* Environmental Toxicology Research Program, Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677; and
Environmental Toxicology Program, Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521
Received May 10, 2001; accepted August 20, 2001
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ABSTRACT |
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Key Words: salinity; aldicarb; FMO; cholinesterase; rainbow trout; striped bass.
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INTRODUCTION |
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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, 1977). 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., 2001
; Perkins and Schlenk, 2000
). 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., 1987
). 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, 1980
; Kulkarni and Hodgson, 1980
). 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, 1991
). 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, 2000
).
Previous studies have indicated that high salinity upregulates FMO activity in Japanese medaka (Schlenk and El-Alfy, 1998), eel (Anguilla japonica), and guppy (Poecilia reticulata; Daikoku et al., 1988
), while low salinity downregulates FMO activity in Atlantic flounder (Paralichthys flesus; Schlenk et al., 1996
). Rainbow trout (Oncorhynchus mykiss) slowly osmoconform to saltwater, whereas hybrid striped bass (Morone saxatilis x chrysops) are rapid osmoconformers (Madsen et al., 1994
). Each species expresses several FMO isoforms in various tissues (Cashman et al., 1990
; Schlenk and Buhler, 1991
). However, FMO activity was lower in striped bass collected from seawater environments compared to freshwater environments (Cashman et al., 1989
, 1990
). In contrast, studies in trout indicated FMO upregulation in response to high salinity (Larsen and Schlenk, 2001
). 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.
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MATERIALS AND METHODS |
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Fish maintenance and treatment.
Juvenile rainbow trout (Oncorhynchus mykiss), weighing 2050 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 1530 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 1011 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, 56 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, 1991).
Microsomal preparation.
Microsomes from dissected tissues were prepared as previously described (Schlenk and Buhler, 1991). Briefly, dissected tissues from 56 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., 1961) adapted for 96-well microplates as previously described (Nostrandt et al., 1993
). 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, 1996
). 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., 1996), 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 µg1 mg), 0.1 M potassium phosphate buffer, pH 8.4, containing 100 mM thiocholine, 0.25 mM NADPH, and 1.2 mM thiourea. After 3060 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 cm1 was used to calculate the catalytic activity (Guo and Ziegler, 1991
). All reaction conditions (i.e., substrate concentrations and incubation times) were determined from previous studies (Schlenk et al., 1996
).
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, 1991). Incubations contained 100 mM 14C-aldicarb (1.0 m Ci/mM), 100 µM NADPH, 0.51.0 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, 1991
). 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, 1991
), 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., 1995), radiolabeled by random primer extension to a specific activity of 106 CPM/ml prehybridization buffer with [a-33P]-dCTP (Feinberg and Vogelstein, 1983
, 1984
). 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 0.249.5 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.
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RESULTS |
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DISCUSSION |
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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, 1977). 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, 1998). 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, 1998).
Earlier studies in our laboratory indicated biotransformation plays a key role in the salinity-induced toxicity of aldicarb (El-Alfy and Schlenk, 1998). 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., 1999
). In mammals, bioactivation of aldicarb to the sulfoxide has been shown to be catalyzed by CYP (Kulkarni and Hodgson, 1980
) and FMOs (Hajjar and Hodgson, 1980
, 1982
). 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., 1999
; Schlenk and Buhler, 1991
). A direct correlation between salinity and FMO expression has been observed in several euryhaline fish species (El-Alfy and Schlenk, 1998
; Larsen and Schlenk, 2001
; Schlenk, 1993
, 1995
, 1998
; Schlenk et al., 1996
), and total CYP does not seem to be influenced by salinity (Schlenk et al., 1996
; Stegeman and Hahn, 1994
). 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, 1991). 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., 1990
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Brecken-Folse, J. A., Mayer, F. L., Pedigo, L. E., and Marking, L. L. (1994). Acute toxicity of 4-nitrophenol, 2,4-dinitrophenol, terbufos and trichlorfon to grass shrimp (Palaemonetes spp.) and sheeepshead minnows (Cyprinodon variegatus) as affected by salinity and temperature. Environ. Toxicol. Chem. 13, 6777.[ISI]
Cashman, J. R., Olsen, L. D., Young, G., and Bern, H. (1989). S-oxygenation of Eptam in hepatic microsomes from fresh- and saltwater striped bass (Morone saxatilis). Chem. Res. Toxicol. 2, 392399.[ISI][Medline]
Cashman, J. R., Olsen, L. D., Nishioka, R. S., Gray, E. S., and Bern, H. A. (1990). S-oxygenation of Thiobencarb (Bolero) in hepatic preparations from striped bass (Morone saxatilis) and mammalian systems. Chem. Res. Toxicol. 3, 433440.[ISI][Medline]
Chomczynski P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156159.[ISI][Medline]
Coppage, D. L. (1977). Anticholinesterase action of pesticidal carbamates in the central nervous system of poisoned fishes. In Physiological Responses of Marine Biota to Pollutants (J. F. Vernberg, Ed.), pp. 93102. Academic Press, New York.
