Effects of Salinity on Aldicarb Toxicity in Juvenile Rainbow Trout (Oncorhynchus mykiss) and Striped Bass (Morone saxatilis x chrysops)

Juan Wang*, Sonja Grisle* and Daniel Schlenk{dagger},1

* Environmental Toxicology Research Program, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677; and {dagger} Environmental Toxicology Program, Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521

Received May 10, 2001; accepted August 20, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fluctuations in several environmental variables, such as salinity, can influence the interactions between organisms and pollutants in aquatic organisms, and, therefore, affect the toxicity of xenobiotics. In this study, after 2 species of fish, rainbow trout (Oncorhynchus mykiss) and hybrid striped bass (Morone saxatilis x chrysops) were acclimated to 4 salinity regimens of 1.5, 7, 14, and 21 ppt for 1 week and then exposed to 0.5 mg/l aldicarb. Mortality, brain, and muscle cholinesterase levels were measured after 96 h. Rates of 14C-aldicarb sulfoxide formation were determined in kidney (trout only), liver, and gill microsomes from each species acclimated to the 4 salinity regimens. Salinity significantly enhanced aldicarb toxicity, cholinesterase inhibition, and 14C-aldicarb sulfoxide formation in rainbow trout but not in striped bass. In vitro incubations with 14C-aldicarb and the cytochrome P450 (CYP) inhibitor, N-benzylimidazole, did not significantly alter aldicarb sulfoxide formation in tissue microsomes from either species of fish, indicating CYP did not contribute to aldicarb sulfoxidation. Salinity increased flavin-containing monooxygenase (FMO) mRNA expression and catalytic activities in microsomes of liver, gill, and kidney of rainbow trout, which was consistent with the salinity-induced enhancement of aldicarb toxicity. Salinity did not alter FMO mRNA expression and catalytic activities in striped bass, which was also consistent with the lack of an effect of salinity on aldicarb toxicity in this species. These results suggest that salinity-mediated enhancement of aldicarb toxicity is species-dependent, and at least partially due to the salinity-related upregulation of FMOs, which, in turn, increases the bioactivation of aldicarb to aldicarb sulfoxide, which is a more potent inhibitor of cholinesterase than aldicarb.

Key Words: salinity; aldicarb; FMO; cholinesterase; rainbow trout; striped bass.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldicarb [2-methyl-2-(methylthio) propanal O-(methylamino) carbonyl oxime; Temik], a carbamate insecticide, is used throughout the world to control insects, mites, and nematodes in numerous agricultural settings including areas adjacent to estuarine shores (Eddleman and Falconer, 2000Go; Tomlin, 1994Go). Due to its high water solubility and moderate half-life, aquatic organisms may be at high risk of being acutely exposed to this chemical especially during agricultural runoff. Several environmental characteristics of water such as temperature, pH, dissolved oxygen content, hardness, and salinity may affect the toxicity of xenobiotics to aquatic organisms (Brecken-Folse et al., 1994Go). Salinity is a critical factor in influencing the distribution and maintenance of aquatic life in estuaries, which are characterized by fluctuations of salinity. Therefore, examining the effects of salinity on the toxicity of water-borne contaminants, including aldicarb, is essential in assessing the risk of these compounds to estuarine organisms.

Aldicarb inhibits various forms of cholinesterase that modulate the neurotransmitter acetylcholine at various locations throughout the body including nerve-cell junctions centrally and peripherally. Increased levels of acetylcholine following cholinesterase inhibition subsequently causes cholinergic overload and disruption of normal neurotransmission (Coppage, 1977Go). Although aldicarb is capable of cholinesterase inhibition, oxidation to the corresponding sulfoxide has been shown to enhance cholinesterase inhibition 150-fold in fish (El-Alfy et al., 2001Go; Perkins and Schlenk, 2000Go). Whereas sulfoxidation to the sulfoxide enhances the toxicity of aldicarb, an additional sulfoxidation forming the sulfone, or hydrolysis to the nitrile or oxime of aldicarb significantly reduces or eliminates toxicity (Risher et al., 1987Go). Generally, both cytochrome P450 (CYP) and flavin-containing monoxygenases (FMOs) are believed responsible for the bioactivation of aldicarb to the sulfoxide in mammals (Hajjar and Hodgson, 1980Go; Kulkarni and Hodgson, 1980Go). In the freshwater-adapted rainbow trout (Oncorhynchus mykiss), the S-oxidation of aldicarb to the sulfoxide was shown to be catalyzed exclusively by one or more forms of FMO (Schlenk and Buhler, 1991Go). Indeed, fish species that possess high levels of FMO activity appear to be more sensitive to aldicarb toxicity than those that have little or no FMO activity (Perkins and Schlenk, 2000Go).

