Environ. Sci. Technol. 2003, 37, 1137-1141
Aquatic Hypoxia Is an Endocrine Disruptor and Impairs Fish Reproduction R U D O L F S . S . W U , * ,† BING SHENG ZHOU,† DAVID J. RANDALL,† NORMAN Y. S. WOO,‡ AND PAUL K. S. LAM† Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, and Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong
There is increasing concern that certain chemicals in the aquatic environment can disrupt endocrine systems, leading to reproductive impairment and threatening survival of wild populations of invertebrates, fish, bird, reptiles, and wildlife. For the first time, we report that hypoxia is also an endocrine disruptor and poses a significant threat to the reproduction and hence sustainability of fish populations. Serum levels of testosterone, estradiol, and triiodothyronine significantly decreased in carp (Cyprinus carpio) upon chronic exposure to hypoxia. These hormonal changes were associated with retarded gonadal development in both male and female carp, reduced spawning success, sperm motility, fertilization success, hatching rate, and larval survival, indicating that adverse effects of hypoxia on reproductive performance resulted from endocrine disruption. Since aquatic hypoxia commonly occurs over thousands of square kilometers in aquatic systems worldwide, our results imply that endocrine disruption and reproductive impairment in fish may be a widespread environmental problem.
Introduction Anthropogenic chemicals, which can disrupt the endocrine systems of animals (e.g., tributyl tin, nonylphenol, oestradiol), are currently a major environmental concern. These chemicals may affect reproductive hormone systems and hence reproductive success of wild populations. For example, endocrine disruption has caused changes of sex in fish and snails, caused reproductive failure in birds, and impaired gonadal development in alligators and polar bears, which eventually cascade into population decline or species extinction (1-5, 13-15). Eutrophication and organic pollution have caused largescale hypoxia (thousands of km2) in aquatic systems all over the world, and this in turn has led to changes in fish species composition, population decline, and decrease in fish biomass (6-10). The observed decline in fish populations and elimination of sensitive fish species may be caused by decreases in reproductive success in response to hypoxia. Reproductive success in fish depends on a number of inter* Corresponding author phone: +852-2788-7401; fax: +852-27887406; e-mail:
[email protected]. † City University of Hong Kong. ‡ The Chinese University of Hong Kong. 10.1021/es0258327 CCC: $25.00 Published on Web 02/11/2003
2003 American Chemical Society
related factors, including gonad development, reproductive behavior, fecundity, gamete quality, and fertilization success as well as larval hatchability and survival. In fish, these important processes are regulated by sex steroid hormones (11-13). The effects of hypoxia on these hormonal mechanisms and on the reproductive process as a whole are completely unknown. For the first time, we report that longterm exposure to hypoxia can impair fish reproduction through changes in endocrine function, egg quality, sperm motility, fertilization, and larval survival in the common carp (Cyprinus carpio).
Materials and Methods Hypoxic Exposure. Immature adult carps (C. carpio, 35.7 ( 5.8 g) were obtained from a single batch of culture from a commercial hatchery and acclimated in fully aerated water in the laboratory for 2 weeks (22.5 ( 0.5 °C; 12-h light and 12-h dark cycle) prior to experimentation. Fish were then divided into two groups. One group was reared in a hypoxic system (1.0 ( 0.2 mg of O2 L-1) and the other in a normoxic system (7.0 ( 0.2 mg of O2 L-1) for 12 weeks (details of the systems were described in ref 14). Fish were daily fed with 2% body wt of a commercial feed (J W Vitra, 35% protein), and 50% of water in the systems was renewed every 2 days. Fish were sampled from the normoxic and hypoxic groups at intervals of 4, 8, and 12 weeks. Levels of reproductive hormones, gonadosomatic index, gonadal histology, spawning success, sperm motility, fertilization success, and larval hatchability of the normoxic and hypoxic groups were compared. To ensure that all experimental fish were at the same maturation stages before the experiment, 13 fish were randomly sampled from both control and hypoxic groups, and developmental stage of gonads were examined by eye, followed by histological examination under a microscope. At the beginning of experiment, all female fish examined contained only oogonia and oocytes, while spermatogonia (SPG) predominated in lobules of all male fish, indicating that female and male fish were in a similar, immature stage prior to experiment. Hormone Assays. Fish were randomly sampled from the hypoxic and normoxic systems after 4 and 8 weeks and immediately anaesthetized with 0.01% neutralized MS222 (pH 7.4) (Sigma, St. Louis, MO). Each