Effect of 17ß-Estradiol and Testosterone on the Expression of Flavin-Containing Monooxygenase and the Toxicity of Aldicarb to Japanese Medaka, Oryzias latipes

Abir T. El-Alfy* and Daniel Schlenk{dagger},1

* School of Pharmacy, Cairo University, Cairo, Egypt; and {dagger} Department of Environmental Sciences, 2268 Geology, University of California, Riverside, California 92521-0424

Received January 9, 2002; accepted March 16, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies in our laboratory indicated gender differences in salinity-enhanced acute toxicity of aldicarb in Japanese medaka with females being more susceptible. In the current study, the effects of the sex steroids, 17ß estradiol (E2) and testosterone (T) on aldicarb toxicity was examined. Adult Japanese medaka were separated by sex and exposed to 100 µg/l E2 or T for 6 days followed by exposure to the 96-h LC50 (0.5 mg/l) of aldicarb. The toxicity of aldicarb to adult males was significantly lowered by E2 and T whereby the mortality percentage was reduced to 23.3 ± 5.8% and 3.3 ± 5.8%, respectively, compared to the fish not receiving steroids (46.7 ± 5.8% mortality). In females, T caused significant reduction in aldicarb toxicity to 16.7 ± 5.8%, while E2 significantly enhanced the toxicity to 96.7 ± 5.8% mortality. Since the flavin-containing monooxygenase (FMO) enzyme system had been shown to play a critical role in aldicarb toxicity, the effect of E2 and T on FMO expression was examined. Gill FMO activity showed a direct correlation with the overall toxicity of aldicarb in both male and female medaka. Expression of FMO1-like protein was significantly reduced by T in male livers and gills, and T did not affect the expression of FMOs in female tissues. In contrast, E2 significantly reduced FMO1-like protein expression in male gills and female livers, as well as FMO3 expression in both male and female livers, but significantly increased gill FMO1 expression in females. Since aldicarb acts by inhibiting the enzyme cholinesterase (ChE), the effect of sex hormones on the activity of this enzyme was also examined. In both male and female medaka, T counteracted the inhibitory effect of aldicarb on muscle ChE. In male fish, E2 had similar effects but did not seem to counteract the ChE inhibition in females. In conclusion, E2 and T modulation of aldicarb toxicity in Japanese medaka seems to be mediated via alteration of gill FMO and ChE actitivies.

Key Words: aldicarb; accumulation; elimination; biotransformation; medaka; salinity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The carbamate insecticide, aldicarb, has been widely used over the last 2 decades in a variety of registered applications including cotton, potato, soybean, sugar cane, sugar beet, ornamental plants, and peanut farming. In the United States, aldicarb ranks seventh among insecticides in terms of total acres treated and fifth in pounds applied (USGS, 1997). The mechanism of action of aldicarb is primarily through the reversible inhibition of cholinesterase (ChE) and subsequent nervous system disruption. The water solubility of aldicarb (4.9 g/l) and its moderate half-life (2–6 weeks in soil and 5–10 days in pond water) create the risk of surface water contamination and hence the potential for exposure of aquatic organisms (Risher et al., 1987Go).

Aldicarb metabolism involves both hydrolytic and oxygenation reactions. The hydrolytic products, including nitrile and oxime derivatives, are detoxification products and lack any anticholinesterase properties (Risher et al., 1987Go). S-oxygenation to the sulfoxide metabolite enhances the anticholinesterase activity several fold in both mammalian and fish species (Baron, 1994Go; El-Alfy and Schlenk, 1998Go; Perkins et al., 1999Go; Risher et al., 1987Go). Oxygenation to the sulfone reduces toxicity (Hastings et al., 1970Go). The bioactivation of aldicarb to aldicarb sulfoxide is catalyzed by either cytochrome P450 (CYP) or flavin monooxygenases (FMOs) but FMOs do not appear to be involved in the further transformation of the sulfoxide to the sulfone (Venkatesh et al., 1991Go). In rainbow trout as well as Japanese medaka, FMOs are responsible for the oxidation of aldicarb to the sulfoxide metabolite (El-Alfy and Schlenk, 1998Go; Schlenk and Buhler, 1991Go). Thus, modulation of the activity of this enzyme system would be expected to potentially alter the acute toxicity of aldicarb.

