In Vivo Acetylcholinesterase Inhibition, Metabolism, and Toxicokinetics of Aldicarb in Channel Catfish: Role of Biotransformation in Acute Toxicity

Everett J. Perkins, Jr.*,1 and Dan Schlenk{dagger}

* Lilly Research Laboratories, Department of Drug Disposition, Eli Lilly and Company, Indianapolis, Indiana 46285; and {dagger} Department of Pharmacology, Environmental Toxicology Research Program, University of Mississippi, University, Mississippi 38677

Received May 17, 1999; accepted August 9, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in mammals, birds, and fish through the inhibition of acetylcholinesterase (AChE), and may present high potential for exposure of aquatic organisms during periods of runoff. Toxicity studies have shown that channel catfish are less sensitive to the acute toxic effects of aldicarb than are rainbow trout or bluegill. An earlier in vitro study suggests that the aldicarb resistance in catfish may be related to a low level of bioactivation to the potent aldicarb sulfoxide. The current study examines the toxicity, AChE inhibition, plasma kinetics, and in vivo metabolism of aldicarb in channel catfish. A 48-h LC50 of 9.7 mg/l was determined for juvenile channel catfish. Mortality was accompanied by dramatic loss of brain AChE. Further characterization of tissue-level effects suggests that muscle AChE plays a causal role in mortality. Aldicarb was metabolized in channel catfish to aldicarb sulfoxide, along with the formation of minor hydrolytic products. The toxicokinetics of aldicarb in catfish are bi-compartmental with rapid elimination (t1/2 = 1.9 h). Plasma AChE was inhibited in a pattern similar to that of the elimination of total aldicarb-derived compounds. A comparison of aldicarb uptake between catfish and rainbow trout showed no difference in compound absorbed in 24 h. The pattern of in vivo metabolism, however, was quite different between these species. Rainbow trout produce significantly more hydrolytic derivatives and have a 3-fold higher aldicarb sulfoxide to aldicarb ratio at 3 h. These data give strength to the hypothesis that a slower rate of bioactivation in the catfish (vs. rainbow trout) is acting as a protective mechanism against the acute toxicity of aldicarb.

Key Words: aldicarb; (TemikTM); 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime; aldicarb sulfoxide; 2-methyl-2-(methylsulfinyl)propionaldehyde-O-(methylcarbamoyl)oxime; channel catfish (Ictalurus punctatus); acetylcholinesterase inhibition; carbamate insecticide; aldicarb sulfoxidation; cytochrome P450; flavin-containing monooxygenase; toxicokinetics.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldicarb (Fig. 1Go) is metabolized rapidly and extensively in most plants and animals. Known metabolites include the oxidative metabolites aldicarb sulfoxide (ASX) and aldicarb sulfone (ASN), as well as hydrolytic oxime and nitrile derivatives of these compounds. While hydrolysis of aldicarb or conversion to ASN can decrease its AChE inhibition, conversion to ASX is considered a process of bioactivation. (Andrawes et al., 1967Go; Black et al., 1973Go; Pelekis and Krishnan, 1997Go; World Health Organization, 1991Go).



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FIG. 1. Structure of aldicarb and metabolites.

 
Few studies have been performed to examine the effects of aldicarb, other than mortality, in fish. Median lethal concentrations (LC50) of aldicarb for most fish species generally range from 0.3–1.0 mg/l (Gallo et al., 1995Go; Montgomery, 1993Go; Spradley, 1991Go). However, an earlier study presented an LC50 value of 45 mg/l for channel catfish fingerlings (Ictalurus punctatus) (Schlenk, 1995Go). The muscle AChE of channel catfish and rainbow trout (Oncorhynchus mykiss) has similar sensitivity to aldicarb and its metabolites, but ASX is 200 fold more potent than the parent compound in both species (Perkins et al., 1999Go). This suggests that dispositional factors may play a role in the resistance of catfish to this compound.