fish was weighed, and blood was collected from the caudal vein with a heparinised syringe (10 mg mL-1). Blood samples were stored on ice and then centrifuged for 10 min at 3000g at 4 °C. Serum was collected and stored at -80 °C for hormone assay. To avoid possible diurnal changes in steroid levels, all blood samples were collected at the same time, and sampling was completed within 1 h. Serum levels of testosterone (T), 17β-estradiol (E2), and triiodothyronine (T3) were assayed using enzyme-linked immunoassays (ELISA), which have been validated for use with fish sera. The testosterone (RE52151) and estradiol (RE52041) ELISA kits were obtained from Immuno-Biological Laboratories GmbH (Hamburg, Germany), and the triiodothyronine ELISA kit (7013) was from BioMerica Inc. (Newport Beach, CA). The detection limits of T, E2, and T3 were 0.1, 0.015, and 0.2 ng/mL, respectively. Gonadosomatic Index (GSI) and Gonadal Histology. The gonad of each individual was dissected and weighed, and the gonadosomatic index (GSI) was calculated by expressing the gonad weight as a percentage of total body weight. A piece of the middle portion of gonad was first fixed in precooled 2.5% glutaraldehyde (0.1 M sodium phosphate buffer, pH 7.2) followed by 1% osmium tetroxide. Tissue was VOL. 37, NO. 6, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. GSI and Serum Hormone Levels (ng mL-1) of Male Carp after Exposure to Normoxia (7.0 mg of O2 L-1) and Hypoxia (1.0 mg of O2 L-1) for 4 Weeksa GSI testosterone estradiol triiodothyronine
7.0 mg of O2 L-1
1.0 mg of O2 L-1
6.04 ( 1.25 4.68 ( 2.88 0.04 ( 0.01 3.62 ( 0.75
5.49 ( 1.55 13.46 ( 5.56* 0.24 ( 0.07** 2.51 ( 0.54*
a Mean ( SD; n ) 4. Asterisk(s) indicate values significantly different from the normoxic control (t-test: *, P < 0.05; **, P < 0.01).
then dehydrated in graded acetone and embedded in Spurr’s resin. Sections (0.5 µm thick) were stained with 0.5% toluidine blue and examined under a light microscope (200×). For the male, cell types in the sections were examined. In each section, 30 testicular lobules were selected at random, and the number of spermatogonia (SPG), cysts containing spermatocytes (SPC), and spermatid (SPD) cells were counted. The diameter of lobules of the testes was measured using the semiquantitative method developed by Jobling et al. (15). Thirty round to semi-round lobules were randomly selected from three different areas for measurement. For the females, different developmental stages were classified by their yolk vacuoles. Oocytes with yolk indicated that development was advanced while previtellogenic oocytes indicated an earlier state of development. Spawning Success and Sperm Motility. Fish were sampled from both the normoxic and hypoxic groups after 12 weeks and injected intraperitoneally with 2 mg kg-1 carp pituitary extract. Eighteen hours after injection, the abdomen of fish was gently pressed to extrude eggs and semen. Spawning was considered successful if semen or free-flowing eggs could be obtained from the fish. Motility of sperm in semen was analyzed under normoxic conditions immediately after collection using a CRISMAS image analysis system (Image House, Copenhagen). The swimming pattern of sperm was recorded for 30 s using a CCD color camera (JVC, Japan) mounted on a microscope immediately after the 20-fold dilution of the semen. Mean curvilinear velocity (VCL), mean straight-line velocity (VSL), and angular path velocity (VAP) of fish sperm from the normoxic and hypoxic groups were analyzed and compared using a t-test (P ) 0.05). Fertilization Success, Hatching Success, and Larval Survivorship. Eggs from individual females exposed to normoxia and hypoxia for 12 weeks were collected and fertilized immediately. A total of 0.4 mL of semen was added into a beaker containing about 500 eggs from each single female and stirred for 5 min. Four replicates were set up for each individual, and each replicate contained about 60 eggs. The eggs were then pipetted into a plastic Petri dish containing normoxic UV-sterilized water. Fertilized eggs were identified by the appearance of an animal pole 2 h postfertilization. The number of fertilized/unfertilized eggs in each sample was counted, and fertilized eggs were isolated and incubated at 27 °C for 72 h (12 h light/12 h dark). The
FIGURE 1. Relationship between GSI and testosterone of male carp after 8 weeks of normoxic and hypoxic exposure. water in the Petri dishes was changed daily with fresh UVsterilized water. The number of successfully hatched eggs was recorded after 72 h, and the number of surviving hatched carp was recorded after another 24 h. Statistical Analysis. Tests for data homogeneity were conducted using Bartlett’s test. Data were arc sin square root transformed if they failed to meet the assumption of normality. A t-test was used to test the null hypothesis that there is no significant difference between the mean of each parameters measured in the hypoxic group and the normoxic group.