Sexual differences in the expression of FMOs have also been reported in certain species, notably rats and mice (Falls et al., 1995Go; Lawton and Philpot, 1993Go; Ripp et al., 1999Go; Tynes and Philpot, 1987Go). Sex steroids, primarily E2, T, and progesterone seem to play a major role in the regulation of FMO expression in different species (Falls et al., 1997Go).

FMO activity is also altered by changes in salinity within euryhaline fish (Schlenk et al., 1996aGo,bGo). We have shown that in Japanese medaka, salinity upregulated gill FMO(s) (El-Alfy and Schlenk, 1998Go). Indeed, salinity significantly enhanced the toxicity of aldicarb in Japanese medaka (El-Alfy and Schlenk, 1998Go) and this phenomenon was gender-selective as females were more sensitive than males to combined exposure of hypersaline conditions and aldicarb (El-Alfy et al., 2001Go). The mechanisms by which salinity modulates the activity of FMOs and hence aldicarb toxicity is still unclear.

Given the previously observed gender difference in salinity-enhanced aldicarb toxicity and the observations that FMO activity is related to salinity in Japanese medaka, the objective of this study was to examine the effect of osmoregulatory hormones, particularly sex steroids, on FMO activity and expression and the toxicity of aldicarb to the euryhaline fish, Japanese medaka.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Aldicarb (98.8% purity) was generously provided by Rhone-Poulenc Inc. (Research Triangle Park, NC). Sodium dodecyl sulfate (SDS) and polyacrylamide were obtained from BioRad (Hercules, CA). Antihuman FMO1 and FMO3 antibodies were a gift from Dr. Allen Rettie at the University of Washington. Alkaline phosphatase-linked antirabbit IgG antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All other reagents were purchased from Sigma Chemical Company (St. Louis, MO).

Animals.
Adult sexually mature medaka (8-weeks-old) of both sexes (0.25–0.48 g) were obtained from the Japanese medaka culture at the University of Mississippi Ecotoxicology Research laboratory. The fish were kept in glass aquaria with the temperature maintained at 25°C and a light cycle of 18 h light:6 h dark. They were fed cultured Artemia and tetramen flakes twice a day. Fish were separated by sex based on distinct sexual morphological characteristics. The fish were fed during the hormonal treatment and feeding was stopped 24 h prior to aldicarb treatment to prevent ammonia production during exposure. Dissolved oxygen ranged from 7.5–8.3 mg/l; unionized ammonia was 0.002–0.010 mg/l; and pH ranged between 6.6 (low salinity) to 7.6 (high salinity); alkalinity was 34.3–36.7 mg/l calcium carbonate; hardness was 82.5–94.1 mg/l calcium carbonate.

Animal treatment.
Adult sexually mature medaka were separated by sex and 10 individuals were placed in 2-liter silanized glass beakers. Each treatment was replicated in triplicate (n = 3). The tanks were fully aerated, filled with filtered dechlorinated water and the fish were left for 24 h to acclimate prior to hormonal treatment. Water was then treated with 100 µg/l of either E2 or T dissolved in ethanol. The volume of ethanol added did not exceed 0.1 % of the total water volume. Solvent control-treated fish were also used for comparison. The fish were exposed to the sex hormones for 6 days with static renewal of the water every 48 h. Using this identical treatment system, exposure of medaka to T and E2 in aqueous solutions at 100 µg/l significantly increased serum levels of the hormones (Thompson, 2000Go). Half of the fish were sacrificed by decapitation, with the subsequent dissection of livers, gills, and skeletal muscle. The tissues were immediately frozen between blocks of dry ice then stored at –80°C until further analysis. The remaining fish were exposed to the previously determined 96-h LC50 of aldicarb (0.5 ppm) and mortality was recorded daily. Following 96 h, the surviving fish were sacrificed by decapitation, skeletal muscle dissected, and ChE activity measured as described below.