The biotransformation of aldicarb, both in vitro and in vivo, has been previously examined in rainbow trout (Schlenk and Buhler, 1991Go; Schlenk et al., 1992Go). It appears that primarily the flavin-containing monooxygenase system (FMO), and to a lesser extent the cytochrome P450 system (CYP), are involved in the conversion of aldicarb to ASX. In contrast to trout, catfish do not appear to express FMO. Consequently, catfish liver microsomes have a slower maximal rate (Vmax) of aldicarb sulfoxidation in vitro (Perkins et al., 1999Go). A slower rate of aldicarb bioactivation in vivo could result in a smaller percentage of the parent compound being bioactivated, leading in turn to the observed lower acute toxicity. However, the in vivo metabolism and disposition of aldicarb in channel catfish has not been characterized.

The objective of this study is to examine the metabolic, toxicokinetic, and toxicodynamic processes involved in the resistance of channel catfish to aldicarb toxicity. Better understanding the mechanisms of piscine pesticide resistance/sensitivity is needed to help reduce uncertainty in ecological risk assessment, and may be useful in the development of new pesticides and piscicides.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
Radiolabeled (S-methyl 14C) aldicarb (23 mCi/mmol) was donated by Rhône Poulenc, Inc. (Research Triangle Park, NC). Aldicarb was diluted to 1 µCi/µmol with unlabeled compound (99% purity, ChemService, West Chester, PA) and stored at –20°C until use. Prior to dosing, aldicarb was re-purified using a Waters Sep-Pak C18 solid phase extraction column and a MeOH step gradient. Purity was assessed to be greater than 98% by radio-HPLC. HPLC grade solvents and Scinti-SafeTM scintillation cocktail were obtained from Fisher Scientific (Pittsburgh, PA). Analytical grade aldicarb, aldicarb sulfoxide, and aldicarb sulfone were purchased from ChemService (West Chester, PA). Standards for oxime and nitrile derivatives of aldicarb and metabolites were produced by base-catalyzed hydrolysis of aldicarb (Payne et al., 1966Go). All other reagents were purchased from Sigma Chemical Company (Saint Louis, MO).

Animals
Channel catfish (1–3 years post-hatch) were obtained from the USDA National Aquaculture Center in Stuttgart, AR, and held in 400-L fiberglass aquaria with flow-through dechlorinated tap water at 18–25°C for at least 1 week prior to exposure. Catfish were fed commercial fish chow once per day during the holding period.

Juvenile rainbow trout ( 80–180 g) were obtained from the Greer's Ferry National Fish Hatchery and U.S. Fish and Wildlife Service (Heber Springs, AR). Trout were held in a fiberglass Living Stream (Frigid Units, Toledo, OH) at 10°C for 2 weeks before experimental use.

LC50 Determination
Six juvenile catfish (1 year old, {approx}100 g) were placed in each of 12 glass aquaria containing 25 l dechlorinated, aerated H2O. Fish were left to acclimate under subdued lighting for 12 h. After 12 h, the water in each tank was changed, and aldicarb was added at the following nominal concentrations: 0, 0.1, 1.0, 10.0, 33.0, or 100.0 mg/l. Actual water concentrations were calculated to be 0, 0.1, 0.93, 10.7, 44.6, and 113 mg/l after HPLC analysis of stock solutions. EtOH was used as a carrier (final concentration = 0.1% in all tanks). Each concentration group consisted of 2 tanks (12 fish). At 12-h intervals, 80% of the water was changed and fresh aldicarb stock was added to maintain nominal concentration. The test was carried out for 48 h, and dead fish were removed as the test proceeded. The entire brain was immediately removed from dead fish during the test as well as from surviving fish (killed by decapitation) at 48 h. Brains were frozen in liquid nitrogen and stored at –80°C. The trimmed Spearman-Karber method was used to calculate the LC50 value.