Results Hormonal Changes. A significant increase in serum T and E2 and a significant decrease in T3 were clearly evident in males exposed to hypoxia for 4 weeks (Table 1). After 8 weeks of exposure to hypoxia, T and T3 levels were significantly reduced, but E2 levels increased significantly in male carp (Table 2). Serum testosterone concentrations showed a significant correlation with the maturational state (GSI) of the testis (Figure 1). Female carp exposed to hypoxia for 8 weeks showed a significant reduction in serum testosterone, E2, and T3 levels (Table 2). GSI and Gonadal Histology. Gonadosomatic Index (GSI) remained unchanged after exposure to hypoxia for 4 weeks (Table 1), but a significant decrease in GSI was found in both male and female carp after hypoxic exposure for 8 weeks (Table 2). Despite the fact that all stages of spermatogenesis can be observed in the testes of both normoxic and hypoxic fish at week 8, the number of SPC and SPD in the testicular lobules of hypoxic fish was significantly reduced with a significantly higher number of SPG being found in hypoxic males (P < 0.01, Figure 2A), indicating that testicular growth and sperm production was inhibited by hypoxia. Likewise, a significant reduction in the diameter of testes lobules was also observed in the hypoxic males after 8 weeks (Figure 2B).
TABLE 2. GSI and Serum Hormone Levels (ng mL-1) of Carp after Exposure to Normoxia (7.0 mg of O2 L-1) and Hypoxia (1.0 mg of O2 L-1) for 8 Weeksa male 7.0 mg of O2 GSI testosterone estradiol triiodothyronine
L-1
10.23 ( 2.17 9.83 ( 2.32 0.033 ( 0.01 3.37 ( 0.35
female 1.0 mg of O2
L-1
4.26 ( 2.02*** 2.22 ( 2.39*** 0.063 ( 0.019*** 1.60 ( 0.53*
7.0 mg of O2
L-1
18.51 ( 5.77 8.23 ( 3.04 0.99 ( 0.92 3.59 ( 0.39
1.0 mg of O2 L-1 6.09 ( 4.69** 0.73 ( 0.69*** 0.19 ( 0.31** 3.08 ( 0.31*
a Mean ( SD; n ) 7-11. Asterisk(s) indicate values significantly different from the normoxic control (t-test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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FIGURE 2. (A) Number of spermatogonia (SPG), spermatocytes (SPC), and spermatid (SPD) in the testes. (B) Lobule diameter of testes of C. carpio upon exposure to 7.0 and 1.0 mg of O2 L-1 for 8 weeks. Values significantly different from the control are indicated by asterisks (n ) 7-11, mean ( SD) (**, P < 0.01; ***, P < 0.001). Similar retardation of gonad development was also found in female carp, with smaller gonads and less yolk in each egg. Stage III oocytes were found in 83.3% of all hypoxic females, but only 16.7% hypoxic females carried stage IV oocytes. None of the hypoxic females produced stage V oocytes. In contrast, eggs were visually observed in all normoxic females, and histological examinations further revealed that oocytes in 57.1% of normoxic females reached stage V. Spawning Success and Sperm Motility. All the normoxic and hypoxic male carp could be induced to spawn using carp pituitary extract, which was consistent with our histological observations that mature spermatozoa were present in all fish. A Z-test showed a significantly higher percentage of spawning success in the normoxic group (71.4%) as compared with the hypoxic group (8.3%) (P < 0.01). All sperm motility parameters (i.e., VCL, VSL, and VAP) significantly decreased after exposure to hypoxia for 12 weeks (Table 3), indicating that sperm quality was impaired. Fertilization Success, Hatching Success, and Larval Survival. Percentage fertilization success was 99.4% and 55.5% in the normoxic and hypoxic groups respectively (P < 0.001; Figure 3). Fish with a higher GSI produced larger size larvae with higher rate of survival (16). A total of 98.8% of the fertilized eggs produced by the normoxic group hatched to larvae, while only 17.2% of fertilized eggs produced by the hypoxic group hatched to larvae (P < 0.01; Figure 3). A total
TABLE 3. Sperm Motility of Carp after Exposure to Normoxia (7.0 mg of O2 L-1) and Hypoxia (1.0 mg of O2 L-1) for 12 Weeksa VCL VSL VAP
7.0 mg of O2 L-1
1.0 mg of O2 L-1
77.42 ( 29.13 38.83 ( 21.01 47.69 ( 5.38
46.25 ( 10.83* 10.65 ( 3.89* 21.12 ( 11.41*
