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(P049) ALAD characterization and sensitivity studies in common wildlife test species.
McFarland, Craig*,1, McBride, Tobias1, Hooper, Michael1, 1 The Institute of Environmental and Human Health (TIEHH), Lubbock, Tx, USA
ABSTRACT- Inhibition of delta-aminolevulinic acid dehydratase (ALAD; EC 188.8.131.52) activity in erythrocytes is used as a biomarker in evaluating Pb exposure and effects in humans and in animals because it is sensitive to, and specific for, blood Pb concentrations. This study characterized erythrocyte ALAD activity, estimated median inhibitory Pb concentrations (IC50s), and reports the results of inhibition reversibility experiments in prairie voles (Microtus ochrogaster), American kestrels (Falco sparverius), European starlings (Sturnus vulgaris), and the domestic pig (Sus domesticus, used as a control blood source). Kinetic analysis of erythrocyte ALAD activity revealed differences in substrate pH optima and activity values. Optimal pH and uninhibited activity levels (in nmol ALA/(min*ml RBC-1) were, respectively, 6.6 and 19.4 in the prairie vole; 6.4 and 119 in the American kestrel; and 6.5 and 37.9 in the domestic pig. In vitro incubation of erythrocyte ALAD with increasing concentrations of lead acetate (PbOAc) for 10 minutes resulted in sigmoidal inhibition curves of the ALAD activity. The Pb IC50 concentrations ranged from 5E-07M to 1E-05M. Incubation of lysed erythrocytes with 1E-05M PbOAc followed by 7.5- to 150-fold dilution demonstrated reactivation of inhibited ALAD activity. ALAD levels (inhibited 90%) recovered over a quarter of their original activity with maximal dilution. Excessive erythrocyte dilution appears to allow reactivation of inhibited enzyme and should be avoided when assessing inhibition of this enzyme.
Key words: alad, enzyme, inhibition, erythrocyte
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