Previous studies have indicated that high salinity upregulates FMO activity in Japanese medaka (Schlenk and El-Alfy, 1998Go), eel (Anguilla japonica), and guppy (Poecilia reticulata; Daikoku et al., 1988Go), while low salinity downregulates FMO activity in Atlantic flounder (Paralichthys flesus; Schlenk et al., 1996Go). Rainbow trout (Oncorhynchus mykiss) slowly osmoconform to saltwater, whereas hybrid striped bass (Morone saxatilis x chrysops) are rapid osmoconformers (Madsen et al., 1994Go). Each species expresses several FMO isoforms in various tissues (Cashman et al., 1990Go; Schlenk and Buhler, 1991Go). However, FMO activity was lower in striped bass collected from seawater environments compared to freshwater environments (Cashman et al., 1989Go, 1990Go). In contrast, studies in trout indicated FMO upregulation in response to high salinity (Larsen and Schlenk, 2001Go). Consequently, the hypothesis of this study is that aldicarb toxicity will be enhanced by hypersalinity in rainbow trout because FMO will be induced and activate aldicarb to a more potent cholinesterase inhibitor. In contrast, striped bass should be resistant against salinity-enhanced toxicity.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
TRI REAGENT was obtained from Molecular Research Center, Inc. (Cincinnati, OH). Analytical standard grade aldicarb, aldicarb sulfoxide, was purchased from ChemService (West Chester, PA). 14C-Aldicarb was donated by Rhone-Poulenc, Inc. (Research Triangle Park, NC) and was repurified on a C18 Prep-sep cartridge (Fisher Scientific, Pittsburgh, PA) using a step elution with 25 and 100% methanol. Acetonitrile was purchased from Fisher Scientific. Sodium dodecylsulfate and acrylamide were obtained from BioRad (Hercules, CA). [a-33P]-Deoxy-cytosine triphosphate (dCTP) was obtained from NEN-Dupont (Boston, MA). Acetylthiochlone iodide, glutathione, P-nicotinamide adenine dinucleiotide phosphate (reduced form, NADPH), phenylmethylsulfonylfluoride (PMSF), anti-rabbit IgG alkaline phosphatase, diethyl pyrocarbonate (for RNA extraction and Northern Blots), molecular weight markers for SDS-PAGE, 0.24–9.5 kb RNA ladders for Northern Blots, and other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

Fish maintenance and treatment.
Juvenile rainbow trout (Oncorhynchus mykiss), weighing 20–50 g, were obtained from the U.S. Fish and Wildlife Hatchery, at Heber Springs, Arkansas, and juvenile hybrid striped bass (Morone saxatilis x chrysops), weighing 15–30 g, were from the Keo Fish Farm, Keo, Arkansas. After 1 week of acclimation in freshwater, rainbow trout were divided into 4 groups (1.5 ppt as control, 7 ppt, 14 ppt, and 18 ppt as 3 salinity regimens). Each treatment was replicated 3 times with 10–11 fish in each aquarium. Fish from the 3 salinity groups (7, 14, and 18 ppt) were exposed to a stepwise increase of salinity (3 ppt per day) until the final salinity for each group was reached, minimizing osmotic stress. All fish were maintained for 2 weeks at their respective salinity regimen. Salinity was adjusted by mixing dechlorinated tap water with sea salt (Coralife Marine Salt; That Fish Place, Lancaster, PA). After 1 week of exposure, 5–6 fish were sampled for FMO catalytic activity and FMO mRNA expression. Fish were euthanized by cervical dislocation and the gill, liver, trunk kidney, muscle (from the lateral surface), and brain were dissected. Tissues were immediately frozen in liquid nitrogen, and stored at –80°C until analysis. The remaining fish were treated with 0.5 mg/l aldicarb (dissolved in ethanol with the final ethanol concentration being 0.01%, v/v). After 96 h, mortality percentages were determined. Muscles and brains of surviving fish were dissected for cholinesterase activity. Striped bass were treated in a similar manner, except that 21 ppt was used as the highest salinity regimen. Since the bass were smaller than the trout, only gill, liver, muscle, and brain were dissected. Aldicarb doses were arbitrarily chosen based on a reported LC50 of 0.8 mg/l in rainbow trout and subsequent range-finding studies in each species (Vittozzi and De Angelis, 1991Go).

