3.1. Acute toxicity
Aqueous chlorpyrifos concentrations in exposure containers showed ~20–25% variability between measured and dosed concentrations (). These data agreed with previous results in similar exposure systems that showed a <20% variability between nominal and measured concentrations (unpublished results). Exposure of juvenile Chinook salmon to a range of chlorpyrifos concentrations elicited a dose-dependent acute response with 100% mortality observed at 81 μg/l, 20% mortality at 7.3 μg/l and 0% mortality at 1.2 μg/l. Fish exposure to a range of esfenvalerate concentrations resulted in 100% mortality at 1.0 μg/l (nominal) or 0% mortality at 0.01 and 0.1 μg/l(nominal). All subsequent enzyme assays were performed on fish that were exposed to sublethal pesticide doses.
Juvenile Chinook salmon mortality following pesticide exposure
3.2. Acetylcholinesterase activity
AChE activity varied with pesticide concentration and tissue type (). Solvent (methanol) exposure altered AChE activity in the brain (11% decrease, P <0.001), but not in muscle, relative to untreated controls. Significant suppression of AChE activity by chlorpyrifos treatment occurred only at the highest chlorpyrifos dose. Relative to solvent controls, exposure to low doses of chlorpyrifos (1.2 μg/l) did not suppress brain AChE, and even slightly elevated muscle AChE levels (112%, P <0.05). In contrast, high dose chlorpyrifos exposure (7.3 μg/l) reduced AChE activity by 85% (brain) and 92% (muscle) compared to solvent controls, and by 84% (brain) and 93% (muscle) relative to low dose fish (P <0.001).
Acetylcholinesterase activity in pesticide-exposed juvenile Chinook salmona
Esfenvalerate exposure did not affect AChE activity in either tissue at the lowest dose, 0.01 μg/l. However, at 0.1 μg/l, brain AChE activity increased by 10% and muscle AChE activity decreased by ~10%. These differences, though slight, were significantly different from solvent controls (0.05 > P > 0.01). Activities at the two esfenvalerate exposure concentrations were not statistically different from each other.
Inter-individual variability in AChE activity was generally less in brain (~10%) than in muscle (20%). The ratio in activity between the individual with the highest level of activity to the individual with the lowest ranged from 1.1 for esfenvalerate at 0.1 μg/l in muscle to 2.2 at 0.01 μg/l esfenvalerate in brain. The majority of the exposures showed no significant effect upon the range of inter-individual activity, with the exception of activity in the muscle after chlorpyrifos exposure. This sample showed a steady increase in the range of activity with treatment. However, the overall change was still relatively small (from 1.2- to 2-fold).
3.3. Carboxylesterase assays
Pesticide exposure affected carboxylesterase activity in a compound and dose-dependent fashion (). All three substrates examined produced very similar results, with hydrolysis activity profiles for each substrate responding identically to pesticide exposure. Increasing concentrations of chlorpyrifos caused significant decreases in carboxylesterase activity (). An identical inhibition pattern was observed using an α-naphthyl acetate carboxylesterase activity stain in a native gel (). Significant reductions in carboxylesterase activity at the highest chlorpyrifos dose (7.3 μg/l) were detected by all three substrates, with reductions of 79% (PNPA), 52% (α-cyano acetate) and 55% (α-cyano butyrate), relative to solvent controls. Only one substrate, PNPA, detected a significant reduction (56%) in carboxylesterase activity at the low chlorpyrifos dose (1.2 μg/l; P <0.001). Solvent exposure had a significant effect upon carboxylesterase activity for all three substrates, with reductions ranging from a 44% decrease in activity for the α-cyanoacetate to 23% for PNPA, relative to untreated controls. The α-cyano acetate and α-cyano butyrate substrates exhibited a wider range of inter-individual carboxylesterase activities (2.4–3.0- and 1.3–3.9-fold, respectively), than did PNPA (1.3–1.8-fold).
Carboxylesterase activity in liver cytosol from pesticide-exposed juvenile Chinook salmona
Fig. 3. Carboxylesterase activity in liver cytosol from chlorpyrifos-treated juvenile Chinook salmon. Activity was visualized in a 12% tris-glycine native gel stained with α-naphthyl acetate. Lane 1: chlorpyrifos-exposed at 10 μg/l; lane 2: chlorpyrifos-exposed (more ...)
Exposure to esfenvalerate had very little effect upon carboxylesterase activity. All concentrations tested were essentially identical to solvent control values for all substrates, and no significant solvent effects were observed (). Exposure to 0.01 and 0.1 μg/l esfenvalerate inhibited carboxylesterase activities relative to untreated controls (P <0.05) when measured with the α-cyano acetate substrate, however values were not significantly different from the solvent control. The range in inter-individual variation in carboxylesterase activity in response to esfenvalerate exposure was similar to that observed with chlorpyrifos. Generally, the range in activity decreased with increasing pesticide concentration with all three substrates. The only exception occurred with the butyrate substrate in which the lowest concentrations of chlorpyrifos and esfenvalerate elicited the greatest range of activity.