Acetylcholinesterase Inhibition
A time-course study of AChE inhibition in catfish muscle, serum, and brain was performed to assess the profile of tissue level effects. For the tissue-response time-course, aquaria were set up as for the LC50 determination (6 fish/tank, 25 l H2O), but only 0, 0.1 and 10 mg/l concentrations were used. Exposure water was replenished every 12 h. Moribund fish were removed and dissected immediately, and 3 live fish were sampled from each concentration at 3, 12, 24, and 48 h. Blood samples were taken prior to dissection. Serum was removed by centrifugation and frozen at –80°C. Brain and muscle tissues were frozen on dry ice and stored at –80°C. Immediately prior to analysis, frozen tissues were thawed, homogenized in 2 volumes of 1.15% KCl, and centrifuged at 10,000 x g for 5 min. The activity of AChE was measured in the supernatant using a modification of the Ellman spectrophotometric assay (Ellman et al., 1961Go), adapted for microplates as previously described (Nostrandt et al., 1993Go). Acetylthiocholine iodide was used as the substrate (0.5 mM) and 5,5`-dithio-bis-2-nitrobenzoic acid as the chromagen. Protein concentration was determined using a microplate adaptation of the Bradford method (1976).

In Vivo Metabolism and Toxicokinetics
Sexually mature channel catfish (700–800 g) were anesthetized with MS-222. After anesthesia, a polyethylene cannula (PE50) was inserted into the dorsal aorta using a modification of the method of Garey (1969). The cannula was sutured into place at the point of insertion and at a second, more rostral position in the mouth. After passing the cannula through a small hole in the lip fold, it was sutured to the top of the head at a point between the eyes. Heparinized saline (75 U/ml) was used to flush and fill the cannula. Cannulated fish were then placed in a plastic-coated, wire-mesh restraining cage (40 x 10 x 11 cm) and allowed to recover for 24 h in flow-through dechlorinated H2O.

For plasma uptake studies, two catfish were cannulated and individually placed in glass aquaria containing 20 l of aerated dechlorinated water after the recovery period. Four µCi of purified 14C-aldicarb stock was added to each tank, giving a final concentration of 38-µg/l (0.2 µM) aldicarb. A 600-µl blood sample was withdrawn from each fish at time points ranging from 0 to 12 h. Sampled blood was replaced with an equal volume of heparinized saline. Blood samples were placed on ice and centrifuged to isolate plasma. An aliquot (330–350 µl) of the plasma was combined with 4 ml Scinti-SafeTM cocktail for scintillation counting. Plasma concentrations of aldicarb equivalents were calculated from DPM/ml.

The toxicokinetics of aldicarb elimination were examined after a bolus dose of 0.32-mg 14C-aldicarb was delivered through the aortic cannula of 4 mature channel catfish (777±66 g). Blood samples ranging from 0.5–1.0 ml were taken for 48 h. Sampled blood was replaced with an equal volume of heparinized saline. Plasma was removed after centrifugation and stored at –20°C until HPLC analysis. The 4 plasma samples from each time point were pooled for HPLC analysis, and profiles of both total 14C and unaltered aldicarb were assessed. The data was fit to a classical 2- or 3-compartment model using the nonlinear curve fitting functions of Graph Pad Prism 2.0.1 (GraphPad Software Inc., San Diego). Toxicokinetic parameters were determined using the equations listed in Table 1Go.


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TABLE 1 Equations Used for Determination of Toxicokinetic Parameters
 
Catfish–Trout Comparisons
One-year-old channel catfish and rainbow trout ( 80–180 g) were used to compare aldicarb uptake rates and metabolite profiles directly between the two species. Whole-body uptake was determined by disappearance of 14C-labled compound from the tank water. Fish (n = 4) were placed in glass chambers (fitted with drainage valves) that contained 3.0 l dechlorinated H2O at the appropriate temperature (10–12°C for trout, 18–20°C for catfish). The tank water was aerated and the fish were allowed to acclimate in the chambers overnight before exposure. At the beginning of the exposure period, the tank water was changed and 1.0 µCi of 14C-aldicarb was added to each tank (final concentration = 63 µg/l). Water samples (1.0-ml) were taken from each tank at the following time points: 0, 15, 30, 60, 90, min and 3, 5, 12, 18, and 23 h. Samples were mixed with 4.0 ml Scinti-SafeTM cocktail and total radioactivity was determined by liquid scintillation counting.