Microsomal preparation.
Microsomes from dissected tissues were prepared as previously described (Schlenk and Buhler, 1991Go). Briefly, dissected tissues from 5–6 fish were pooled and homogenized in 4 x (w:v) of 100 mM Tris-HCl, pH 7.6, containing 0.15 M KCl, 1 mM EDTA, and 0.1 mM PMSF. The tissue homogenates were initially centrifuged at 10,000 x g for 30 min and the supernatant was recentrifuged at 100,000 x g for 90 min. The microsomal pellets were resuspended in 100 mM potassium phosphate, pH 7.4, containing 20% (v/v) glycerol and 1 mM EDTA. All sample procedures were carried out at 4°C. The microsomal suspensions were either used immediately for enzyme activities or stored at –80°C until assayed. Microsomal protein concentrations were determined using the Pierce protein staining kit (Pierce), with bovine serum albumin as the standard.

Cholinesterase activity determination.
Approximately 0.5 g of muscle and brain dissected from fish were homogenized in 1.15% KCl and homogenates were centrifuged at 5000 rpm for 5 min to pellet tissue debris. The supernatants were used to measure cholinesterase activity. The activity was measured following a modification of the Ellman spectrophotometric assay (Ellman et al., 1961Go) adapted for 96-well microplates as previously described (Nostrandt et al., 1993Go). Acetylthiocholine iodide was used as the substrate (0.5 mM) and 5, 5'-dithio-bis-nitrobenzoic acid as the chromagen. Eserine sulfate (10 M dissolved in methanol) was used as a blank to correct for noncholinesterase mediated hydrolysis (Boone and Chambers, 1996Go). Glutathione was used as the standard.

Thiourea-dependent thiocholine oxidation assay.
The catalytic activities of FMOs were measured using the thiourea-dependent oxidation of thiocholine assay (Schlenk et al., 1996Go), which is a modification of the procedure described by Guo and Ziegler (1991). Reactions were carried out in a total volume of 1 ml and consisted of microsomal protein (500 µg–1 mg), 0.1 M potassium phosphate buffer, pH 8.4, containing 100 mM thiocholine, 0.25 mM NADPH, and 1.2 mM thiourea. After 30–60 min of incubation, 0.8 ml aliquots were transferred to tubes on ice with 0.08 ml 3.0 M trichloroacetic acid. Precipitated protein was separated by centrifugation at 10,000 x g for 10 min, and the resulting supernatant was treated with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB). The absorbance was measured at 412 nm and was compared to the absorbance from incubations without NADPH. A millimolar absorptivity of 13.6 cm–1 was used to calculate the catalytic activity (Guo and Ziegler, 1991Go). All reaction conditions (i.e., substrate concentrations and incubation times) were determined from previous studies (Schlenk et al., 1996Go).