Microsomal preparation.
Livers and gills excised from fish were homogenized in ice-cold buffer (0.1M Tris, 0.15M KCl, 1 mM EDTA, pH 7.4) containing phenylmethylsulfonyl fluoride (10 µM) and centrifuged at 10,000 x g for 30 min. Microsomes were isolated from the supernatant by ultracentrifugation (100,000 x g for 1.5 h) and were resuspended in 10 mM phosphate buffer (pH 7.4) containing 20% glycerol. All preparative steps were carried out at 4°C.

Western blotting.
Microsomal proteins were separated by denaturing polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970Go) using an 8% polyacrylamide separating gel. Proteins were transferred onto nitrocellulose sheets (Towbin et al., 1979Go) using a Biorad TransBlot apparatus (Hercules, CA). Antibodies used to probe protein blots were polyclonal antihuman FMO1 and FMO3 antibodies (Yeung et al., 2000Go). These antibodies have been used previously (Larsen and Schlenk, 2001Go) and were readily available. An alkaline phosphatase-linked secondary antibody was used for detection. Bands were quantified by scanning densitometry using NIH image software (version 1.61).

FMO activity.
FMO activity was measured in hepatic and gill microsomes using the thiourea dependent thiocholine oxidation assay (Guo and Ziegler, 1991Go) as modified by Schlenk et al.(1995). This assay is not isoform specific as FMO1 and FMO3 have thiourea oxygenase activities. An incubation mixture of 0.5 ml consisted of 0.5 mg microsomal protein, 0.1M phosphate buffer, pH 8.8, 0.1 mM thiocholine, 0.1 mM NADPH, and 1.2 mM thiourea. After 60 min incubation at room temperature, 0.4 ml was removed, placed in a tube containing 0.05 ml of 3.0 M trichloroacetic acid, and centrifuged at 10,000 x g for 10 min. The supernatant was then transferred to a tube containing 0.5 ml of 1.0 M phosphate buffer, pH 7.5, 0.3 ml of 18 {Omega} nanopure water, and 0.025 ml of 10 mM dithionitrobenzoate (DTNB). The concentration of thiocholine was determined using a millimolar absorptivity of 13.6 cm–1 at 412 nm and compared against incubations that did not contain NADPH (Schlenk et al., 1996aGo).

ChE activity assay.
Previous studies in Japanese medaka indicated that muscle ChE and lethality were closely associated (El-Alfy et al., 2001Go). Muscle tissue dissected from the fish was homogenized in 1.15% KCl (1:4 w/v) using an electrically driven tissue tearer and the homogenate was centrifuged at 5000 rpm for 5 min to pellet tissue debris. The supernatant was used to measure ChE activity. The activity was measured following 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. Eserine sulfate (10–5 M dissolved in methanol) was used as a blank to correct for noncholinesterase mediated hydrolysis (Nostrandt et al., 1993Go).

Statistical analysis.
All treatments were performed in 3 replicates (n = 3). Each replicate consisted of 10 individuals. Following Bartlett’s test for homogeneity, treatment group differences were determined using Kruskal-Wallis nonparametric ANOVA with Steel’s Multiple treatment–control Rank sum test p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FMO Expression and Activity
Each of the antibodies used (anti-FMO1 and anti-FMO3) recognized 1 band in liver and gill tissues of male and female medaka. The bands identified were approximately 57 and 59 kDa when probed with anti-FMO1 and anti-FMO3 antibodies, respectively. T significantly reduced the expression of the FMO1-like protein in male liver and gill but had no effect in females in either tissue. Gill FMO1 was reduced by E2 from 14.7 ± 1.8 to 11.5 ± 0.9 Optical Density Units (ODU). In females, E2 significantly reduced hepatic FMO1-like expression from 19.5 ± 1.3 to 7.2 ± 5.3 ODU. E2 led to a significant increase in gill FMO1-like protein from 20.1 ± 0.5 to 45.3 ± 15.3 ODU.

