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Changes in the timing of reproduction following chronic exposure to ibuprofen in Japanese medaka, Oryzias latipes
Aquatic Toxicology 81 (2007) 73–78
Changes in the timing of reproduction following chronic exposure to ibuprofen in Japanese medaka, Oryzias latipes Jennifer L. Flippin a,1 , Duane Huggett b , Christy M. Foran a,∗ a b
Department of Biology, West Virginia University, Morgantown, WV 26506-6057, United States Department of Biological Sciences, University of North Texas, Denton, TX 76203, United States
Received 6 September 2006; received in revised form 3 November 2006; accepted 4 November 2006
Abstract Effluents from wastewater treatment plants and untreated sewage constitute a low concentration but continuous source of pharmaceutical products to the aquatic environment. One such drug, ibuprofen, is a non-steroidal anti-inflammatory agent that primarily acts through inhibition of cyclooxygenase (COX) activity. Oryzias latipes (Japanese medaka) were exposed for 6 weeks via water to three concentrations of ibuprofen (1–100 g/L nominal concentrations) and a water control. Reproductive parameters, including frequency of spawning, fecundity, egg size, and rate of fertilization, were measured for each pair of adult medaka following 6 weeks of exposure. Livers homogenates from exposed individuals were assayed for COX activity and whole individuals were histologically examined for tissue damage. Increasing exposure to ibuprofen significantly increased the number of eggs per reproductive event, but decreased the number of spawning events per week. Liver tissue collected from females had less variability in COX activity with increasing concentration of ibuprofen exposure, and tended to have elevated hepatosomatic indices. No pathological damage was evident the in the gills, livers and head kidneys of animals from the highest exposure group. The results of this experiment begin to show that exposure to chronic low levels of ibuprofen alter the pattern of reproduction and may produce sex-specific responses in teleosts. © 2006 Elsevier B.V. All rights reserved. Keywords: Reproduction; Cyclooxygenase; Personal care products; NSAID
1. Introduction Effluents from wastewater treatment plants and untreated sewage may constitute a low but continuous source of pharmaceutical products to the aquatic environment. The concentration of these compounds is generally detectable in lake and river waters in North America and Europe. A pharmaceutical product is designed to alter a specific biologic process, so exposure to a single drug, its metabolites, or a mixture of various environmental contaminants may be sufficient to cause system disruption in an exposed organism (Daughton and Ternes, 1999). Ibuprofen is an over the counter analgesic and antiinflammatory product classified as a non-steroidal anti∗ Corresponding author at: Department of Biology, 53 Campus Drive, Suite 3139, P.O. Box 6057, West Virginia University, Morgantown, WV 26506-6057, United States. Tel.: +1 304 293 5201x31537; fax: +1 304 293 6363. E-mail address: [email protected] (C.M. Foran). 1 Current address: Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633, United States.
