* School of Veterinary Medicine, Department of Anatomy, Physiology, and Cell Biology, and
College of Agricultural and Environmental Sciences, Department of Environmental Toxicology, University of California at Davis, Davis, California 95616
Received November 9, 2000; accepted February 7, 2001
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ABSTRACT |
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Key Words: organophosphate; developmental sensitivity; teleost.
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INTRODUCTION |
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Diazinon is widely used to control pests in residential settings as well as commercial agriculture. Due to its varied and widespread use, diazinon release from nonpoint sources is significant (for a review see Larkin and Tjeerdema, 2000). Locally, diazinon has been detected in the Sacramento and San Joaquin Rivers, their delta, and the upper San Francisco Bay following dormant spray usage and at levels exceeding National Academy of Science guidelines as far down as the Upper San Francisco Bay (Kuivila and Foe, 1995). Our laboratory monitored the toxicity of these ambient waters using tests with the water flea, Ceriodaphnia dubia (Werner et al., 2000
), and toxicant identification evaluations indicated diazinon as the causative agent in assays with surface waters containing runoff from urban and agricultural areas in the watershed (Kuivila and Foe, 1995
).
OP toxicity results from inhibition of acetylcholinesterase (AChE), and metabolic conversion of OPs to their oxon metabolite results in formation of potent AChE inhibitors. Metabolic conversion of diazinon to diazoxon is mediated by the cytochrome P450 monooxygenase system of fish (Fujii and Asaka, 1982; Hogan and Knowles, 1972
). Limited studies to date demonstrate rapid increases in the activity of cytochrome P450mediated enzyme activity in fish as hatching approaches (Binder and Stegeman, 1984
; Binder et al., 1985
; Wisk and Cooper, 1992
). Interestingly, the limited studies of phosphorothionate OPs, using parent compounds, indicate that embryos are less sensitive than larvae (Anguiano et al., 1994
; Takimoto et al., 1984a
). Studies of juvenile and adult fishes have concluded that sensitivity to OPs is determined by AChE sensitivity, as measured by in vitro AChE inhibition by oxon metabolites (see review, Chambers and Carr, 1995). However, factors affecting sensitivity of els fishes, including uptake, bioactivation, and the sensitivity of AChE, remain undefined.
Information on developmentally related changes in uptake, biotransformation, and resultant toxic effect of OPs is needed. Medaka (Oryzias latipes), a surrogate species in the present study, are small fish native to Japan and other countries of Southeast Asia. Because of their transparent chorion, precise staging of the developmental processes is possible without invasion, making medaka particularly advantageous for embryological and developmental investigations (Iwamatsu, 1994; Kirchen and West, 1976
). Here we report on factors affecting the sensitivity of embryo-larval medaka to diazinon.
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MATERIALS AND METHODS |
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Stock preparation.
Diazinon was weighed in a glass boat and transferred to a volumetric flask containing embryo-rearing medium (ERM) (Kirchen and West, 1976), and its concentration was confirmed by gas chromatographic analysis (Aston and Seiber, 1996
). Measured concentration was within 5% of the nominal concentration. Dilutions of the defined stock solution were used for tests described below.
Embryo collection and culture.
Culture conditions for medaka have been described (Hamm et al., 1998). Briefly, broodstock were maintained in water reconditioned to U.S. Environmental Protection Agency moderately hard conditions (Horning and Weber, 1985
) at 25°C under a 16L:8D photoperiod and fed a purified, casein-based diet (DeKoven et al., 1992
) supplemented with brine shrimp nauplii. Embryos were collected from females and individuals were separated by rolling clusters between fingertips to break connective filaments (Marty et al., 1990
). After cleaning, embryos were placed in ERM, aerated, and maintained at 25°C until exposure.
AChE measurement.