In vivo metabolism was compared using the same chambers and water temperatures, but fish were injected intraperitonealy (i.p.) with 1 µCi 14C-aldicarb dissolved in 160–200 µl DMSO. Fish were placed back into individual chambers after injection and the tank water was changed twice during the 3 hr experimental period. After 3 hr, fish were anesthetized with MS-222 and 2–3 ml of blood was withdrawn with a heparinized syringe from the caudal vein. Blood samples were placed on ice, and plasma was removed by centrifugation. The plasma samples were extracted immediately and metabolite profiles were determined by radio-HPLC.

Plasma Extraction and Analysis
Plasma samples were extracted using Oasis SPE columns (Waters Corporation, Milford, MA). Each SPE column was prepared according to the manufacturer's instructions and 1.0–1.7 ml of plasma was applied. Bound compounds were eluted using 1.0 ml MeOH. The elution fraction was concentrated to dryness under a stream of nitrogen, and samples were re-dissolved in 200 µl of 11% acetonitrile. Extraction efficiency averaged 68% as determined by liquid scintillation counting.

Separation of metabolites was performed using a Luna C8 column (Phenomenex, Torrance, CA) attached to a Waters HPLC system equipped with Waters Millennium 32 chromatographic software. A ß-RAM flow-scintillation detector (INUS Systems, Tampa, FL) was used to detect and quantitate 14C peaks. The column was equilibrated with 9% acetonitrile before sample injection, and a gradient was used for elution. Elution conditions were 9% acetonitrile for 5 min, followed by a 10-min increase to 60% acetonitrile, which was held for 3 min before returning to initial conditions. Identity of metabolites was determined by co-elution with standards.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Toxicity
The acute toxicity of aldicarb to channel catfish was assessed by the determination of the 48-h LC50 value for juvenile fish. The calculated LC50 was 9.75 mg/l with a 95% confidence interval of 5.6–17 mg/l. Although some hyperactivity and rapid ventilation was noted at 0.9 mg/l, no mortality occurred at this concentration or lower. Brain-AChE activity was determined in both morbid and surviving fish from this study (Fig. 2Go). At the lowest concentration (0.1 mg/l), activity was inhibited 63%. At the next higher concentration (1.0 mg/l), where no mortality was seen, almost 90% of the brain AChE was inhibited. No significant difference ({alpha} = 0.05) was detected between the living fish at 1.0 and 10 mg/l and the dead fish at 33 and 100 mg/l.



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FIG. 2. Inhibition of channel catfish brain AChE activity during LC50 determination (mean ± SD; n = 7–12). Brains from 0–10-mg/l groups were taken from live fish at 48 h; those from 33- and 100-mg/l groups were taken immediately after death (2–5 h).

 
Because brain cholinesterase appeared to be very sensitive to inhibition by aldicarb, the AChE activity in muscle and serum was subsequently measured after aldicarb exposure. In order to assess the time-dependent profile of inhibition, tissues were taken from living fish at set time-points and from dead fish immediately after mortality (Figs. 3A–3CGo). No mortality occurred in the control or low concentration (0.1 mg/l) groups, but all fish were dead or had been sampled in the high concentration (10.0 mg/l) group by 12 h. By 3 h, the activity of AChE in all tissues was essentially abolished in both living anddead individuals from the 10-mg/l group. Distinct differences appeared, however, in the patterns of inhibition among tissues of the lower-concentration group. In this group, a time-dependent decrease in brain AChE occurred that was significantly different from controls by 12 h (Fig. 3AGo). Serum AChE was rapidly and significantly decreased at 0.1 mg/l, but never reached the level of inhibition measured at 10.0 mg/l (Fig. 3BGo). However, muscle AChE was not significantly affected at any time point in the low-concentration group (Fig. 3CGo).