0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.11.002
inflammatory drug (NSAID). The compound is used for the treatment of headaches and general pain (Sycha et al., 2003). NSAIDs partially hinder the inflammatory response by inhibiting one or two isoforms of the cyclooxygenase (COX) enzyme. Ibuprofen is a nonspecific COX inhibitor and is shown to inhibit both COX-1 and COX-2 (Van Hecken et al., 2000). The inhibition of COX serves as the rate limiting step in the formation of prostoglandins which are found at the site of inflammation. The COX-1 enzyme is expressed in many tissue types including those in the gastrointestinal tract, and it is responsible for producing prosanoids that function to form cytoprotection in the gastric system. Inhibition of the COX-1 enzyme may lead to the formation of ulcers in the gastrointestinal tract. Meanwhile, COX-2 is present in fewer cell types but is a key mediator in the inflammation cascade and the pain and fever response (Cryer and Kimmey, 1998; Simmons et al., 2004). COX-1 activity is known to play an important role in development in zebrafish (Cha et al., 2005) and some mammals (Cappon et al., 2003a,b). As well, COX mediated production of prostaglandins is important for ovulation in mammals (Gaytan et al., 2006)
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and fishes (Mercure and Van Der Kraak, 1996; Sorbera et al., 2001). Wastewater processing studies in Europe show that between 20 and 90% of various drugs are removed from wastewater during treatment, but the percentages for individual drugs dramatically decrease if disturbances take place during the sludge activation process (Fent et al., 2006). Ibuprofen is biodegraded to carboxylated and hydroxylated metabolites, and between 52 and 90% of ibuprofen is removed depending on treatment conditions (Paxeus, 2004). Studies have identified environmental ibuprofen, along with other NSAIDS, diclofenac, acetylsalicylic acid, and ketoprofen in quantities exceeding l g/L (Fent et al., 2006). Untreated sewage was shown to contain up to75 g/L of ibuprofen; a mean of 4 g/L and maximum of 24.6 g/L was detected in treated effluents (Metcalfe et al., 2003). Treated and untreated wastewater both enter aquatic ecosystems and carry these pharmaceutical products into the environment (Fent et al., 2006). Ibuprofen literature indicates that changes in function of several physiological systems are possible with long-term exposure. Exposure has resulted in histopathological changes in the gills and kidney of fish (Schwaiger et al., 2004; Triebskorn et al., 2004). In mammals, NSAIDS alter ovulation and development (Cappon et al., 2003a,b; Dawood, 1993). Furthermore, longterm treatment in humans frequently produces inflammatory lesions in the gastrointestinal tract (Bjorkman, 1998), and have been associated with in increased risk of heart disease in women (Egan et al., 2004; Grosser et al., 2006). The purpose of this study is to begin to determine the reproductive and physiological effects of chronic, low-level ibuprofen exposure in Oryzias latipes, Japanese medaka. Years of characterization and development of biomarkers make medaka an ideal model species of the assessment of the reproductive and developmental consequences of exposure (Metcalfe et al., 1999). 2. Materials and methods 2.1. Medaka culture Juvenile medaka aged approximately 2 months post-hatch were obtained from laboratory culture, originally stocked with eggs from the University of Mississippi Environmental Toxicology Research Program. Culture and test animals were maintained in balanced salt solution (BSS; Yamamoto, 1975) made with Nanopure® filtered water. Water quality parameters for the culture average pH 6.5, 22 ◦ C, >5.0 mg/L dissolved oxygen, 6.0 mg/L general hardness, and 1.5 g/L salinity. Ammonia and nitrite were generally undetectable. Culture and experimental animals were maintained on a 16:8 light:dark cycle. All fish were fed twice daily with freshly hatched brine shrimp and once daily with commercial flake food (TetraMin© ).
control “stock” was 1 L water. Other ibuprofen stock solutions were made by adding 80, 800, or 8000 g of the drug to water. Stocks were stored in the dark at 4 ◦ C. New stocks were made every 2 weeks. Thirty-two quart jars were filled with 790 mL of BSS and randomly placed in a room temperature water bath maintained at 26 ± 1 ◦ C; five fish were distributed to each container. Each water bath was lit with overhead fluorescent lights and a single 18 in. fluorescent fixture (510 lumens) mounted on the wall approximately 60 cm above the jars. Each jar was then dosed with 10 mL of ibuprofen solutions (at 4 ◦ C) to obtain final water concentrations of 0, 1, 10 and 100 g/L. These concentrations were chosen to overlap mean and maximal reported ibuprofen concentrations in sewage and effluent (Metcalfe et al., 2003). Each day, 100% of water was changed and a fresh dose of ibuprofen solution was administered to each jar. Nominal concentrations were not confirmed analytically. However, current Material Safety Data Sheets indicate that ibuprofen is stable and extremely soluable in water (>2 mg/mL at 25 ◦ C). Huggett et al. (2004) measured concentrations of ibuprofen from a 96 h flow through experiment