The objective of this proposal is to evaluate the effect of chronic lead exposure (water-borne divalent lead) on sodium, potassium ATP-ase (Na+K+-ATPase, sodium pump) activity in the gill of Pimephales promelas (fathead minnow) and how this enzymatic activity is affected by lead-induced peroxidation of gill lipids. Our working hypothesis is that lead affects Na+K+-ATPase by promoting formation of isoprostanes (iPs), prostaglandin-like fatty acid derivatives, within gill membranes by a free radical-mediated mechanism. Our initial studies, using isolated gill preparations, indicate that concentrations of lead near 1 ppm cause pronounced stimulation of Na+K+-ATPase activity. Priority will be given to gaining a more complete picture of the dose and time-dependence of this in vitro effect. Subsequently, we will evaluate the possible dependence of the stimulatory effect on protein phosphorylation. The in vivo influence of lead on gill Na+K+-ATPase activity will be examined in aquarium studies wherein adult fathead minnows are exposed to selected concentrations of lead (as lead nitrate) for up to 3 months. As part of this work, the tissue distribution of lead will be determined (e.g., gills, skin and muscle, viscera, skeleton) as well as the temporal relationships between accumulation of lead in the gill and sodium pump activity. Similar experiments will be carried out to follow the time course for the release of gill iPs in fish exposed to lead in the aquarium water. Gill lipids will be extracted, and analyzed by LC/MS-Electrospray, with structural confirmation with authentic and deuterated homologous standards. In some experiments iP release will be measured after adding lead to in vitro preparations (isolated gills and membrane fractions) from control fish. In both the in vivo and in vitro studies, we will determine the lead concentration of gill membrane fractions enriched in sodium pump activity. Data will be analyzed to determine if membrane lead levels correlate with Na+K+-ATPase activity and/or iP generation. Later in the project, we will evaluate the hypothesis that chronic lead exposure may result in a compensatory rise in levels of reduced glutathione (GSH) within the gill. Also, in the last year, renal proximal tubules will be isolated from rats administered lead in the drinking water. Suspensions of tubules will be incubated to assess iP biosynthesis, sodium pump activity, and GSH status. Results will be analyzed to determine if lead causes analogous iP-dependent changes in proximal nephron and gill epithelium.
| Spokas, Eric G; Harshman, Scott; Cohen, Glenn M et al. (2008) Release of the lipid peroxidation marker 8-epi-prostaglandin F2 alpha from isolated gill pavement cells. Environ Toxicol Chem 27:1569-75 |
| Spokas, Eric G; Spur, Bernd W; Smith, Holly et al. (2006) Tissue lead concentration during chronic exposure of Pimephales promelas (fathead minnow) to lead nitrate in aquarium water. Environ Sci Technol 40:6852-8 |
| Spokas, E G; Crivellone, M D; Kemp, F et al. (2002) Characterization of sodium, potassium, ATPase activity in the gills of Pimephales promelas (fathead minnow): influence of in vitro exposure to lead. Bull Environ Contam Toxicol 69:384-92 |
| Spokas, E G; Spur, B W (2001) Rapid measurement of low levels of sodium, potassium-ATPase activity by ascorbic acid reduction without strong acid. Anal Biochem 299:112-6 |