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FIG. 3. Inhibition of AChE in brain (A), serum (B), and muscle (C) after lethal and non-lethal exposure of channel catfish to aldicarb. Dashed line indicates mean AChE activity of dead fish. *Significantly different from control ({alpha} = 0.05).

 
Aldicarb Uptake and Elimination
The waterborne uptake of 14C-aldicarb by catfish occurred fairly rapidly, as plasma concentrations reached an apparent plateau by 6 h (Fig. 4Go). A classical 2-compartment model (Fig. 5AGo) described the elimination of the parent compound after a bolus iv injection. The toxicokinetic parameters derived from this model are presented in Table 2Go. Aldicarb was rapidly distributed and eliminated, with a terminal half-life of 1.9 h. This rapid elimination was due in part to the metabolism of aldicarb to ASX, which accounted for the entire plasma radioactivity by 12 h (Fig. 5BGo). Although ASX was the major metabolite detected, trace amounts of aldicarb oxime and ASX oxime were detected in some samples. Because both aldicarb and ASX are capable of causing toxicity, the elimination of total 14C-aldicarb equivalents was also modeled (Fig. 5CGo). A 3-compartment model, with a terminal elimination half-life of 44.7 h, best described the total 14C data. Total 14C had a rather large volume of distribution (4.4 l/kg), which may be related to storage of compounds bound to AChE in tissues. The clearance of total aldicarb-derived compounds (64.9 ml/h/kg) was slightly higher than the estimated cardiac output of catfish (42 ml/h/kg) (Nichols et al., 1996Go). The inhibition of plasma AChE was fit to a 2-phase nonlinear model (Fig. 5DGo), which resembled most closely the pattern of total 14C elimination in these fish.



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FIG. 4. Concentration of 14C-aldicarb equivalents in plasma of channel catfish (n = 2) during uptake form exposure water (38 µg/l).

 


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FIG. 5. Aldicarb toxicokinetics in channel catfish. (mean ± SEM; n = 4). (A) Elimination profile of 14C-aldicarb. (B) Concentration of aldicarb sulfoxide in plasma. (C) Elimination profile of total 14C-aldicarb equivalents (parent and metabolites). (D) Inhibition of AChE in plasma during aldicarb elimination.

 

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TABLE 2 Model-Derived Toxicokinetic Parameters for Aldicarb in Channel Catfish
 
In order to directly compare uptake between trout and catfish, juvenile fish were placed in small chambers and whole-body uptake was measured as disappearance of aldicarb equivalents from the exposure water (Fig. 6Go). The initial phase of uptake was more rapid in channel catfish, but total uptake at 23 h. was not different between the two species. Aldicarb metabolism, in vivo, was also compared using fish of similar size and age. The plasma metabolite profiles were determined at 3 h after an ip injection of 14C-aldicarb. The pattern of metabolites in juvenile catfish was very similar to that observed in larger, mature animals at the same time point, with ASX as the major metabolite (Fig. 7Go). Rainbow trout exhibited a metabolite profile much different from that of catfish. Although ASX was also the major metabolite in trout plasma, the ratio of ASX to aldicarb was 3 times higher than in catfish plasma (Table 3Go). In addition, there were significantly more of the hydrolytic products, aldicarb oxime and ASX oxime. The larger percentage of available parent compound converted to ASX demonstrates a more rapid rate of aldicarb sulfoxidation in trout.



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FIG. 6. Comparison of whole-body uptake of aldicarb by channel catfish and rainbow trout (n = 4). Uptake was determined by disappearance of 14C from exposure water.

 


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FIG. 7. Typical chromatograms of 14C-aldicarb metabolites in plasma of channel catfish (A) and rainbow trout (B). Blood samples were taken 3 h after ip injection of aldicarb. Peak identification: 1 = ASX oxime, 2 = ASX, 3 = aldicarb oxime, 4 = aldicarb.