at 900 g/L recovered 920 g/L. In this experiment, recoveries of fortified water samples at 700 and 900 g/L were 100%. Research has shown that photolysis of ibuprofen is undetectable in sunlight (Packer et al., 2003). We expect nominal concentrations of ibuprofen to be representative of the actual exposure. After 4 weeks, O. latipes in the same exposure group were separated into 80 jars with single sex pairs and acclimated to 28 ± 1 ◦ C. We have observed that housing medaka in same sex groups prior to reproductive assessment increases the consistency of egg production and fertilization once the animals are switched to male–female pairs. Reproductive adult pairs were switched within treatments to male–female pairs after 1 week and allowed to breed. Over the course of 7 days, eggs were collected from reproductively active pairs every 24 h during water renewal and ibuprofen exposure. The frequency of reproduction, total clutch size, number of fertilized eggs, and average egg size were recorded. Animals were sacrificed following 1 week of reproductive monitoring. All animals were anesthetized with MS-222 (Sigma, 3-aminobenzoic acid ethyl ester, methanesulfonate salt), weighed and measured. Livers were removed from 20 males and 20 females in each treatment for mass determination, hepatosomatic index (HSI) calculation, and later used in a cyclooxygenase activity assay. Animals were decapitated and livers removed and weighed. Livers were placed in a microcentrifuge tube with 5 L of PMSF solution and stored at −80 ◦ C. The remaining animals were fixed in 10% neutral buffered formalin (VWR Scientific, West Chester, PA) for histological analysis. 2.3. COX enzyme activity
2.2. Exposure Stock solutions for the exposure were prepared by dissolving ibuprofen (Sigma Chemical, St. Louis, MO) into 1 L of Nanopure® water. One stock was made for each treatment. The
COX activity assays were performed on whole liver tissue homogenate using the Cayman Chemical (Ann Arbor, MI) COX activity assay. Livers were thawed on ice, homogenized and centrifuged at 4 ◦ C with 10,000 rpm (11,180 × g)
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for 30 min. The supernatant was removed and protein content measured using Bio-Rad protein assay protein dye and standards of bovine serum albumin (BSA; Sigma, cold alcohol precipitated, frac V; 0.0625–0.5 mg/mL). Liver supernatant was assayed in a 96-well plate with appropriate COX activity reagents. This assay measures the peroxidase activity of both COX enzymes, and the COX-1 and COX-2 specific activity was not determined. Samples were incubated with assay buffer and a colorometric substrate and the appearance of tetramethylp-phenylenediene (TMPD) was compared to an ovine COX-1 standard. Background absorbance was measured from tissue after enzyme activity was destroyed by boiling, and this background value was subtracted from each sample. The plate was allowed to develop for 15 min at room temperature and COX activity was determined with absorbance at 595 nm for each sample. The specificity of the assay was determined with the addition of inhibitors for both COX-1 and COX-2 enzymes (SC-560 and DuP-697, respectively). No COX activity was detected in these samples. Reported COX activities are normalized to the protein content (mg) in the homogenized liver supernatant. 2.4. Data analysis Data for reproduction and COX activity were analyzed by nonparametric statistical tests (Mann–Whitney U tests, Kruskal–Wallis tests, and Kendall rank correlations) unless samples met requirements for normality. Many of the endpoints exhibited unequal variance between treatments. All analyses were conducted using StatView (nonparametric tests) or SAS 9.0 (regressions) with statistical significance defined at an α-level of p ≤ 0.05.
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3. Results 3.1. Reproduction Long-term ibuprofen exposure had a concentration dependent effect on the pattern of reproduction in exposure pairs. With increasing exposure, pairs spawned less frequently during the week and produced more eggs when they did spawn. The number of days that O. latipes produced eggs during 1 week of reproduction depended on treatment concentration. The total number of eggs produced by pairs over the week of assessment did not differ with exposure (Kruskal–Wallis test, H = 1.792, p = 0.62). As ibuprofen treatment increased, the frequency of egg production decreased (Kendall rank correlation, τ = −0.138, p = 0.03; Fig. 1). Increasing ibuprofen exposure also had a significant effect on the number of eggs produced by a pair per spawning event (Kendall rank correlation, τ = 0.248, p = 0.01). The greatest number of eggs per day was produced by breeding pairs exposed to the highest concentration of ibuprofen. On days when they do reproduce, pairs exposed to 100 g/L of ibuprofen produced nearly twice as many eggs as those in the control group (Fig. 1). The rate of fertilization was generally greater than 90% for all treatment groups; no difference was observed in the rate of fertilization between treatment groups (Kruskal–Wallis test, H = 2.278, p = 0.52). Mean egg diameter was 1.25 mm, and did not differ with ibuprofen treatment (Kruskal–Wallis test, H = 1.402, p = 0.71).