a Mean ( SD; n ) 6. The velocity is expressed as µm S-1. Asterisk indicates values significantly different from the control (t-test: *, P < 0.05). VCL, mean curvilinear velocity; VSL, mean straight-line velocity; VAP, angular path velocity.
of 93.7% of hatched larvae survived in the normoxic group, while larval survival decreased to 46.4% in the hypoxic group 24 h post-hatching (P < 0.001; Figure 3). Overall, the survival of fertilized eggs through 24-hour-old larvae was 92.3% in the normoxic group but only 4.4% in the hypoxic group (P < 0.001; Figure 3).
Discussion Endocrine disruption by chemical pollutants has been related to the decline in many fish, reptile, bird, and wildlife populations (20-23) and therefore has rapidly become a major concern in the past decade. The present study documents for the first time that hypoxia can also cause endocrine disruption in fish. Our results provide clear VOL. 37, NO. 6, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Percentage of fertilization, hatching rate, larval survivorship, and overall survivorship (fertilized egg to 24 h post-hatching) after 12 weeks of normoxic and hypoxic exposure of the parent adult carp (mean ( SD, n ) 6) (t-test: **, P < 0.01; ***, P < 0.001). evidence that hypoxia not only disrupts levels of T, E2, and T3 but also affects key reproductive processes such as gametogenesis and gonad maturation and reduces GSI and gonad size, quality of gametes, fertilization and hatching successes, and larval survival. The endocrine disruption and reproductive impairments observed implicate that hypoxia poses a significant threat to the survival and sustainability of natural fish population. Importantly, hypoxia affects very large areas in aquatic systems worldwide (6-9), which implies that endocrine disruption in fish may be a wide spread phenomenon, and the problem of endocrine disruption caused by hypoxia is potentially much more serious than that caused by known anthropogenic chemicals. It is worth noting that only adult (parental) fish were exposed to hypoxia in this study, and sperm motility and larval survival were determined from sperm and larvae produced by hypoxic fish under normoxic conditions. In the natural environment, it is likely that the sperm, eggs, and larvae would be exposed to the hypoxic insult as well, which may cause a further reduction in survival below the already low 4.4% observed in this study. Changes of sex steroids in response to stress may vary according to species and type of stress. For example, handling and sampling stress resulted in depressed serum androgen levels in male trout, but estradiol levels were not affected (24). In contrast, sea breams infected by Vibrio show marked elevations in T but lowered E2 levels in serum (25). The observed differential changes in serum sex steroid in hypoxic carp (i.e., elevated E2 but lowered T) appears to be a unique response to hypoxic stress. T acts as a precursor for E2 (3). The reduction in T associated with hypoxia could be due, at least in part, to the increased serum levels of E2 observed in male fish. In male fish, T plays an important role in stimulating and maintaining spermatogenesis (3, 11). E2 normally occurs only in trace amounts in male fish, levels of which are ineffective in regulating spermatogenesis (3), and higher levels of estradiol can inhibit spermatogenesis (17). Thus, both low levels of T and high levels of E2 in male fish may have contributed to the reduction in spermatogenesis following hypoxic exposure. In female fish, previous studies have found a positive correlation between increased plasma level of E2 and the growth of vitellogenic oocytes in many species (3, 11, 12). Although serum E2 levels were not significantly correlated with GSI in either hypoxic or normoxic female carp (r ) 0.36, P ) 0.59; r ) 0.69, P ) 0.09), the decrease in E2 could have impaired vitellogenesis and reduced yolk deposition and hence lower the quality of eggs in hypoxic fish. T3 may be involved in oocyte growth and maturation in female fish, and inhibition of T3 retards testicular growth in developing male fish (18). Sperm motility is a major factor 1140