Determination of AChE activity followed the method of Ellman et al. (1961) as modified for a 96-well microplate reader (see Hamm et al., 1998 for details). Briefly, following homogenization in assay buffer (0.1 M sodium phosphate buffer, pH 8.0), samples were incubated for 15 min with 0.115 mM tetraisopropyl pyrophosphoramide (iso-OMPA) to inhibit nonspecific cholinesterases (BChE). Following incubation, 30 µl acetylthiocholine (10.7 mM) substrate was added, and with DTNB as chromogen, activity was determined. Substrate blanks and tissue blanks were used to standardize activity, and this value was normalized to protein concentration (see below).
Developmental pattern of AChE.
Embryos and larvae were pooled by developmental stage (Kirchen and West, 1976), snap-frozen, and stored at 80°C until analysis. Numbers of animals used for a homogenate varied depending on amount of activity present, i.e., stage of development, ranging from 5 larvae to 20 early-stage embryos with three replicates per stage.
Sensitivity
LC50 determination in embryos and larvae.
Embryos at stages 11, 29, or 34 and 24-h-old larvae were used. These were placed in 20-ml borosilicate vials (Fisher Scientific, Pittsburgh, PA) containing 2 mls ERM at pH 7.27.3 or a solution of diazinon in ERM. Replicates (n = 5) per concentration with at least five concentrations of diazinon were tested. At 24-h intervals, vials were monitored for dead organisms, solutions were withdrawn, and new solution was added. Dead embryos or larvae were removed daily. At 96 h, exposures were terminated, and the concentration required to kill 50% of the organisms was calculated.
Examination of developmental changes in the degree of AChE inhibition.
Embryos at stages 31, 32, 33, 34, and 35, and larvae that were 24 h or 7 days old were exposed to diazinon for 24 h. Replicates (n = 5) of five individuals each were placed in 2 ml ERM for control or test concentrations of 0.088, 0.88, 3.53, 17.6, 44.1, and 88.2 µmolar diazinon. After 24 h, embryos were transferred to cryovials, snap-frozen in liquid nitrogen, and stored at 80°C until analysis of AChE activity (see above).
Determination of the in vitro sensitivity of AChE.
A total of 100 stage 34 embryos and 100 24-h-old larvae were pooled and homogenized in 2 ml assay buffer using an ice-chilled teflon glass homogenizer and brief sonication as above. Homogenate (100 µl) was placed into glass test tubes with 900 µl assay buffer to yield three replicates per concentration. Sufficient diazoxon in 1 µl hexane was added to yield final µmolar concentrations of 0.1, 0.5, 1, or 10. Incubations with homogenate only and homogenate with 1 µl hexane were run as controls. Incubations were run at 25°C for 15 min on a rotary shaker. Following incubation, AChE activity of homogenates was determined (see above).
Uptake
Uptake of diazinon.
Embryos at stages 29, 33, and 35 or 24-h larvae were exposed to ERM or radiolabeled diazinon at 0.88, 3.53, or 17.6 µmolar concentrations. In addition, 24-h-old larvae were exposed to the above concentrations of labeled diazinon with 2.0 x 105 molar piperonyl butoxide (PBO) in order to determine if PBO affected uptake. At 24 h, embryos and larvae were removed from solution, rinsed twice, and homogenized in 500 µl of 0.1 M sodium phosphate buffer. From the homogenate, 40 µl was transferred to a 7-ml scintillation vial containing scintillation fluid (UniverSolTM, ICN, Costa Mesa, CA) and counted on a Beckman LS-5801 scintillation counter (Beckman Instruments, Inc., Irvine, CA).
In vivo exposure to diazoxon.
Replicates (n = 5) of stage 34 and 24-h-old larvae were exposed to 0.01, 0.10, 1, 10, and 100 µmolar diazoxon for 24 h, and AChE activity was analyzed.
Metabolism
Piperonyl butoxide (PBO) studies.
PBO, an inhibitor of P450, was used to inhibit bioactivation of diazinon. A range-finding experiment was run using stage 34 embryos and 24-h-old larvae exposed to 0.88 µmolar diazinon in the presence or absence of 0, 2.7 x 107, 2.0 x 106, or 2.0 x 105 molar PBO (three replicates per treatment). AChE activity was determined after exposure for 24 h.