2.5. Histology For histological analysis, medaka were anesthetized until opercular movement stopped and no response was observed to a pinch of the caudal peduncle. Ten control animals (5 male and 5 female) and 10 animals from the highest exposure group were analyzed. Fish were immersion fixed in 10% neutral buffered formalin for at least 1 week. Prior to slicing, each fish was decalcified in 50% Cal-Ex (Fisher Scientific, Fair Lawn, NJ) for 24 h and rinsed in phosphate buffered saline for 24 h. Each animal was placed at 4 ◦ C in 30% sucrose made in phosphate buffer and allowed to absorb the sucrose overnight or until the animal sank to the bottom of the vial. Whole medaka were embedded in Cryo-M-Bed (VWR) and frozen using dry ice. Embedded frozen tissue was mounted in a Leica cryostat and sliced into 10-m thick sagital sections which were melted onto glass slides. Sections were washed in phosphate buffer and stained in hemotoxylin and eosin. Slides were then dehydrated through a series of ethanol washes (50–100%), soaked twice in xylene, and coverslipped under Permount (VWR). Each section was viewed at 100× magnification for visual damage. Tissues (gills, liver, gonads, and gastrointestinal track) from each animal were viewed at 400×. No quantitative histological measurements were made.
Fig. 1. Changes in the pattern of reproduction for pairs of Japanese medaka exposed to ibuprofen for 6 weeks. The number of days that pairs produced eggs declined with increasing ibuprofen exposure concentrations (p = 0.03). On days that pairs reproduced, the number of eggs released (clutch size) increased with exposure concentration (p = 0.01). Bars represent mean values for the group ± standard error.
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Fig. 2. Hepatosomatic index (HSI ± standard error), the relative weight of the liver, is shown for females and males following ibuprofen exposure. Although no significant differences were detected from controls, female HSI (gray bars) tended to increase while male HSI (white bars) tended to decrease. Female HSI are significantly higher than males for each exposure concentration (all p > 0.04).
Over the week that reproduction was monitored, the average number of eggs produced was related to the weight of the female (regression ANOVA, F = 7.573, R2 = 0.18, p = 0.01). The residual difference in the relationship between size (weight) and egg production was not significantly different between treatment groups (Kruskal–Wallis test, H = 6.643, p = 0.08). Although the variability in residuals is greatest in the 10 and 100 g/L ibuprofen treatment groups. 3.2. Hepatosomatic index The relative liver weight of ibuprofen exposed animals was not different than controls for males or females (Kruskal–Wallis tests, both p > 0.48). However, the HSI of females tended to increase with exposure to ibuprofen, while male HSI seemed to decrease (Fig. 2). When compared directly, the HSI of males and females differs significantly for each exposure concentration (1 g/L: Mann–Whitney U, Z = −2.03, p = 0.04; 10 g/L: Mann–Whitney U, Z = −3.55, p > 0.01; 100 g/L: Mann–Whitney U, Z = −2.50, p = 0.01) but not controls (Mann–Whitney U, Z = −0.68, p = 0.49). The pattern of HSI changes indicates that males and females may respond differently to ibuprofen exposure. 3.3. Hepatic COX activity The COX activity in homogenized liver tissue was highly variable in control groups, and a decrease in this variability was the primary effect of ibuprofen exposure. Females in the control treatment exhibited the most variability in individual COX activity (Fig. 3). Females showed a significant decline in this variability following ibuprofen exposure at 10 and 100 g/L (F0,10 = 8.43, p0,10 = 0.02; F0,100 = 21.54, p0,100 > 0.001; F1,100 = 8.65, p1,100 = 0.02). Therefore, ibuprofen exposure changed the range of activity but did not significantly alter the mean value for COX activity. COX activity in male liver samples was the most variable in controls. However, variability in male COX activity was significantly dif-
Fig. 3. Cyclooxygenase (COX) activity normalized to the amount of protein (mg) in liver homogenates (±standard error) is shown for males and females following ibuprofen exposure. In both sexes, variability of COX activity was highest in control samples. For females, variability in enzyme activity declines following 10 and 100g/L exposure (all p > 0.02). A change in variability was detected in males between tissues from controls and those exposed to 1.0 g/L (p = 0.01).