In vitro aldicarb metabolism.
To determine the role of FMOs on aldicarb biotransformation, in vitro metabolism studies were conducted as described previously (Schlenk and Buhler, 1991Go). Incubations contained 100 mM 14C-aldicarb (1.0 m Ci/mM), 100 µM NADPH, 0.5–1.0 mg microsomal protein, and 50 mM Tris, pH 8.0, containing 1 mM MgCl2, 5 mM KCl, and 0.1 mM EDTA. To inhibit the CYP450 enzyme system, 3 mM N-benzylimidazole was allowed to preincubate for 5 min prior to the addition of 14C-aldicarb (Schlenk and Buhler, 1991Go). Reactions were initiated by the addition of 14C-aldicarb and stopped with addition of half the volume of 30% trichloroacetic acid. The precipitated proteins were pelleted by centrifugation at 9000 x g for 5 min. The supernatant was filtered using 0.45mm PVDF filter units (Midwest Scientific, St. Louis, MO) prior to injection of 50 ml on to a Beckman ultrasphere C-8 HPLC column (4.6 mm int. diam. x 15 cm) utilizing a Waters 600E HPLC system equipped with Waters Millennium 32 chromatographic software. As described earlier (Schlenk and Buhler, 1991Go), the solvent system was 11% acetonitrile, 89% 50 mM phosphate buffer, pH 7.0, for 7 min with the gradient changing to 50% acetonitrile, 50% phosphate buffer over a period of 4 min. The gradient was maintained for 3 min and then returned to the initial system over 5 min for a period of 7 min. Recovered radioactivity was compared with retention times of known standards detected by UV absorption at 210 nm.

Northern blot analysis.
Total RNA fish samples were isolated by a single-step method of Chomezynski and Sacchi (1987) using phenol/guanidine thiocyanate/chloroform/ethanol extraction (TRI REAGENT). RNA concentrations were estimated from the A260 and the integrity of the RNA was confirmed by examining the 18S and 28S rRNA bands for lack of degradation. After electrophoresis, RNA was then transferred onto a nylon membrane (Hybond-N, Amersham International), and immobilized by UV cross-linking for 10 min. The membrane was hybridized at 42°C overnight with a full-length cDNA probe (1.6 kb) encoding rabbit liver FMO1 (Shehin-Johnson et al., 1995Go), radiolabeled by random primer extension to a specific activity of 106 CPM/ml prehybridization buffer with [a-33P]-dCTP (Feinberg and Vogelstein, 1983Go, 1984Go). Digitized images were analyzed with the aid of OptiQuant software (a Packard BioScience company), and normalized to the corresponding 28S band density. The approximate size of the hybridization band was evaluated by including a 0.24–9.5 kb RNA ladder (Gibco BRL, Gaithersburg, MD) and a plasmid containing FMO1 cDNA provided by Dr. Ron Hines (Medical College of Wisconsin) was used as a positive control.

Statistics.
All the experiments were performed in 3 replicates. Values were presented as mean ± SD. SigmaStat 2.0.3 software and Microsoft Excel software were used to test differences among sample means and perform correlation analysis. To determine the differences among groups, data were analyzed using 1-way ANOVA, followed by the Student-Neuman-Keul or Kruskal-Wallis test, depending on equal variance test, or Student's t-test as appropriate; p < 0.05 was accepted as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of Salinity on Aldicarb Acute Toxicity to Juvenile Rainbow Trout and Striped Bass
Salinity significantly increased the acute toxicity of aldicarb (0.5 mg/l) to juvenile rainbow trout. Raising the salinity from 1.5 to 14 ppt increased 96-h mortality from 29.7 ± 12.7% to 63.9 ± 2.4%. At 18 ppt, another significant increase in mortality was observed (91.7 ± 11.8%, Fig. 1Go). Significant differences were also seen between the 7 ppt and 18 ppt, 14 ppt and 18 ppt groups. Salinity did not significantly alter the toxicity of aldicarb to juvenile striped bass. In all the salinity regimens, mortality was consistently 60–70% after 96-h of exposure to aldicarb (Fig. 1Go).



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FIG. 1. Mortality percentage after 96-h aldicarb (0.5 mg/l) treatment following a 2-week exposure to different salinity regimens (trout were exposed to 18 ppt and striped bass to 21 ppt). Data points represent the mean ± SD of 3 replicates. Each replicate consisted of 9–11 individuals. **Significantly different from control at p < 0.05. ##Significantly different from 7 ppt salinity group at p < 0.05.