Daikoku, T., Murata, M., and Sakaguchi, M. (1988). Effects of intraperitoneally injected and dietary trimethylamine on the biosynthesis of trimethylamine oxide in relation to seawater adaptation of the eel Anguilla japonica and the guppy Poecilia reticulata. Comp. Biochem. Physiol. 89A, 261264.
Dyer, S. D., Coats, J. R., Bradbury, S. P., Atchison, G. J., and Clark, J. M. (1989). Effects of water hardness and salinity on the acute toxicity and uptake of fenvalerate by bluegill (Lepomis macrochirus). Bull. Environ. Contam. Toxicol. 42, 359366.[ISI][Medline]
Eddleman, B. R., and Falconer, L. (2000). Assessment of Surface Water Quality from Agricultural Croplands in the Odem Ranch Watershed. Texas Natural Resource Conservation Commission, Austin, TX.
El-Alfy, A., and Schlenk, D. (1998). Potential mechanisms of the enhancement of aldicarb toxicity to Japanese medaka, Oryzias latipes, at high salinity. Toxicol. Appl. Pharmacol. 152, 175183.[ISI][Medline]
El-Alfy, A., Grisle, S., and Schlenk, D. (2001). Characterization of salinity-enhanced toxicity of aldicarb to Japanese medaka: Sexual and developmental differences. Environ. Toxicol. Chem. 20, 20932098.[ISI][Medline]
Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961). A new and rapid colorimetric method for the determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 8895.[ISI][Medline]
Feinberg, A. P., and Vogelstein, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 613.[ISI][Medline]
Feinberg, A. P., and Vogelstein, B. (1984). Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137, 266267.[ISI][Medline]
Guo, W. X. A., and Ziegler, D. M. (1991). Estimation of flavin-containing monooxygenase activities in crude tissue preparations by thiourea-dependent oxidation of thiocholine. Anal. Biochem. 198, 143148.[ISI][Medline]
Hall, L. W., Ziegenfuss, M. C., Anderson, R. D., Spittler, T. D., and Leichtweis, H. C. (1994). Influence of salinity on atrazine toxicity to a Chesapeake Bay copepod (Eurytemora affinis) and fish (Cyprinodon variegatus). Estuaries 17, 181185.[ISI]
Hajjar, N. P., and Hodgson, E. (1980). Flavin-adenine dinucleotide-dependent monooxygenase: Its role in the sulfoxidation of pesticides in mammals. Science 209, 11341136.[ISI][Medline]
Hajjar, N. P., and Hodgson, E. (1982). Sulfoxidation of thioether-containing pesticides by the flavin-adenine dinucleotide-dependent monooxygenase of pig liver microsomes. Biochem. Pharmacol. 31, 745752.[ISI][Medline]
Katz, M. (1961). Acute toxicity of some organic insecticides to three species of salmonids and to three-spine stickle back. Trans. Fish. Soc. 90, 264269.