ferent only between controls and the 1.0 g/L exposure group (F0,10 = 49.09, p0,10 = 0.01). Measurements of COX activity, along with HSI, indicate that male and female medaka respond differently to ibuprofen exposure. 3.4. Histology Serial sections from the gills, head kidneys, and livers of exposed animals resembled those of controls. Gills filaments were normal in length and distribution, without degradation or fusion. Head kidneys appeared to have normal tubule shape and numbers. Livers exhibited no evidence of hepatocyte degradation. The liver of one female exposed to 100 g/L ibuprofen contained acellular solid deposits, although deposits were not detected in any other animal. 4. Discussion Six weeks of exposure to aqueous ibuprofen produced a change in the pattern of reproduction. With increasing concentration, pairs of Japanese medaka reproduced less frequently and produced a greater number of fertilized eggs when they did spawn. NSAID-related changes in reproduction are not unprecedented in the literature. NSAIDS, specifically COX-2 inhibitors, are known to inhibit ovulation in mammals (Gaytan et al., 2006). Human reproductive studies show that ibuprofen may increase the time interval between the luteinizing hormone surge and follicular collapse (Uhler et al., 2001). In teleosts, prostaglandins have been reported to be produced in the ovaries (Goetz et al., 1989; Knight et al., 1995), and are known to play a role in oocyte maturation in sea bass (Sorbera et al., 2001). Indomethacin, which is a general inhibitor of COX-1 and -2, repressed human gonadotropin-induced maturation of harvested oocytes but not spontaneous maturation of oocytes (Sorbera et al., 2001). Therefore, limiting the induction of COX activity in the ovary may slow the rate of oocyte maturation or ovulation in medaka.
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HSI changes with exposure were divergent for males and females. Females exhibited hepatic growth relative to males with exposure. The observed sexually dimorphic response may be related to changes in reproduction. Since the liver is site of production of vitellogenin, a glycoprotein precursor to yolk (Lazier and MacKay, 1993), the size of the liver may increase with the number of eggs maturing or spawned in a single event. HSI has been shown to increase with measures of vitellogenin production (Pereira et al., 1993) and decrease with ovarian development (Yoneda et al., 2001). The liver is also the site of a major portion of metabolism and detoxification of contaminants (Treinen-Moslen, 2001). Eicosanoids can alter the production of steroid hormones and steroid metabolizing enzymes (Gravel and Vijayan, 2006; Mercure and Van Der Kraak, 1996). Exposure of rainbow trout to diclofenac caused a visual depletion of glycogen from liver hepatocytes (Triebskorn et al., 2004). Thus, growth of hepatocytes may be altered by their change in function with NSAID exposure. Hepatic COX activity was found to be less variable among males than females. Neither males nor females showed a significant inhibition of COX activity following 6 weeks of exposure. One explanation is that ibuprofen is ineffective in its inhibition of teleost COX activity, as evidenced by a study of sunfish (Cavallaro and Burnside, 1988). Another possibility is that 6 weeks of exposure allowed recovery of COX activity, either through an increase in the synthesis of COX enzymes or through increased metabolism of ibuprofen. However, we found no evidence in the literature of changing effective concentrations of ibuprofen with long-term use or exposure. Another possibility is that the natural variability of hepatic COX activity in female medaka precludes the detection of inhibition of activity; measurements of hepatic COX activity from exposed females are similar to lower values measured from female controls. In this study, we did not observe histological changes in the gill or head kidney following 6 weeks of ibuprofen exposure. One animal displayed solid, acelluar deposits in the liver. Fish exposure to environmentally relevant levels of another NSAID, diclofenac, has been shown to change organ structure (Schwaiger et al., 2004; Triebskorn et