 
Effects of Salinity on Aldicarb Inhibition of Cholinesterase Activity in Muscle and Brain of Juvenile Rainbow Trout and Striped Bass
In juvenile rainbow trout, following exposure to aldicarb (0.5 mg/l), cholinesterase activity in muscle of surviving fish decreased in a salinity-dependent manner. Cholinesterase inhibition in muscle was greatest in fish acclimated to the highest salinity (Fig. 2Go). Cholinesterase activities in unexposed freshwater trout were 250 ± 12 and 101 ± 10 nmol/min/mg in muscle and brain, respectively. Salinity did not alter overall cholinesterase activities in either species (data not shown). However, there were significant differences in cholinesterase inhibition by aldicarb among the fish residing at 7 ppt, 14 ppt, and 18 ppt salinities. Due to high interindividual variability, no significant differences were detected between the 1.5 ppt group and 7 ppt or 14 ppt groups, but a significant decrease in the cholinesterase activity in fish residing at 18 ppt salinity compared with 1.5 ppt fish was observed. In brain, however, there were no statistical differences in cholinesterase inhibition among different salinity regimens following aldicarb exposure. In the striped bass, no salinity-dependent cholinesterase inhibition by aldicarb was detected. Cholinesterase activities in muscle and brain of unexposed freshwater bass were 290 ± 22 and 150 ± 9 nmol/min/mg, respectively. Among all salinity regimens, there were no significant differences in the cholinesterase activities of both muscle and brain (Fig. 3Go). To determine if salinity had any direct effect on cholinesterase activity, muscular cholinesterase activities were measured in fish acclimated to different salinity regimens but not exposed to aldicarb. Raising the salinity from 1.5 to 18 ppt did not have any effect on the cholinesterase activity in the muscle (data not shown).



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FIG. 2. Specific activity of cholinesterase in muscle and brain tissues of surviving rainbow trout after aldicarb treatment (0.5 mg/l) following exposure to different salinities for 2 weeks. Data points represent the mean ± SD of 3 replicates. **Significantly different from corresponding control (1.5 ppt salinity) at p < 0.05. ##Significantly different from corresponding 7-ppt salinity group at p < 0.05.

 


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FIG. 3. Specific activity of cholinesterase in muscle and brain tissues of surviving striped bass after aldicarb treatment (0.5 mg/l) following exposure to different salinities for 2 weeks. Data points represent the mean ± SD of 3 replicates.

 
Effects of Salinity on in Vitro Aldicarb Metabolism in Juvenile Rainbow Trout and Striped Bass
To understand the role of FMOs in aldicarb metabolism and the effect of salinity on this biotransformation, 14C-aldicarb metabolism was measured in gill, liver, and kidney microsomes of animals residing at various salinities (Table 1Go). In gill and liver microsomes from trout residing at 1.5 ppt salinity, no detectable level of 14C-aldicarb sulfoxide was observed. However, the major metabolite formed in incubations with gill, liver, and kidney microsomes from fish acclimated to higher salinity regimens was aldicarb sulfoxide (93, 95, and 90% of total metabolites in kidney, gill, and liver microsomes, respectively). Raising the salinity from 1.5 ppt to 7 ppt increased the formation of aldicarb sulfoxide in gill and liver microsomes. In gill and liver microsomes from 14 ppt and 18 ppt animals, a significant increase in sulfoxide production was observed compared to the 1.5 ppt group. Although sulfoxide formation was observed at 1.5 ppt, salinity did not alter sulfoxide levels in kidney microsomes of trout. In striped bass, aldicarb sulfoxide was also the major metabolite in liver microsomes (90% of total metabolites) but the formation of aldicarb sulfoxide was not affected by salinity. Gill microsomes of striped bass did not show formation of detectable levels of aldicarb sulfoxide from animals maintained at any salinity regimen. Under all conditions, the formation of aldicarb sulfoxide was dependent upon NADPH (data not shown). N-benzylimidazole had no significant effect on the formation of aldicarb sulfoxide in microsomes from liver, gill, and kidney of rainbow trout or liver of striped bass at any salinity regimen (Table 1Go).