The FMO3-like band significantly decreased in intensity from 7.3 ± 0.6 to 3.6 ± 0.5 ODU in male livers and from 19.4 ± 6.1 to 7.1 ± 1.9 ODU in female livers after T treatment. A significant reduction in liver FMO3 in males was observed after E2 treatment with the band intensity decreasing from 7.3 ± 0.6 to 5.0 ± 0.1 ODU. Figures 1 and 2GoGo summarize the results of hormonal effects on FMO expression. Figure 3Go compares expression of each FMO isoform and activity between males and females.



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FIG. 1. Effect of 17ß-estradiol and testosterone on FMO1-like expression in liver (A) and gill (B) microsomes of adult Japanese medaka. Fish were exposed to 100 ppb either estradiol or testosterone for 6 days. Data points represent mean ± SD of 3 replicate experiments. Each replicate consisted of pooled tissues from 10 individual animals. Each lane was loaded with 0.02 mg of microsomal protein. *Denotes statistical significance from control at p < 0.05.

 


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FIG. 2. Effect of 17ß-estradiol and testosterone on FMO3-like protein expression in liver (A) and gill (B) microsomes of adult Japanese medaka. Fish were exposed to 100 ppb either estradiol or testosterone for 6 days. Data points represent mean ± SD of 3 replicate experiments. Each replicate consisted of pooled tissues from 10 individual animals. Each lane was loaded with 0.02 mg of microsomal protein. *Denotes statistical significance from control at p < 0.05.

 


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FIG. 3. Sex and tissue distribution of FMO expression (isoforms 1 and 3) and activity (thiourea S-oxygenase activity) in Japanese medaka. *Denotes statistical significance from males at p < 0.05.

 
Figure 4Go shows the effect of sex hormones on hepatic and gill FMO activity as measured by the thiourea oxidase assay. The male sex hormone T significantly downregulated FMO activity in liver and gill microsomes of both male and female medaka. In males, FMO activity was reduced by T to 81.46 and 81.25% of the control levels in livers and gills, respectively. In female livers and gills, T caused a significant 66.7 and 89.2% reduction in FMO activity, respectively. Similarly, the female sex hormone, E2 led to a significant 44.4, 68.75, and 76.16% decrease in FMO activity in male livers, gills, and female livers, respectively. In female gills, E2 led to a significant 237.9% increase in FMO activity.



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FIG. 4. Effect of 17ß-estradiol and testosterone on FMO activity in liver (A) and gill (B) microsomes of Japanese medaka. Data points represent mean ± SD of 3 replicate experiments. *Denotes statistical significance from control at p < 0.05.

 
Effect of Sex Hormones on Aldicarb Toxicity
As shown in Figure 5Go, pretreatment with T led to significant reduction in aldicarb toxicity in both male and female medaka. Mortality dropped from 46.7 ± 5.8% in control fish to 3.3 ± 5.8% and 16.7 ± 5.8% in male and female medaka, respectively. E2 caused a significant reduction in mortality in males; however, it caused a significant increase in mortality in female fish.



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FIG. 5. Effect of 17ß-estradiol and testosterone on aldicarb toxicity to adult Japanese medaka. Adult fish were exposed to 100 ppb either estradiol or testosterone for 6 days followed by exposure to the 96-h LC50 of aldicarb and fish mortality was recorded. Each value represents the mean of 3 replicate exposures ± SD. *Denotes statistical significance from controls using Fisher’s Exact Test at p < 0.05.