al., 2004). Studies involving rainbow trout, Oncorhynchus mykiss, found 28 days of exposure produced kidney damage, specifically damage to tubular epithelial cells (Schwaiger et al., 2004). In one study, gill damage was evident in the secondary lamellae following exposure (Schwaiger et al., 2004). Ultrastructual analysis using electron microscopy revealed further damage to hepatocytes and glycogen depletion in the liver (Triebskorn et al., 2004). These investigations determined the lowest observed effect concentration (LOEC) for “cytological alterations in the liver, kidney, and gills” to be l g/L (Triebskorn et al., 2004). By comparison, histological damage was not observed in Japanese medaka following 6 weeks of ibuprofen exposure. This disparity may be due to differences in the COX inhibition between ibuprofen and diclofenac, differences in species sensitivity, or the use of more sensitive ultrastructral comparisons by Triebskorn et al. (2004). In vitro COX potencies of diclofenac and ibuprofen differ significantly. For instance, the COX-2 IC50 for diclofenac is 0.06 M,
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while the IC50 for ibuprofen is 19 M (Blain et al., 2002). These data suggest that diclofenac at equivalent concentrations would be more portent than ibuporofen in in vivo systems. Further studies should look at the relationship between the specificity of inhibition of COX-1 and -2 and histopathological damage from environmental exposure. Disruption of COX-1 expression disrupts development in zebrafish embryos but not mammalian embryos (Cappon et al., 2003a,b; Cha et al., 2005; Grosser et al., 2002). Although the different patterns of COX expression in fish and mammals have important implications for risk assessment for fishes, we saw no evidence that exposure during sexual maturation would inhibit either reproductive output or hepatic COX activity. However, studies have reported developmental and reproductive consequences of exposure in fish (Cha et al., 2005; Sorbera et al., 2001) and mammals (Cappon et al., 2003a,b; Gaytan et al., 2006). Developmental or reproductive impairment mediated by COX inhibitors could have a significant impact on fish populations. More research is needed to completely characterize the risk of environmental NSAID exposure. 5. Conclusion Environmental pharmaceuticals are frequently detected in wastewater and surface water. These contaminants, although expected to be found in relatively low concentrations, may be biologically potent. In this study we examined the long-term reproductive and physiological consequences of ibuprofen exposure. As expected, no acute toxicity was observed. Six weeks of exposure did produce a change in the temporal pattern of spawning in pairs of Japanese medaka. With increasing concentrations, pairs were more likely to spawn less frequently and produce more eggs when they did spawn. Timing of spawning is not likely to impair population growth in a laboratory species. However, if a similar delay is possible in species with highly coordinated spawning, such as cod (Scott et al., 2006) and salmon (de Gaudemar and Beall, 1998), fertilization and offspring survival may be compromised. Females and males responded differently to long-term ibuprofen exposure. Females exhibited more variable COX activity, and that variability decreased with exposure concentration. The relative liver weight of exposed females also tended to increase with exposure, potentially related to the change in spawning and vitellogenin production. Sex differences in the response to NSAIDS have been reported in the clinical and mammalian literature (Egan et al., 2004; Grosser et al., 2006; Reese et al., 2001). However, we present the first evidence of sexspecific changes in a teleost. Although the mammalian studies indicate estrogen upregulates COX-2, the potential for steroids to regulate expression of these genes in fish is unknown. Acknowledgments The authors would like to thank E. Anderson, Dr. J.R. Ripley, and T. Steuckle for their assistance, and Dr. K. Willett for providing O. latipes fry. This research was funded in part by West Virginia University’s Eberly College of Arts and Sciences.