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TABLE 1 Effects of Benzylimidazole on Formation of Aldicarb Sulfoxide in Vitro in Gill, Liver, and Kidney Microsomes in Rainbow Trout and Gill and Liver Microsomes in Striped Bass following a 2-Week Exposure to Different Salinity Regimens
 
Effects of Salinity on FMO Catalytic Activity (Thiourea-Dependent Thiocholine Oxidation) in Juvenile Rainbow Trout and Striped Bass
In juvenile rainbow trout, following 2 weeks of salinity exposure, FMO catalytic activity increased significantly in gill, liver, and kidney (Fig. 4Go). This "salinity dependency" was found most clearly in the gill. Compared to the 1.5 ppt treatment, the activity in gill was enhanced 40- and 80-fold (0.224 ± 0.091 and 0.414 ± 0.101 nmol/min/mg) in 14 ppt and 18 ppt salinity regimens, respectively. In the liver, the catalytic activity of fish residing in 14 ppt increased 7.5-fold compared to 1.5 ppt fish. Raising the salinity to 18 ppt led to an 8.6-fold increase of hepatic FMO activity compared to 1.5 ppt. In the trunk kidney, 2.2- and 3.6-fold increases of activity relative to 1.5 ppt were found in the 14 and 18 ppt groups, respectively. In contrast, salinity did not influence FMO catalytic activity in juvenile striped bass. Among all the salinity regimens, FMO catalytic activity in gill remained approximately 0.027 nmol/min/mg while the average activity in liver was 0.404 nmol/min/mg (Fig. 5Go).



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FIG. 4. Specific activity of FMOs in gill, liver, and trunk kidney from rainbow trout following a 2-week exposure to different salinities. Each point represents the mean ± SD of 3 replicates. Each replicate consisted of pooled samples of 5–6 individuals. **Significantly different from corresponding control (1.5 ppt salinity) at p < 0.05. ##Significantly different from corresponding 7-ppt salinity group at p < 0.05.

 


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FIG. 5. Specific activity of FMOs in gill and liver microsomes from striped bass after a 2-week exposure to different salinities. Each point represents the mean ± SD of 3 replicates. Each replicate consisted of a pool of 5–6 individuals.

 
Effects of Salinity on FMO Transcriptional Expression in Juvenile Rainbow Trout and Striped Bass
Northern blot analysis revealed a single hybridizing mRNA band of approximately 2.5 kb, in the whole-tissue RNA extractions of liver (14 and 18 ppt), gill (7, 14, and 18 ppt), and trunk kidney (all salinity regimens) of rainbow trout and liver of striped bass. FMO mRNA correlated with the catalytic activity with a salinity-dependent expression in gill, liver, and trunk kidney of juvenile rainbow trout (Fig. 6Go). FMO mRNA expression in the kidneys of fish residing at both 14 and 18 ppt were significantly elevated above 1.5 ppt fish. A representative Northern blot of trout kidney FMO mRNA is shown in Figure 7Go. In the gill, FMO mRNA was significantly higher at the 7 ppt salinity regimen compared to 1.5 ppt. In striped bass, salinity did not affect FMO transcriptional expression. In the liver, there were no significant differences in FMO mRNA expression levels in different salinity regimens (Fig. 8Go). In the gill, there was no detectable FMO mRNA expression in the salinity regimens, which was consistent with the lower catalytic activities versus trout.



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FIG. 6. Transcriptional expression of FMOs in gill, liver, and kidney whole tissue RNA extractions from rainbow trout after a 2-week exposure to different salinity regimens. Each point represents the mean ± SD of 3 replicates. The result is standardized by corresponding 28S band density. **Significantly different from corresponding control at p < 0.05.

 


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FIG. 7. Representative Northern blot of trout kidney FMO mRNA after a 2-week exposure to different salinity regimens. Lane 1, FMO1 Plasmid; Lane 2, 1.5 ppt; Lane 3, 3 ppt; Lane 4, 7 ppt; Lane 5, 18 ppt.

 


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FIG. 8. Transcriptional expression of FMOs in liver whole tissue RNA extractions from striped bass after a 2-week exposure to different salinity regimens. Each point represents the mean ± SD of 3 replicates.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a widely used carbamate insecticide, aldicarb is applied on crops that are frequently grown adjacent to estuaries, which are characterized by fluctuations in salinity. Understanding how fluctuating salinity might affect the toxicity of this compound is of critical importance to euryhaline organisms, which are at high risk of being acutely exposed to aldicarb because of its high water solubility (4.9 g/l) especially during an agricultural runoff event (Risher et al., 1987Go). Although the relationship between salinity and toxicity of xenobiotics is still relatively unclear, a series of studies indicated that several pesticides seem to have an increased toxicity at high salinity. The toxicity of several organophosphate insecticides increased with increasing salinity (Brecken-Folse et al., 1994Go). Azinophosmethyl, coumaphos, and chlorinated hydrocarbons (dieldrin and aldrin) proved to be more toxic to three-spine stickle-back (Gasterosteus aculeaus) at high salinity (Katz, 1961Go) and the herbicide atrazine caused higher morality to Cyprinodon. variegatus larvae residing at high salinity levels (Hall et al., 1994Go). Aldicarb caused greater toxicity to Japanese medaka (Oryzias latipes) following hypersalinity exposure (El-Alfy and Schlenk, 1998Go).