 
ChE Activity
As shown in Table 1Go, neither treatment with T nor E2 altered the ChE activity in male or female medaka. Following exposure to aldicarb, the surviving fish that were not treated with sex hormones had significantly reduced ChE activity (53.4 ± 4.8 and 55.1 ± 0.6 nmol/min/mg in male and female fish, respectively). Treatment with both sex hormones slightly reduced the inhibitory effect of aldicarb in male medaka whereby the ChE activity was not significantly different from that of untreated fish. In females, T treatment offered the same counteractive effect on the inhibitory action of aldicarb on ChE. However, E2-treated females that were also exposed to aldicarb had a significantly reduced ChE activity compared to animals receiving only aldicarb.


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TABLE 1 Effect of Estradiol and Testosterone on Muscle Cholinesterase (ChE) Activity and Degree of ChE Inhibition in Japanese Medaka
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aldicarb sulfoxide is the primary metabolic product of aldicarb for most species studied and exhibits much greater toxicity (DePass et al., 1985Go; Dorough and Levi, 1968Go; Foran et al., 1985Go; Pelekis and Krishnan, 1997Go; Perkins et al., 1999Go). Consequently, the degree of aldicarb biotransformation to the sulfoxide metabolite may have great influence on the ultimate toxicity of this compound. Perkins and Schlenk (2000) reported that the low rate of aldicarb sulfoxidation in channel catfish as compared to rainbow trout might be responsible for resistance exhibited by channel catfish to aldicarb toxicity. The differences in sulfoxide production in these 2 species were attributed to the finding that FMOs are not catalytically active in channel catfish (Schlenk et al., 1993Go). The lack of FMOs in channel catfish reduces the animal’s ability to bioactivate aldicarb to the more potent sulfoxide. In Japanese medaka, we have previously shown that sulfoxide production is catalyzed by FMOs rather than CYP, as carbon monoxide, a CYP inhibitor, did not affect the level of in vitro sulfoxide production (El-Alfy and Schlenk, 1998Go). Aldicarb has also been observed in estuaries of the Gulf of Mexico demonstrating exposure in organisms that simultaneously undergo tidal and salinity fluctuations (Colbert et al., 1999Go).

Because of their role in aldicarb biotransformation, the expression of FMOs were examined in both gill and hepatic microsomes of medaka, using two isoform specific FMO antibodies (antihuman FMO1 and FMO3 antibodies). Each of the antibodies recognized a single band in Japanese medaka in both liver and gill tissues. The FMO1 antibody recognized a 57 kDa protein while the FMO3 antibody bound a protein of slightly higher molecular weight (59 kDa). These molecular weight (MW) values are consistent with measurements previously observed in trout and mammals (Schlenk and Buhler, 1993Go). Mammalian FMO1 antibodies have been widely used for the detection of FMO-like proteins in numerous fish species including sharks (Squalus acanthius and Carcharhinus falciformis), rainbow trout (Oncorhynchus mykiss), Atlantic flounder (Platichthys flesus), and turbot (Scophthalmus maximus; Peters et al., 1995Go; Schlenk and Buhler, 1993Go; Schlenk and Li-Schlenk, 1994Go; Schlenk et al., 1995Go). This is the first report of a positive response to anti-FMO3 in fish (Schlenk, 1998Go). In humans, FMO3 is the predominant hepatic FMO isoform, but FMO1 expression is more pronounced in rat and pig liver as well as human kidney (Hines et al., 1994Go). The relevance and regulation of species and tissue-specific expression of FMOs is unclear and deserves further study.