An additional P450 inhibitor, 1-aminobenzotriazole, was used to confirm findings with PBO. Replicates (n = 3) of five larvae (24 h old) were exposed to 0.88 µmolar diazinon for 24 h in the presence or absence of 0, 1 x 103, 104, or 105 M 1-ABT. Following exposure, larvae were handled as above for AChE analysis.
The role of metabolic differences accompanying degree of development was estimated, replicates (n = 5) of stage 31 or 34 embryos or five 24-h-old larvae were exposed to diazinon for 24 h in the presence or absence of 2.0 x 105 molar PBO, and AChE activity was determined. Diazinon concentrations were: 0.016, 0.088, 0.88, 3.53, 17.6, and 44.1 µmolar.
To ensure the effects of PBO were not due to changes in uptake or distribution of diazinon, 24-h-old larvae were exposed to the active metabolite diazoxon with or without PBO. Replicates (n = 4) of five larvae (24 h old) were exposed to 0, 0.1, 0.5, 1, and 10 µmolar diazoxon in the presence or absence of 2.0 x 105 molar PBO for 24 h and treated as above for AChE analysis.
In vitro bioactivation of diazinon.
Bioactivation was measured by incubating diazinon with microsomes and indirectly measuring levels of oxon produced by determining resultant AChE inhibition. The use of AChE inhibition to estimate levels of oxon metabolites gives comparable measures to gas chromatographic analysis (Mirer et al., 1975) and has been used extensively (Forsyth and Chambers, 1989
). Our methods followed this approach with the following exceptions: a) magnesium chloride (5 mM) was eliminated because it interfered with AChE determinations. As magnesium is a cofactor for P450, metabolism in our system should be lower than in others. b) The concentration of NADP was decreased from 7.5 mM to 375 µM, with no recorded change in bioactivation. c) Incubation temperature was set at 35°C after initial tests showed little to no metabolism in incubations at 25 or 30°C. d) 2-methoxyethanol replaced ethanol as solvent for P450 inhibitors due to AChE inhibition with ethanol.
Using the above conditions, incubations (n = 3 per developmental stage) were prepared on ice in a final volume of 500 µl using 0.1 M Tris-HCl (pH 7.4). Incubations consisted of microsomal protein (150 µg) from either stage 31 embryos or 24-h-old larvae, 10 µl of the exogenous AChE source (see below), an NADPH-generating system [NADP (375 µM), glucose-6-phosphate (7.5 mM), glucose-6-phosphate dehydrogenase (0.5 I.U.)] and diazinon (50 µM) in 5 µl 2-methoxyethanol. A series of controls was incubated in conjunction with each experiment. All controls had the AChE, NADPH-generating system and one of the following: solvent, diazinon, or microsomes. Following set-up, incubation tubes were transferred to a shaking incubator for 30 min. After incubation, 30-µl samples (in triplicate) were rapidly placed in individual wells of a 96-well plate for AChE determination.
Microsomal preparation.
Microsomal preparation followed a modification of Buckpitt and Warren (1983). Adult medaka, 6 months old, were sacrificed by decapitation, and livers were quickly removed and transferred to ice-cold 0.02 M Tris-1.15% KCl, pH 7.4 buffer. All subsequent procedures were performed on ice. Pooled livers were removed from buffer, blotted, and weighed. Homogenization was in three volumes of ice-cold buffer (0.02 M Tris, 1.15% KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 15% glycerol, pH 7.4), and resultant homogenate was centrifuged at 10,000 x g for 30 min; the supernatant was then removed and centrifuged at 105,000 x g for 1 h. The microsomal pellet was resuspended in Tris/KCl buffer in approximately half the original volume and repelleted at 105,000 x g for 1 h. The final microsomal pellet was resuspended in 0.1 M sodium phosphate buffer, pH 7.4. Protein content was determined as described below.