3.4. Determination of carboxylesterase kinetic constants in different species
The carboxylesterase kinetic constants varied with species and substrate. It is not appropriate to directly compare species in this study as the tissues were prepared differently and thus limited conclusions can be drawn from the data. For medaka and splittail, whole body homogenates were used, for salmon, liver homogenates were used, and for porcine carboxylesterase, measurements were made using a partially purified commercial esterase. Because purified carboxylesterases were not used for any species, kinetic constants are reported as apparent values. The Michaelis constant (Km app) for PNPA varied from a low of 95 μM for medaka to a high of 578 μM for split-tail (a six-fold range) as shown in . Results for the α-cyanoacetate and butyrate substrates were more similar to each other than to PNPA. Km app values were ~30 μM for the acetate for all species except split-tail (5.8 μM), whereas butyrate values ranged from 7.8 to 16.8 μM. Interestingly, values for the salmon and porcine enzymes fell within the range observed for medaka, splittail and trout, even though the salmon and porcine enzymes were prepared from single organs (salmon) or were partially purified (porcine) and had higher specific activities.
Kinetic constants for substrates used in this studya
In contrast to the Michaelis constant, the variation in Vmax app
was much greater among the different species examined. This variability tracked carboxylesterase concentration in the preparation very closely, as Vmax app
values are normalized to unit protein. The two samples that were prepared identically (whole body homogenates) had similar Vmax app
values for PNPA, 198,000 nmol/min/mg for medaka and 129,600 nmol/min/mg for splittail, whereas the samples that contained a higher degree of specific activity (salmon liver or porcine esterase) had Vmax app
values that were up to 10-fold higher. Similar results were reported for studies with rainbow trout liver preparations, which reported a Vmax app
for PNPA of 672,000 nmol/min/mg (Barron et al., 1999
). Similar trends were observed with both the α-cyanoacetate and butyrate substrates.
3.5. Measurement of inhibitor potency
The OP pesticides diazinon and chlorpyrifos, and their oxon-derivatives, were examined for their ability to inhibit carboxylesterase activity in four species using three different substrates (). The IC50’s (concentration of enzyme required to reduce enzyme velocity by 50%) for diazinon and chlorpyrifos were greater than 100 μM for all three substrates in all four species, indicating that these two pesticides do not significantly inhibit carboxylesterase activity. In contrast, the oxon forms of both pesticides were significant carboxylesterase inhibitors, with IC50 values in the low nM range for all substrates in all species examined, except splittail. In splittails, diazinon-oxon did not inhibit α-cyano acetate hydrolysis at any concentration examined (IC50 > 100 μM) while chlorpyrifosoxon mediated inhibition was as much as 1000-fold less potent than in the other species tested.
Inhibition concentrations (IC50) for selected organophosphatesa
Of the two oxons tested, chlorpyrifos-oxon was the more potent carboxylesterase inhibitor, being on average ~10-fold more potent than diazinon-oxon. However, this number varied considerably with substrate and species. For medaka and the porcine esterase, the IC50s for the two different oxons did not vary greatly with substrate. However, for the Chinook salmon esterase, the two acetate-containing substrates, PNPA and α-cyano acetate, had IC50s more similar to one another than to the IC50 for the α-cyano butyrate substrate. This observation is in spite of the fact that the alcohol moiety of α-cyano acetate is quite different from that of PNPA, being similar to α-cyano butyrate (see Figs. and for a description of acid and alcohol nomenclature and substrate structures).
3.6. Pyrethroid hydrolysis
Pyrethroid surrogate hydrolysis was not observed with any of the fish species examined in this study. Only the porcine enzyme significantly hydrolyzed α-cyanoesters of pyrethroid acids as shown in . A number of assay conditions were varied in an attempt to measure hydrolysis activity. The temperature of the assay was increased up to 37 °C and the pH up to 9; however none of these conditions was sufficient to increase pyrethroid hydrolysis activity to quantifiable levels. Unless otherwise stated, the substrates were a mixture of isomers. The hydrolysis rates of the eight different pyrethroid surrogates examined did not vary by more than ~10-fold across all compounds (3.78-38.16 nmol/min/mg). For compound 1, an esfenvalerate mimic, hydrolysis was ~2.5-fold slower than for the corresponding R-analog (compound 2). The cypermethrin surrogate (compound 3) was hydrolyzed at the same rate as its dimethyl analog (synthesized from chrysanthemic acid, compound 7); however substitution of the dichloro moiety by dibromo (deltamethrin surrogate) decreased hydrolysis by 7-fold (com-pound 4). The λ-cyhalothrin surrogates~exhibited a 4-fold difference in hydrolysis rate, with the trans isomer (compound 5) hydrolyzed faster than the cis (com-pound 6).
Hydrolysis activity of synthetic pyrethroid surrogate substrates
3.7. CYP1A levels
CYP1A protein expression was slightly, but significantly (P <0.05), suppressed (1.4-fold) in salmon liver cytosol from the high dose (7.3 μg/l) chlorpyrifos-treated group relative to those treated with solvent carrier (). However, there was no significant effect of chlorpyrifos at either dose relative to untreated controls. Esfenvalerate treatment had no effect on hepatic CYP1A protein expression at either dose (). No significant trends were observed in the range of CYP1A levels in response to the different pesticide exposures. The range varied from a low of 1.6-fold for the highest dose of esfenvalerate (similar to the control value of 1.7) to a high of 4.3 for the highest dose of chlorpyrifos. However, variability in response to low dose esfenvalerate (4.2) was similar to that provoked by the high dose of chlorpyrifos (4.3), suggesting salmon response to these pesticides was generally similar. The variability in response to solvent exposure was moderate, with a range of 2.5 and 2.6 for esfenvalerate- and chlorpyrifos-exposed fish, respectively, ranges higher than the response range for untreated controls (1.7).
Relative levels of CYP1A protein in liver cytosol from pesticide-exposed Chinook salmona