We have previously shown that salinity upregulates FMOs expression and activity in Japanse medaka (El-Alfy and Schlenk, 1998Go). Similar findings have been reported in rainbow trout (Larsen and Schlenk, 2001Go). In each of these species, higher levels of FMO-catalyzed aldicarb sulfoxide were formed at higher salinities (El-Alfy and Schlenk, 1998Go; Wang et al., 2001Go). Induced by salinity in these species, FMOs appear to play an important physiological role in osmoregulation and protein stabilization in marine elasmobranchs and teleosts through the N-oxidation of trimethylamine (TMA) to the trimethylamine oxide (TMAO). TMAO acts as an organic osmolyte and protects proteins against the protein denaturing effects of urea, which is elevated in response to salinity (Larsen and Schlenk, in pressGo) and in high concentrations in marine elasmobranches (Pang et al., 1977Go; Van Warade, 1988Go). In mammals, FMO3 is the primary isoform that catalyzes the oxidation of TMA to TMAO (Lang et al., 1998Go) although FMO1 may also catalyze the reaction but with lower affinity for TMA. It is still unclear which FMO isoform is responsible for this activity in fish, and how salinity alters expression of FMOs.

One group of modulating compounds that have been shown to not only affect FMO expression, but also alter osmoregulation in fishes are sex steroids (McCormick, 1995Go). Since previous studies indicated gender-related differences in salinity-enhanced toxicity of aldicarb in Japanese medaka (El-Alfy et al., 2001Go), one of the objectives of this study was to examine whether FMOs in Japanese medaka were affected by exposure to sex steroids. Treatment with T significantly downregulated the expression of FMO1-like protein in male livers and gills but had no effect in females. However, FMO activity was significantly reduced by T treatment in female gill tissue indicating the possibility of an additional isoform that has not yet been identified. The effect of the female sex hormone, E2, seems to be gender as well as isoform specific. While E2 downregulated hepatic FMO3-like protein expression and gill FMO1-like protein expression in male medaka, it significantly lowered FMO1-like protein expression in female livers but significantly upregulated gill FMO1-like protein (see Fig. 6Go). The effects of both hormones on FMO activity parallel the total effect on FMO expression. With the exception of E2 upregulation of female gill FMO3-like protein, both sex steroids negatively regulate FMO expression and activity in medaka. Previous studies in trout and mice have shown that T negatively regulates FMO1 and FMO3 expression and is responsible for the sex differences in FMO activity in mice (Duffel et al., 1980Go; Falls et al., 1995Go; Schlenk et al., 1997Go; Wirth and Thorgeirsson, 1978Go). In rats, pretreatment with E2 in ovarectomized animals led to significant reduction in FMO S-oxygenase activity in liver and kidney but caused large increases in the activity in lung microsomes (Cashman et al., 1989Go). Similarly, rabbit lung FMO2 activity has been reported to be upregulated by E2 during pregnancy (Williams et al., 1985Go). These results suggest that E2 regulation of FMO in female medaka gill mimics that of FMO2 regulation in mammals.



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FIG. 6. Represenative Western blot of female medaka microsomes from gill analyzed using antihuman FMO1 (A) and liver microsomes from female analyzed using antihuman FMO3 (B). Each lane was loaded with 0.02 mg of microsomal protein, except positive controls and molecular weight standards. For Blot A: Lane 1 is Molecular Weight Markers; Lane 2 is positivie control (+) 5 pmol FMO1; Lanes 3–5 microsomes of untreated (control) animals; Lanes 5–7 are microsomes from E2-treated animals (note doublet in last lane). For blot B: Lane 1 is Molecular Weight Markers; Lanes 2–4 are microsomes from untreated female animals; Lanes 5–7 are microsomes from E2-treated animals; Lane 8 is 2 pmol of purified FMO3.

 
The peculiar tissue and sex dependent regulation of FMOs by E2 is intriguing. It appears that one of the gill FMOs in female medaka is specifically affected by the female sex hormone. Female medaka demand high amounts of calcium because they produce eggs daily. E2 is known to control calcium influx through the gills in salmonid fish (Bjornsson et al., 1989Go). A previous study has shown that rabbit FMO2 forms a complex with calreticulin, a calcium binding protein (Guan et al., 1991Go). The role of the E2 upregulation of this gill FMO in medaka and its relationship with calcium metabolism is certainly worth further investigation.