For determination of developmental differences in in vitro bioactivation, 500 stage 31 embryos and 500 24-h-old larvae were separately pooled and microsomes were prepared as above, with the exception that microsomes were resuspended in a final volume of 100 µl.
Preparation of exogenous AChE.
Thirty adult medaka were decapitated and their brains were removed and pooled in ice-cold 0.1 M Tris-HCl (pH 7.4). Tissues were transferred to 3 ml 0.1 M Tris-HCl and homogenized using 57 passes of a chilled teflon glass homogenizer. Aliquots of 100 µl were snap-frozen in liquid nitrogen and transferred to a 80°C freezer for storage until use.
Determination of protein concentration.
Protein concentration was measured in homogenates using a simplified procedure of Smith et al. (1985), the bicinchoninic acid protein assay (Sigma Chemical Company; St. Louis, MO) with bovine serum albumin as standard.
Statistics.
Levels of statistical significance were analyzed by ANOVA, followed by a Scheffé's F-test as a post hoc test to compare means between the different treatment groups. Differences were considered significant if p < 0.05. LC50 values at 96 h with 95% confidence intervals were calculated using probit analysis.
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RESULTS |
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Incubation of microsomes derived from early life stage medaka resulted in 7.3 ± 2.7 and 28.3 ± 1.1% inhibition, for stage 31 embryos and 24-h-old larva, respectively. These results indicate a greater ability of the larval preparation to generate the oxon metabolite in vitro.
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DISCUSSION |
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During the period of development examined, AChE activity rose rapidly, and this pattern appears similar to that reported for rainbow trout (Uesugi and Yamazoe, 1964). Previous results from this laboratory showed that histochemical staining of acetylcholinesterase activity appears in neural tissue along with heavy staining of skeletal musculature (Hamm et al., 1998
). Higher levels of cholinesterase activity have been reported to account for decreased sensitivity to OP pesticides in insects (Fournier et al., 1993
). However, in the current study, increasing levels of AChE were associated with greater sensitivity. It is possible that in early life stage teleosts the rapid development of the cholinergic system fosters a dependence on this system, for example neurotransmission controlling gill movement during respiration, that results in higher sensitivity to the lethal effects of anticholinesterase pesticides. We showed that with the development of the cholinergic system in the retina, cell death appeared at sites of cholinesterase activity following exposure to diazinon (Hamm et al., 1998
).
Comparison of vertebrates by species, sex, and age groups reveals differences in sensitivity to OP compounds. These differences in sensitivity have been correlated to metabolic bioactivation and/or detoxification, A-esterase levels, and the sensitivity of AChE to inhibition (for a review see, Chambers and Carr, 1995). Benke and Murphy (1975) reported that age-dependent changes in OP sensitivity of rat pups was correlated to changes in P450-mediated detoxification, levels of glutathione, A-esterase levels, and binding to nontarget molecules.
In addition to the factors listed above, exposures of fish embryonated eggs must consider the role of uptake in toxicity. Because these life stages are surrounded by the chorion, a protective yet semipermeable barrier (for a review see Weis and Weis, 1989), the question of uptake is even more important. Helmstetter and Alden (1995) reported that the rate of uptake of agents topically applied to medaka embryonated eggs was proportional to their lipophilicity. OPs, including diazinon, with low water solubilities should be readily absorbed (Bowman and Sans, 1983). Medaka embryonated eggs exposed to radiolabeled fenitrothion showed rapid uptake, and subsequent autoradiography revealed distribution within internal organs and yolk sac of the embryo (Takimoto et al., 1984b
). Marty et al. (1990) studied the uptake of a series of radiolabelled compounds of varying hydrophobicity. No differences were seen until immediately prior to hatch, when uptake increased. In contrast to this observation, our exposures to 14C-diazinon resulted in significantly greater uptake by more developed embryos (see Fig. 3
; compare stage 29 to more developed embryos). In addition, these differences in uptake occurred at diazinon concentrations that resulted in substantial differences in AChE inhibition between the developmental stages tested. Finally, addition of 2.0 x 105 M PBO, which significantly decreases the toxicity of diazinon, to media containing 14C-diazinon did not alter the total radioactivity in larvae. This lack of effect by PBO on uptake, demonstrated herein, is in contrast to earlier reports that PBO altered toxicity due to decreased uptake (Kuo et al., 1983
; Sriram et al., 1995
); however, in the previous cases, the investigators were not studying organophosphates but instead worked with ionized compounds.