 

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TABLE 3 Comparison of in Vivo Metabolism of Aldicarb between Channel Catfish and Rainbow Trout
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is generally accepted that the acute toxicity of aldicarb and other carbamate pesticides is caused by inhibition of AChE. Therefore, any factor that reduces the amount of inhibition should subsequently decrease toxicity. Several mechanisms exist by which in vivo AChE inhibition can be reduced. Among these are lower sensitivity of the enzyme, increased hydrolysis of the active compound by non-specific esterases, increased elimination, reduced uptake, and in some cases, decreased bioactivation.

Channel catfish are less sensitive to aldicarb than rainbow trout (96-h LC50 = 0.88 mg/l) or bluegill (96-h LC50 = 0.1–1.5 mg/l), with a 48-h LC50 determined in this study to be approximately 10 mg/l (Montgomery, 1993Go). However, earlier studies have shown that the sensitivity of muscle AChE to aldicarb inhibition in vitro does not differ between catfish and trout (Perkins et al., 1999Go). If AChE inhibition is actually the ultimate mechanism of acute toxicity, it may be assumed that some essential pool of AChE must reach a critical threshold of inhibition before mortality occurs. The primary cause of AChE inhibition-induced death in mammals is generally regarded to be respiratory failure (Chambers and Carr, 1995Go), which is likely to be a problem for fish as well. Most examinations of AChE inhibition in fish, however, have focused on activity in the brain. (Keizer et al., 1995Go; Qadri et al., 1994Go; Wang and Murphy, 1982Go; Zinkl et al., 1987Go). Catfish brain AChE appears to be quite sensitive to inhibition by aldicarb, but fish with as much as 90% of neuronal activity diminished were still able to survive with only moderate outward signs of toxicity. There was no difference in the extent of AChE inhibition between living and dead fish at concentrations higher than 1 mg/l, suggesting that reduced brain AChE activity alone is not sufficient to cause mortality. Upon closer examination of tissue-level AChE effects, it was noted that although serum and brain AChE was substantially inhibited at a sublethal concentration of aldicarb (0.1 mg/l), muscle AChE was not affected. This result is similar to that described by Straus and Chambers (1995) in response to phosphorothionate exposure.

The inhibition of serum/plasma AChE is proportional to the plasma concentration of the inhibiting substance. Therefore, the enzyme activity in catfish plasma appears to regenerate at a rate similar to that of the elimination of aldicarb and its metabolites. During static exposure, however, serum AChE is quickly inhibited and remains at a constant level throughout the exposure. Regeneration of AChE after static exposure was not examined in this study, but does occur in fish (Straus and Chambers, 1995Go; Zinkl et al., 1987Go).

Together, these data suggest that inhibition of muscle AChE is the more important factor in the ultimate mortality of catfish exposed to aldicarb. Muscle AChE represents the largest pool of cholinesterase in the body, and is important in controlling muscular function. The loss of muscular control can cause multiple problems for fish, including loss of swimming control and cessation of opercular movement. As the fish loses the ability to effectively move water across the gills, a situation occurs that is analogous to respiratory failure in mammals. The result is reduced oxygenation of the blood, which can lead to hypoxia-induced death (Zinkl et al., 1987Go).

The uptake of aldicarb by catfish is rapid, reaching maximal plasma concentration by 6 h. The distribution of aldicarb from the plasma compartment to the tissue compartment also occurs very quickly, based on the {alpha} half-life. A comparison of total aldicarb uptake in rainbow trout and catfish demonstrated that although catfish absorbed the compound more quickly in the initial phase, no difference existed in the total amount of compound accumulated over 24 h.

Distribution and elimination of unaltered aldicarb was biphasic and rapid, while total aldicarb-derived compounds (primarily ASX) followed a triphasic pattern with a relatively large volume of distribution and a much longer half-life. Because the labeled compounds remaining in the circulation after 12 h consisted almost completely of ASX, the inhibition of plasma AChE was also prolonged. The slower elimination of ASX, as well as the large volume of distribution, would be expected, due to high affinity binding to tissue AChE. A similar pattern of elimination has been described in rats dosed with 14C-carbaryl (Fernandez et al., 1982Go), which also showed tricompartmental kinetics for total labeled compounds but bicompartmental kinetics for the parent. In rats, as in catfish, AChE inhibition was more closely related to the elimination of total carbamate-derived 14C. Although no data exists for plasma kinetics of aldicarb in trout, an earlier study examined the elimination of aldicarb into the aqueous environment after ip injection (Schlenk et al., 1992Go). If aldicarb is more rapidly eliminated in catfish than in trout, one would expect the elimination half-life of total 14C in catfish to be shorter than the published values (25.3–28.4 h) for elimination from trout. The opposite is true, however, with the catfish half-life more than double that of trout, suggesting that elimination rate should not be a factor in the aldicarb resistance of catfish.