In Japanese medaka, females had significantly higher expression of both isoforms compared to males (Fig. 3Go). However, FMO activity, as measured by thiourea oxidase, does not exhibit such gender differences. Discrepancies between expression and activity might indicate the involvement of other FMO isoforms, which are not recognized by the antihuman FMO antibodies, but catalyze thiourea oxidase activity. Similar sexual differences in the expression of FMOs have been previously reported in rainbow trout (Schlenk and Buhler, 1993Go). Gender differences in hepatic FMO expression and activity have been shown to exist in other species, particularly mice and dogs (Ripp et al., 1999Go). Female mice show significantly higher dimethylaniline N-oxide formation compared to males (Falls et al., 1995Go). Based on protein and mRNA levels, FMO forms responsible for the gender difference in activity in mice were FMO1 and FMO3 (Falls et al., 1995Go).

Aldicarb toxicity in medaka treated with E2 and T paralleled FMO expression in each gender. E2 and T significantly reduced aldicarb toxicity in male Japanese medaka. In females, while T reduced aldicarb toxicity, E2 significantly enhanced its toxicity. These results were similar to earlier studies in medaka that demonstrated a direct relationship between FMO expression, aldicarb sulfoxide formation, ChE inhibition, and acute toxicity (El-Alfy and Schlenk, 1998Go; El-Alfy et al., 2001Go). Similar results were also observed in Sarotherodon mossambicus in which T reduced the toxicity of the carbamate thiobencarb (Babu et al., 1989Go). With the recent identification of E2 and T and their mimics in surface waters, the toxicity of compounds activated by FMO may be altered providing either protection (males) or enhancing susceptibility (females). More studies are necessary to explore the effects of these mixtures in aquatic organisms.

ChE is thought to be the primary target and an indicator of acute aldicarb toxicity (Baron, 1994Go; Risher et al., 1987Go; WHO, 1991Go). Thus, it was important to determine whether E2 or T altered activities of ChEs without aldicarb treatment. Muscular ChE of Japanese medaka (not treated with aldicarb) was unaffected by E2 or T. However, both hormones, with the exception of E2-treated females, diminished the inhibitory action of aldicarb on muscle ChE in medaka. E2 caused an increase in blood plasma AChE activity during estrus in dairy cattle (Ramachandra et al., 1992Go). Grisaru et al.(1999) have shown that human AChE mRNA is differentially upregulated during osteogenesis. Similarly, E2 significantly increased AChE activity in several areas of female rat brain (Iramain et al., 1980Go). Similar studies have shown that T stimulated AChE synthesis at the endplate region (Godinho et al., 1994Go). Treatment of the female songbird, Syrinx, with T increased total acetylcholine receptor number 2.1-fold and AChE activity 5.0-fold (Bleisch et al., 1984Go). Thus, it may be possible that E2 or T may augment other ChEs besides that of muscle ChE and provide protection from aldicarb toxicity.

In conclusion, this study has shown that sex hormones, which play a role in osmoregulation in euryhaline fish, regulate the expression and activity of FMOs in both male and female Japanese medaka. Such regulation is both tissue and gender dependent. This study also shows that modulation of FMOs can have a significant impact on the response of aquatic organisms to xenobiotic exposure. Despite increasing evidence implicating hormones as major factors in regulating FMO-mediated metabolism, the precise mechanisms remain unknown.


    ACKNOWLEDGMENTS
 
This study was supported by the U.S. Environmental Protection Agency Exploratory Grants Program (grant number R826109-01-0). The authors acknowledge the Environmental Toxicology Research Program at the University of Mississippi for partial support of A.E.-A. Special thanks are also extended to Erica Marsh for her technical help in maintaining fish cultures.


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


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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