Following uptake and distribution, a key component in the toxicity of phosphorothionates is bioactivation to oxon metabolites. Differences in the capacity to bioactivate these compounds affects toxicity (Chambers and Carr, 1995). Ma and Chambers (1995) demonstrated that, in rat tissues, parathion was readily bioactivated by desulfuration, whereas chlorpyrifos was readily detoxified by dearylation; these metabolic differences correspond to the lower toxicity of chlorpyrifos. Compared with mammalian species, fish are known to have a low ability to metabolize OPs. Hitchcock and Murphy (1971) studied bioactivation of parathion and guthion by rat and by two fish species, bullhead (Ictalurus melas) and winter flounder (Pseudopleuronectes americanus) and noted that rat tissues bioactivated both compounds 2- to 3-fold more than either fish species. However, when fish species are compared, data demonstrate that toxicity differences are related to host metabolism. Keizer et al. (1995) demonstrated that guppy (Poecilia reticulata), the most sensitive of several species they studied, had the highest capacity to bioactivate diazinon, whereas carp (Cyprinus carpio) were insensitive and had a low ability to bioactivate diazinon. In addition, alternative pathways of metabolism that produce more polar metabolites, presumably hydroxylated metabolites other than the oxon, were reported to account for the insensitivity to diazinon of adult medaka versus loach (Misgurnus anguillicaudatus) (Oh et al., 1991
).
Our work demonstrated that as early life stage medaka developed (from fertilization to larvae), equimolar concentrations of diazinon caused increasing amounts of AChE inhibition. However, when larvae were exposed to diazinon in combination with either of two P450 inhibitors, piperonyl butoxide (Anders, 1968) or 1-aminobenzotriazole (Knickle and Bend, 1992
; Meschter et al., 1994
), levels of AChE inhibition were greatly decreased. These data suggest that metabolism is present in these stages and, once formed, continues to be important in later stages. Further, PBO provided greater protection from AChE inhibition with further development. Ankley et al. (1991) reported that aqueous exposures with PBO decreased toxicity of four metabolically activated OPs, including diazinon, but did not alter the toxicity of three OPs not requiring bioactivation. Mirer et al. (1977) demonstrated that PBO inhibited bioactivation and P450-mediated detoxification of methyl parathion in vitro and resulted in a 40-fold decrease in toxicity following in vivo exposure. In contrast, PBO did not affect the in vivo toxicity of the active metabolite methyl paraoxon. In the present study, PBO had no effect on IC50 values generated from in vivo exposures of early stage medaka to diazoxon (Table 3
), suggesting that inhibition of P450-mediated detoxification steps has little influence on sensitivity changes. PBO's lack of effect on diazoxon was important because PBO is a nonselective inhibitor of P450 and inhibits the oxidative reactions that both bioactivate and detoxify diazinon (Smith et al., 1974
).