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Mercure, F., Van Der Kraak, G., 1996. Mechanisms of action of free arachidonic acid on ovarian steroid production in the goldfish. Gen. Comp. Endocrinol. 102, 130–140. Metcalfe, C.D., Gray, M.A., Kiparssos, Y., 1999. The Japanese medaka (Oryzias latipes): an in vivo model for assessing the impacts of aquatic contaminants on the reproductive success of fish. In: Rao, S.S. (Ed.), Impact Assessment of Hazardous Aquatic Contaminants. Lewis Publishers, Boca Raton, pp. 29–52. Metcalfe, C.D., Koenig, B.G., Bennie, D.T., Servos, M., Ternes, T.A., Hirsch, R., 2003. Occurrence of neutral and acidic drugs in the effluents of Canadian sewage treatment plants. Environ. Toxicol. Chem. 22, 2872–2880. Packer, J.L., Weaver, J.J., Latch, D.E., McNeill, K., Arnold, W.A., 2003. Photochemicals fate of pharacueticals in the environment: naproxen, diclofenac, clofibric acid, and ibuprofen. Aquat. Sci. 65, 342–351. Paxeus, N., 2004. Removal of selected non-steroidal anti-inflammatory drugs (NSAIDs), gemfibrozil, carbamazepine, beta-blockers, trimethoprim and triclosan in conventional wastewater treatment plants in five EU countries and their discharge to the aquatic environment. Water Sci. Technol. 50, 253– 260. Pereira, J.J., Mercado-Allen, R., Kuropat, C., Luedke, D., Sennefelder, G., 1993. Effect of cadium accumulation on serum vitellogenin levels and hepatosomatic and gonadosomatic indices of winter flounder (Pleuronectes americanus). Arch. Contam. Toxicol. 24, 427–431. Reese, J., Zhao, X., Ma, W.G., Brown, N., Maziasz, T.J., Dey, S.K., 2001. Comparitive ananlysis of pharmacologic and/or genetic disruption of cyclooxygenases-1 and cyclooxygenase-2 function in female reproduction in mice. Endocrinology 142, 198–206. Schwaiger, J., Ferling, H., Mallow, U., Wintermayr, H., Negele, R.D., 2004. Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part I. Histopathological alterations and bioaccumulation in rainbow trout. Aquat. Toxicol. 68, 141–150. Scott, B.E., Marteinsdottir, G., Begg, G.A., Wright, P.J., Kjesbu, O.S., 2006. Effects of population size/age structure, condition and temporal dynamics of spawning on reproductive output in Atlantic cod (Gadus morhua). Ecol. Modell. 191, 383–415. Simmons, D.L., Botting, R.M., Hla, T., 2004. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol. Rev. 56, 387–437. Sorbera, L.A., Asturiano, J.F., Carrillo, M., Zanuy, S., 2001. Effects of polyunsaturated fatty acids in prostaglandins on oocyte maturation in a marine teleost, the European sea bass (Dicentrarchus labrax). Biol. Reprod. 64, 382–389. Sycha, T., Gustorff, B., Lehr, S., Tanew, A., Eichler, H.G., Schmetterer, L., 2003. A simple pain model for the evaluation of analgesic effects of NSAIDs in healthy subjects. Br. J. Clin. Pharmacol. 56, 165–172. Treinen-Moslen, M., 2001. Toxic responses of the liver. In: Klaasen, C.D. (Ed.), Casarett and Doull’s Toxicology. McGraw-Hill, New York, pp. 471–489. Triebskorn, R., Casper, H., Heyd, A., Eikemper, R., Kohler, H.R., Schwaiger, J., 2004. Toxic effects of the non-steroidal anti-inflammatory drug diclofenac. Part II. Cytological effects in liver, kidney, gills and intestine of rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 68, 151–166. Uhler, M.L., Hsu, J.W., Fisher, S.G., Zinaman, M.J., 2001. The effect of nonsteroidal anti-inflammatory drugs on ovulation: a prospective, randomized clinical trail. Fertil. Steril. 76, 957–961. Van Hecken, A., Schwartz, J.I., Depre, M., De Lepeleire, I., Dallob, A., Tanaka, W., Wynants, K., Buntinx, A., Arnout, J., Wong, P.H., Ebel, D.L., Gertz, B.J., De Schepper, P.J., 2000. Comparative inhibitory activity of rofecoxib, meloxicam, diclofenac, ibuprofen, and naproxen on COX-2 versus COX-1 in healthy volunteers. J. Clin. Pharmacol. 40, 1109–1120. Yamamoto, T.S., 1975. Medaka (Killifish): Biology and Strains. Keigaku Publishing Co., Tokyo, Japan. Yoneda, M., Tokimura, M., Fujita, H., Takeshita, N., Takeshita, K., Matsuyama, M., Matsuura, S., 2001. Reproductive cycle, fecundity, and seasonal distribution of the anglerfish Lophius litulon in the East China and Yellow seas. Fish. Bull. 99, 356–370.