Metabolism of aldicarb in vivo by rainbow trout produces ASX, ASX oxime, aldicarb oxime, and aldicarb nitrile as excreted products (Schlenk et al., 1992Go). The same metabolites, minus the nitrile, are detected in catfish plasma after dosing with aldicarb. The current study shows, however, that the proportion of these metabolites in the plasma is much different between these two species. In trout, a greater percentage of the circulating 14C exists as ASX and its oxime derivative, suggesting more rapid sulfoxidation and hydrolysis than in catfish. This study supports earlier in vitro metabolism experiments by demonstrating that aldicarb bioactivation in vivo (to ASX) is slower in catfish than in rainbow trout.

Rapid sulfoxidation of aldicarb in the trout should lead to a greater tissue dose of ASX during the initial phase of distribution out of the plasma compartment. Because the critical period for mortality appears to occur very soon (<3h) after exposure to aldicarb, the potentially higher level of ASX exposure in trout tissues may contribute substantially to the acute toxic response. By demonstrating the more rapid sulfoxidation of aldicarb in trout, the data presented supports the hypothesis that increased ASX production is involved in the greater toxicity of aldicarb compared to channel catfish. Because FMO is responsible for much of the sulfoxidation in both trout and rats (Pelekis and Krishnan, 1997Go; Schlenk and Buhler, 1991Go), the lack of FMO in catfish is probably responsible for its decreased sensitivity to aldicarb. Other factors, such as enzyme sensitivity, uptake/elimination rates, and carboxylesterase activity do not appear to play a substantial role in catfish resistance.

To summarize, the 48-h LC50 for aldicarb in channel catfish was determined to be 9.7 mg/l, with acute toxicity accompanied by decreased AChE activity in brain, serum/plasma, and muscle. Serum AChE responded most rapidly and sensitively to inhibition, and activity was regenerated as aldicarb and ASX were eliminated. Brain AChE was also greatly inhibited at sublethal concentrations but does not appear to be the major factor in aldicarb-induced mortality. Muscle AChE, on the other hand, was only inhibited at a lethal concentration of aldicarb, suggesting that significant inhibition of muscle AChE may be a final step in acute cholinesterase poisoning. As is the case with mammals, cholinergic overload in muscle would lead to loss of motor and respiratory function. Cardiac effects were not determined in this study, but might also play a role. Aldicarb is rapidly absorbed in channel catfish and quickly distributed out of plasma. Metabolism of aldicarb in catfish occurs primarily through the CYP-mediated conversion to ASX; however, the proportion of ASX to aldicarb is several times higher in rainbow trout. Aldicarb depuration in catfish is rapid, presumably owing to both excretion and metabolism, but the elimination of all aldicarb-derived compounds is much slower. The elimination half-life of aldicarb and its metabolites in catfish is twice as long as that in trout, but a greater amount of ASX exposure to trout muscle during the early critical window of exposure may lead to greater mortality. The data presented help to confirm the hypothesis that decreased bioactivation in the catfish is acting as a protective mechanism against the acute toxicity of aldicarb. A similar mechanism may play a role in determining species sensitivity to other FMO-activated toxicants in fish.


    NOTES
 
1 To whom correspondence should be addressed at Lilly Research Laboratories, Lilly Corporate Center, Drop Code 0170, Indianapolis, IN 46285. Fax: (317) 433-2473. E-mail: eperkins{at}lilly.com. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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