The hypothesis of the current study was that osmotic pressure affects FMO expression such that hypersalinity would increase the toxicity of chemicals bioactivated by FMO. In this regard, we observed that salinity significantly enhanced the acute toxicity of aldicarb to rainbow trout but not striped bass. Aldicarb, like other carbamates, exerts its toxic effect primarily by inhibiting cholinesterase activity, thereby disrupting the neuromuscular system (Coppage, 1977Go). In order to correlate the acute toxicity of aldicarb with cholinesterase inhibition, the cholinesterase activities of muscle and brain tissues in surviving fish after aldicarb treatment were measured. In rainbow trout, salinity-dependent cholinesterase inhibition was indicated in muscle but not in brain. These results suggest a potential causative relationship between the acute toxicity of aldicarb and muscular cholinesterase inhibition. Thus, the extent of cholinesterase inhibition in muscle rather than brain might be an essential factor in the ultimate mortality of fish exposed to aldicarb. These results are consistent with the studies by Perkins and Schlenk (2000) in channel catfish showing that lethal concentrations of aldicarb inhibited brain cholinesterase activity to a lesser extent than muscle cholinesterase.

Salinity did not significantly change aldicarb-mediated cholinesterase inhibition in either muscle or brain tissues in striped bass. These results were consistent with the absence of salinity-enhanced toxicity of aldicarb in striped bass. Several potential mechanisms of salinity-enhanced toxicity exist: (1) salinity increases the uptake and accumulation of xenobiotics; (2) salinity alters the dispositional fate of xenobiotics by either inhibiting detoxification pathways, enhancing bioactivation pathways, or reducing elimination; (3) salinity increases the sensitivity or susceptibility of target molecules to the toxicants. Previous studies in our laboratory have shown that salinity had no effect on aldicarb accumulation in Japanese medaka (El-Alfy and Schlenk, 1998Go). In studies with pentachlorophenol, Tachikawa et al. (1991) even reported a reduction in the amount of chemical accumulated by seawater-adapted medaka. Dyer et al. (1989) also showed that salinity had no effect on the uptake of the pyrethroid insecticide fenvalerate by bluegill (Lepomis macrochirus). These findings suggest that salinity does not appear to significantly impact the uptake of organic chemicals.

In order to exclude the possibility that salinity diminishes cholinesterase activity, subsequently enhancing aldicarb toxicity, muscular cholinesterase activity of rainbow trout was measured with and without 2 weeks of salinity exposure. The results indicated that salinity alone did not change the cholinesterase activity statistically, which is consistent with the findings in Japanese medaka (El-Alfy and Schlenk, 1998Go).