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contributing to successful fertilization and production of fish larvae (3, 19); higher sperm motility tended to produce larger larvae at hatching (16). The lower motility in sperm from hypoxic fish is clearly indicative of poor sperm quality, which may be due to poor development of gonads and changes in the levels of steroid hormones. Our earlier studies showed that hypoxia significantly reduces food intake as well as energy allocated to somatic growth and reproduction in C. carpio (14). In this study, a significant reduction in gonad size (GSI) and a slower gonad development were found in hypoxic carp. These reproductive impairments could provide an explanation for the changes in serum T and E2 and may also account for the observed poor fertility and hatching success in the hypoxic group since the quality of sperm and eggs as well as offspring viability would depend on GSI and the level of reproductive hormones produced by the gonads during gametogenesis (3, 11, 12). Recently, it has been shown that nonylphenol (a well-known chemical endocrine disruptor) can affect gonadotropin levels in the pituitary gland and plasma of female rainbow trout (26). It would be instructive to further investigate if hypoxia could similarly affect gonadotropins (e.g., FSH and LH) in fish. Time to spawning, egg size, egg viability, fertilization success, time to hatching, and larval survivorship have all been regarded as important attributes leading to reproductive success (27, 28). Fertilization success and the proportion of normally developing embryos and larvae depend on the quality of the eggs and sperm (29). Maternal levels of steroid and thyroid hormones within the eggs, or their metabolism by the embryo, are important in determining hatching success and normal larval development (3, 19). Likewise, factors influencing the maternal levels of T can have a major impact on larval survivorship (29). It is clear from our results that fish exposed to hypoxia produce inferior quality gametes, which in turn probably accounted for the observed low hatching success and high larval mortality. In mammals, hypoxia results in the stabilization of hypoxia inducing factor 1R (HIF-1R), which combines with HIF-1β to form HIF-1, a transcription factor that modulates the expression of a variety of genes (e.g., erythropoietin gene) (30). HIF-1β is also the aryl hydrocarbon receptor nuclear translocator (ARNT). The aryl hydrocarbon receptor (AhR) can be ligand activated to heterodimerize with ARNT, which in turn increases expression of cytochrome P450. Thus both HIF-1R and AhR compete for ARNT, and as a result, hypoxia has been shown to decrease the expression of cytochrome P450 (31, 32). The cytochrome P450 system is involved in steroidogenesis (33), and hypoxia may impair fish reproduction through this pathway. This is, however, only one of several possibilities. In addition, because of the competition between HIF-1R and AhR for ARNT, hypoxia may also enhance the susceptibility of animals to a variety of toxic substances. We conclude that hypoxia reduces overall reproductive success by disturbing endocrine functions, which in turn affect gametogenesis, sexual maturity, gamete quality, fecundity, fertilization success, hatching, and viability of larvae. The present study represents the first report on impairment of fish reproduction by hypoxic stress, which may be an important mechanism accounting for the population decline and changes of fish species composition observed in hypoxic environments.
Acknowledgments We thank the Agriculture, Fisheries and Conservation Department, Hong Kong, for providing the common carp. This research was supported by a Competitive Earmark Research Grant from the Research Grants Council of Hong Kong awarded to R.S.S.W. and D.J.R. (9040387).
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Received for review May 29, 2002. Revised manuscript received December 23, 2002. Accepted January 10, 2003. ES0258327
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