Kulkarni, A. P., and Hodgson, E. (1980). Metabolism of insecticides by mixed function oxidase systems. Pharmacol. Ther. 8, 379475.[ISI][Medline]
Larsen, B. K., and Schlenk, D. (2001). Effects of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B. 171, 421429.[ISI][Medline]
Madsen, S. S., McCormick, S. D., Young, G., Endersen, J. S., Nishioka, R. S., and Bern, H. A (1994). Physiology of seawater acclimation in the striped bass, Morone saxatilis. Fish Physiol. Biochem. 13, 111.[ISI]
Nostrandt, A. C., Duncan, J. A., and Padilla, S. (1993). A modified spectrophotometric method appropriate for measuring cholinesterase activity in tissue from carbaryl-treated animals. Fund. Appl. Toxicol. 21, 196203.[ISI][Medline]
Perkins, E. J., El-Alfy, A., and Schlenk, D. (1999). In vitro sulfoxidation of aldicarb by hepatic microsomes of channel catfish, Ictalurus punctatus. Toxicol. Sci. 48, 6773.
Perkins, E. J., and Schlenk, D. (2000). In vivo acetylcholinesterase inhibition, metabolism, and toxicokinetics of aldicarb in channel catfish: Role of biotransformation in acute toxicity. Toxicol. Sci. 53, 308315.
Risher, J. F., Mink, F. L., and Stara, J. F. (1987). The toxicologic effects of the carbamate insecticide aldicarb in mammals: A review. Environ. Health Perspect. 72, 267281.[ISI][Medline]
Schlenk, D. (1993). A comparison of endogenous and exogenous substrates of the flavin-containing monooxygenases in aquatic organisms. Aquat. Toxicol. 26, 157162.[ISI]
Schlenk, D. (1995). Use of aquatic organisms as models to determine the in vivo contribution of flavin-containing monooxygenases in xenobiotic biotransformation. Mol. Marine. Biol. Biotech. 4, 323330.[ISI][Medline]
Schlenk, D. (1998). Occurrence of flavin-containing monooxygenases in non-mammalian eukaryotic organisms. Comp. Biochem. Physiol. 121C, 185195.[ISI]
Schlenk, D., and Buhler, D. R. (1991). Role of flavin-containing monooxygenases in the in vitro biotransformation of aldicarb in rainbow trout (Oncorhynchus mykiss). Xenobiotica 21, 15831589.[ISI][Medline]
Schlenk, D., and El-Alfy, A. (1998). Expression of Branchial flavin-containing monooxygenase is directly correlated with salinity-induced aldicarb toxicity in the euryhaline fish (Oryzias latipes). Mar. Env. Res. 46, 103106.[ISI]
Schlenk, D., Peters, L. D., and Livingstone, D. R. (1996). Correlation of salinity with flavin-containing monooxygenase activity but not cytochrome P450 activity in the euryhaline fish (Platichthys flesus). Biochem. Pharmacol. 52, 815818.[ISI][Medline]
Shehin-Johnson, S., Williams, D. E., Larsen-Su, S., Stresser, D. M., and Hines, R. N. (1995) Tissue-specific expression of flavin containing monooxygenase (FMO) forms 1 and 2 in the rabbit. J. Pharmacol. Exp. Ther. 272, 12931299.[Abstract]
Stegeman, J., and Hahn, M. (1994). Biochemistry and molecular biology of monooxygenases: Current perspectives of forms, functions and regulation of cytochrome P450 in aquatic species. In Aquatic Toxicology: Molecular, Biochemical, and Cellular Perspectives (D. Malin and G. Ostrander, Eds.), pp. 87204. Lewis, Boca Raton, FL.
Tachikawa, M., Sawamura, R., Okada, S., and Hamada, A. (1991). Differences between freshwater and seawater killifish (Oryzias latipes) in the accumulation and elimination of pentachlorophenol. Arch. Environ. Contam. Toxicol. 21, 146151.[ISI][Medline]
Tomlin C. (1994). The Pesticide Manual. Crop Protection Publication, Farnham, Surrey, UK.
Vittozzi, L., and De Angelis, G. (1991). A critical review of comparative acute toxicity data on freshwater fish. Aquat. Toxicol. 19, 167204.[ISI]