Earlier studies in our laboratory indicated biotransformation plays a key role in the salinity-induced toxicity of aldicarb (El-Alfy and Schlenk, 1998Go). Although aldicarb can inhibit cholinesterase activity without bioactivation, the oxidation of the thioether moiety to the corresponding sulfoxide was shown to enhance cholinesterase inhibition 200-fold in channel catfish (Ictalurus punctatus) and rainbow trout (Oncorhynchus mykiss) (Perkins et al., 1999Go). In mammals, bioactivation of aldicarb to the sulfoxide has been shown to be catalyzed by CYP (Kulkarni and Hodgson, 1980Go) and FMOs (Hajjar and Hodgson, 1980Go, 1982Go). In vitro studies in rainbow trout indicated that one or more forms of FMOs were primarily responsible for the oxidation of aldicarb to its sulfoxide in several tissues (Perkins et al., 1999Go; Schlenk and Buhler, 1991Go). A direct correlation between salinity and FMO expression has been observed in several euryhaline fish species (El-Alfy and Schlenk, 1998Go; Larsen and Schlenk, 2001Go; Schlenk, 1993Go, 1995Go, 1998Go; Schlenk et al., 1996Go), and total CYP does not seem to be influenced by salinity (Schlenk et al., 1996Go; Stegeman and Hahn, 1994Go). To further confirm the role of FMOs in aldicarb biotransformation, in vitro aldicarb metabolism assays were conducted in gill, liver, and kidney microsomes from fish residing in different salinity regimens. Aldicarb sulfoxide was the major metabolite in all the tissue microsomes of rainbow trout, and the formation of aldicarb sulfoxide was salinity-enhanced in gill and liver microsomes (10.6- and 2.3-fold increases, respectively); this was well correlated to thiourea oxidase activity. In contrast to the salinity-induced FMO activity in kidney, the formation of aldicarb sulfoxide in kidney microsomes was salinity-independent. These results appeared to agree with previous studies by Schlenk and Buhler (1991) showing there were no significant differences in sulfoxide formation in kidney microsomes after coincubation of other FMO substrates. It is possible that FMOs may not be involved in aldicarb sulfoxide formation in this organ. Another possibility is that there may be more than one isoform of FMO existing in kidney, and the one that catalyzes aldicarb bioactivation to aldicarb sulfoxide might not be induced by salinity.

In all tissues at each salinity regimen, the formation of aldicarb sulfoxide was NADPH-dependent, indicating a potential role of CYP-catalyzed sulfoxidation. In order to differentiate the effects of CYPs and FMOs, N-benzylimidazole was added to microsomal incubations prepared from liver, kidney, and gill. Earlier studies in liver microsomes from rainbow trout demonstrated reductions in CYP catalytic activity after coincubation with N-benzylimidazole (Schlenk and Buhler, 1991Go). In the current study, N-benzylimidazole failed to alter rates of sulfoxide formation, suggesting that CYP might not play a key role in aldicarb bioactivation in these tissues. In striped bass, aldicarb sulfoxide was still the major metabolite in liver microsomes, and the formation of sulfoxide was also NADPH-dependent and unaffected by N-benzylimidazole. In hepatic microsomal preparations from striped bass, FMOs were primarily responsible for S-oxygenation of thiobencarb (Cashman et al., 1990Go). But in contrast to the rainbow trout, salinity did not alter the formation of sulfoxide in liver microsomes of striped bass; this was consistent with no observed effect of salinity on aldicarb toxicity. Thus, the studies in striped bass provide further evidence for the hypothesis that salinity-enhanced aldicarb toxicity might be partially due to the salinity-dependent upregulation of FMOs. In all salinity regimens, gill microsomes of striped bass did not show formation of detectable levels of aldicarb sulfoxide, which might be due to the relatively low FMO expression level in this tissue and also suggests that the gill FMOs may not play an important part in aldicarb bioactivation in striped bass.

To summarize, a direct correlation between salinity and FMO expression was observed in euryhaline rainbow trout, but not in striped bass. In an attempt to understand the underlying influences of salinity on FMO-mediated xenobiotic metabolisms and toxicological effects, aldicarb acute toxicity, target enzyme cholinesterase activity, and biotransformation were examined in both in vivo models following a 2-week salinity exposure. The results suggest that the salinity-related upregulation of FMO expression might contribute to the salinity-induced enhancement of aldicarb toxicity to rainbow trout by increasing the bioactivation of aldicarb to aldicarb sulfoxide, a more potent cholinesterase inhibitor. Studies are underway to examine the effects of salinity on other potential environmental agents that are activated by FMO.


    ACKNOWLEDGMENTS
 
The authors thank the Rhone Poulenc Chemical Co. for their provision of radiolabeled aldicarb. This research was funded by an Exploratory Research Grant from the U.S. EPA to D.S. (R-823450-01-0) and the Environmental Toxicology Research Program of the Environmental and Community Health Research Division of the Research Institute for Pharmaceutical Sciences at the University of Mississippi.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (909) 787-3993. E-mail: daniel.schlenk{at}ucr.edu. Back


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