Further evidence for the role of metabolic activation in the sensitivity changes was obtained from in vitro metabolism studies. We used an incubation system in which inhibition of an exogenous AChE source is related to metabolic conversion of diazinon to the more potent AChE-inhibiting oxon metabolite. Using adult medaka hepatic microsomes, we showed that bioactivation in this system was inhibited by each of three well-established ways to deactivate cytochrome P450 activity: a) boiling, b) exposure of microsomes to carbon monoxid, or c) removal of NADPH. These results demonstrated the essential requirement of cytochrome P450 activity for conversion of diazinon to a form that inhibits AChE. Microsomes from medaka embryos were capable of bioactivating diazinon, and based on levels of AChE inhibition, 24-h-old larvae had significantly greater capacity for bioactivation than did stage 31 or earlier embryos. This apparent increase in metabolic capacity immediately after hatch mirrors increases in cytochrome P450-mediated enzyme activity occurring in early life stage teleosts at this time (Binder and Stegeman, 1984; Binder et al., 1985
; Wisk and Cooper, 1992
). Similarly, our measurements of ethoxyresorufin-O-deethylase activity, associated with cytochrome P450 1A1 in teleosts (Stegeman, 1989
), show a rapid increase around the time of hatch in medaka (unpublished observations in this laboratory).
Once bioactivated, organophosphate pesticides target AChE, and the sensitivity of this enzyme to in vitro inhibition has been used to explain species sensitivity differences. Combining substantial original research and a review of the literature, Chambers and Carr (1995) assert that in vitro sensitivity of AChE to oxon metabolites largely determines in vivo sensitivity of juvenile and adult fish. Johnson and Wallace (1987) compared species and noted that AChE from rats, a sensitive species, was more sensitive to inhibition by paraoxon than AChE derived from two insensitive species, fathead minnows and rainbow trout. Murphy et al. (1968) compared sensitivity to two OPs and found that fish had higher in vivo sensitivity to gutoxon than paraoxon; this sensitivity paralleled the higher in vitro sensitivity of fish brain cholinesterase to gutoxon. Finally, Keizer et al. (1995) reported that in vitro sensitivity of AChE was a determinant of toxicity differences among four species of fish.
In addition to species-specific differences in AChE, some have suggested that developmental changes in the sensitivity of AChE explain differences in toxicity between age groups. Anguiano et al. (1994) demonstrated that toad embryos were less sensitive than larvae to parathion, and AChE from an embryo homogenate had a higher in vitro IC50 value with paraoxon. AChE exists as several molecular forms, and the distribution of these forms varies with development (for a review see Massoulie and Bon, 1982). Therefore, developmental changes in AChE inhibition could result from differences in the sensitivity of these molecular forms. However, whereas Mortensen et al. (1997) found differences in IC50 values between tissues and during development in crude homogenates incubated with chlorpyrifos-oxon, immunoprecipitation of AChE and subsequent in vitro incubation resulted in similar IC50 values. The use of immunopurified AChE demonstrates that the apparent sensitivity of this enzyme may result from an interaction of oxon with nontarget molecules. Perhaps the tissue preparations used by Anguiano et al. (1994) resulted in a similar interaction with nontarget proteins. In this study, no difference between embryos and larvae in in vitro AChE inhibition by diazoxon was recorded (Table 2), further suggesting that in vitro AChE sensitivity is not a factor in developmental changes. Similarly, Benke and Murphy (1975) reported developmental changes in the sensitivity of rat pups to parathion and methylparathion, but did not detect changes in in vitro sensitivity of AChE.
In conclusion, the present study shows that toxicity of diazinon to a model early life stage teleost increases markedly around the time of hatch. Lower LC50 values and greater AChE inhibition with added development were the evidence of this change. Over the period of development examined, both the uptake of 14C-diazinon and the AChE inhibition following in vivo exposure to diazoxon increased with added development, indicating at least some of the sensitivity was associated with greater uptake of the compound. In addition, further development was associated with enhanced metabolism of diazinon. As medaka developed, P450 inhibitors had an increasing protective effect. Finally, in vitro metabolism studies demonstrated a higher rate of bioactivation with added development. These mechanistic investigations provide an improved understanding of organophosphate toxicity in early life stages of fishes.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Current address: Duke University, Nicholas School of the Environment, Durham, NC 27708-0328.
3 To whom correspondence should be addressed at Nicholas School of the Environment, Duke University, A246, LSRC-Science Dr., Box 90328, Durham, NC 27708-0328. Fax: (919) 613-8077. E-mail: dhinton{at}duke.edu.
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