recently observed that several molecules linked to actin cytoskeleton dynamics were up-regulated in effector vs. naive CD8(+) T cells, leading us to investigate whether T cell differentiation is accompanied by changes in actin-dependent processes. We observed that both naive and effector CD8(+) T cells underwent T cell receptor capping and formed stable conjugates with antigen-specific antigen-presenting cells. However, the characteristics of the immunological synapse were distinct. Whereas accumulation of signaling molecules at the T cell/antigen-presenting cell contact site was detectable in both naive and effector CD8(+) T cells, only effector cells developed a central supramolecular activation cluster as defined by punctate focusing of PKC theta, phospho-PKC theta, and phospho-ZAP70. Extended kinetics, CD28 costimulation, and high-affinity antigenic peptide did not promote PKC theta focusing in naive cells. Nonetheless, naive CD8(+) T cells polarized the microtubule organizing center, produced IL-2, proliferated, and differentiated into effector cells. Our results suggest that the formation of a central supramolecular activation cluster is not required for activation of naive CD8(+) T cells and support the notion that one role of an organized immune synapse is directed delivery of effector function.

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration.

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.

Gene array and protein expression profiles suggest post-transcriptional regulation during CD8+ T cell differentiation.

Peripheral CD8(+) T cells circulate in a quiescent naive state until they are primed by specific antigen and differentiate into effector cells. In the effector state, CD8(+) T cells acquire cytolytic activity and produce increased levels of cytokines such as interferon-gamma. They also exhibit increased T cell receptor sensitivity, decreased CD28 dependence, and become inhibitable by CTLA-4 and other negative regulatory pathways. We hypothesized that one mechanism by which these two states are regulated is via differential expression of specific genes. To this end, basal gene expression profiles of naive and effector 2C TCR transgenic x RAG2(-/-) CD8(+) T cells were analyzed using Affymetrix arrays representing 11,000 genes. Of the 177 differentially expressed known genes, 68 were expressed at higher levels in effector cells, but 109 were more abundant in naive cells, supporting the notion that the naive state is not passive. Expression of genes related to metabolism, actin cytoskeletal dynamics, and effector function increased with priming, whereas expression of putative anti-proliferative genes decreased. Semiquantitative reverse transcription-PCR was utilized as a secondary validation for selected transcripts, and Western blot analysis was used to examine protein expression for molecules of interest. Surprisingly, for 24 genes examined, 12 showed discordant protein versus mRNA expression. In summary, our study indicates that: 1) not only does the expression of some genes in naive CD8(+) T cells become up-regulated upon priming, but the expression of other genes is down-regulated as well and 2) the complexities of T cell differentiation include regulation at the post-transcriptional level.