Chris Lloyd Mills

The effect of copper on osmoregulation in the freshwater amphipod Gammarus pulex

The influence of copper on osmoregulation in the freshwater amphipod Gammarus pulex was determined from the analysis of water permeability, haemolymph sodium concentration, sodium influx and gill Na(+)/K(+) ATPase and Mg(2+) ATPase activity. Exposure to nominal copper concentrations of 100 microg l(-1) or greater caused a significant reduction in both haemolymph sodium concentration and sodium influx within 4 h. Measurements of water permeability, expressed as the half-time of exchange of body water (t(1/2)), excluded structural gill damage as the cause of this fall in haemolymph sodium. Copper at 10 microg l(-1) or above in the assay solution significantly reduced gill Na(+)/K(+) ATPase activity. In contrast gill Mg(2+) ATPase activity was markedly less affected by copper. These differences in enzyme sensitivity were considered with respect to the potential mechanisms of copper toxicity.

Cation regulation and alteration of water permeability in the amphipod Dikerogammarus villosus: an indicator of invasion potential.

The freshwater amphipod Dikerogammarus villosus (Sowinsky) can devastate gammarid communities that it invades. We investigated aspects of osmoregulation in D. villosus to determine whether it could adjust to altered salinity. D. villosus maintained relatively constant haemolymph Na + concentration up to an acclimation sa- linity of 10 ‰, and haemolymph K + , Ca 2+ and Mg 2+ concentration up to a salinity of 16 ‰. Increasing acclimation salinity above 16 ‰ induced sharp increases in haemolymph cation concentrations, suggesting osmoregulatory dys- function. Both sodium infl ux and the half time of exchange of water (t1/2) fell with increasing acclimation salinity. In addition, sudden transfer of freshwater acclimated D. villosus to 20 ‰ produced a rapid reduction in sodium infl ux to the rate found in amphipods acclimated to 20 ‰. This indicates that D. villosus can regulate water permeability and sodium infl ux, both of which are important osmoregulatory mechanisms in euryhaline gammarids. In conclu- sion D. villosus poses a serious invasion risk to areas accessible via transit through brackish water of salinity 20 ‰ or less. It is therefore recommended that complete mid ocean ballast water exchange is practiced, to reduce the risk of introduction of D. villosus to areas currently free of this amphipod, e.g. North America and the Great Lakes.

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 μM CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12 μM CdCl2 for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 μM CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12 μM Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.

Molecular pharmacology : from DNA to drug discovery

This textbook provides a fresh, comprehensive and accessible introduction to the rapidly expanding field of molecular pharmacology. Adopting a drug target-based, rather than the traditional organ/system based, approach this innovative guide reflects the current advances and research trend towards molecular based drug design, derived from a detailed understanding of chemical responses in the body. Drugs are then tailored to fit a treatment profile, rather than the traditional method of ‘trial and error’ drug discovery which focuses on testing chemicals on animals or cell cultures and matching their effects to treatments.

Parasitic infection manipulates sodium regulation in the freshwater amphipod Gammarus pulex (L.).

The acanthocephalan parasite Polymorphus minutus induces both physiological and behavioural effects in its intermediate host, Gammarus pulex. The net effect of parasite infection is to increase the likelihood of transmission to the definitive host. Osmoregulation is an energetically expensive mechanism that allows G. pulex to survive in dilute media. Any factor influencing osmoregulation is thus likely to affect the allocation of resources to other areas. This study investigated whether P. minutus infection alters sodium regulation in G. pulex. Haemolymph sodium concentration, water permeability and sodium fluxes were measured over the salinity acclimation range of G. pulex. Water permeability was unaltered by either acclimation salinity or parasite infection. Acclimation to 12‰ significantly raised the haemolymph sodium concentration, reduced the sodium influx, and increased the sodium efflux, to the same extent in both uninfected and infected G. pulex. However, parasite infection induced a significant increase in haemolymph sodium concentration in G. pulex acclimated to 6‰, which was not observed in uninfected G. pulex acclimated to the same salinity. Also, both sodium influx and sodium efflux were significantly lower in parasitized G. pulex acclimated to 6‰, when compared to uninfected G. pulex acclimated to the same salinity. It was concluded that the parasite induced disturbances to sodium regulation in G. pulex acclimated to 6‰ were a functional consequence of the manipulative strategy employed to alter behaviour, rather than a primary target.

OSMOREGULATION IN HYPOGEAN POPULATIONS OF THE FRESHWATER AMPHIPOD, GAMMARUS PULEX (L.)

Abstract The freshwater amphipod Gammarus pulex is widely distributed in freshwater streams and rivers of Europe. This amphipod also has isolated hypogean populations, which are transparent in appearance, suggestive of adaptation to their cave environment. Since cave habitats are often food limited, physiological adaptations have been observed that reduce the energy expenditure of cave organisms. Osmoregulation is an energetically expensive mechanism that allows gammarids to survive in fresh water. This study tested the hypothesis that differences in osmoregulation existed between hypogean and epigean populations of G. pulex. The osmoregulatory parameters measured were haemolymph cation concentrations, water and sodium fluxes and gill Na+/K+-ATPase activity. The hypogean G. pulex had significantly lower haemolymph sodium and potassium concentrations, but had a significantly higher haemolymph ammonium concentration than the epigean G. pulex. The low food availability in the hypogean environment was considered to be the underlying cause for these differences in haemolymph ion concentrations.