Changes in the timing of reproduction following chronic exposure to ibuprofen in Japanese medaka, Oryzias latipes Jennifer L. Flippin a,1 , Duane Huggett b , Christy M. Foran a,∗ a b
Department of Biology, West Virginia University, Morgantown, WV 26506-6057, United States Department of Biological Sciences, University of North Texas, Denton, TX 76203, United States
Received 6 September 2006; received in revised form 3 November 2006; accepted 4 November 2006
Abstract Effluents from wastewater treatment plants and untreated sewage constitute a low concentration but continuous source of pharmaceutical products to the aquatic environment. One such drug, ibuprofen, is a non-steroidal anti-inflammatory agent that primarily acts through inhibition of cyclooxygenase (COX) activity. Oryzias latipes (Japanese medaka) were exposed for 6 weeks via water to three concentrations of ibuprofen (1–100 g/L nominal concentrations) and a water control. Reproductive parameters, including frequency of spawning, fecundity, egg size, and rate of fertilization, were measured for each pair of adult medaka following 6 weeks of exposure. Livers homogenates from exposed individuals were assayed for COX activity and whole individuals were histologically examined for tissue damage. Increasing exposure to ibuprofen significantly increased the number of eggs per reproductive event, but decreased the number of spawning events per week. Liver tissue collected from females had less variability in COX activity with increasing concentration of ibuprofen exposure, and tended to have elevated hepatosomatic indices. No pathological damage was evident the in the gills, livers and head kidneys of animals from the highest exposure group. The results of this experiment begin to show that exposure to chronic low levels of ibuprofen alter the pattern of reproduction and may produce sex-specific responses in teleosts. © 2006 Elsevier B.V. All rights reserved. Keywords: Reproduction; Cyclooxygenase; Personal care products; NSAID
1. Introduction Effluents from wastewater treatment plants and untreated sewage may constitute a low but continuous source of pharmaceutical products to the aquatic environment. The concentration of these compounds is generally detectable in lake and river waters in North America and Europe. A pharmaceutical product is designed to alter a specific biologic process, so exposure to a single drug, its metabolites, or a mixture of various environmental contaminants may be sufficient to cause system disruption in an exposed organism (Daughton and Ternes, 1999). Ibuprofen is an over the counter analgesic and antiinflammatory product classified as a non-steroidal anti∗ Corresponding author at: Department of Biology, 53 Campus Drive, Suite 3139, P.O. Box 6057, West Virginia University, Morgantown, WV 26506-6057, United States. Tel.: +1 304 293 5201x31537; fax: +1 304 293 6363. E-mail address: [email protected] (C.M. Foran). 1 Current address: Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC 27695-7633, United States.
0166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2006.11.002
inflammatory drug (NSAID). The compound is used for the treatment of headaches and general pain (Sycha et al., 2003). NSAIDs partially hinder the inflammatory response by inhibiting one or two isoforms of the cyclooxygenase (COX) enzyme. Ibuprofen is a nonspecific COX inhibitor and is shown to inhibit both COX-1 and COX-2 (Van Hecken et al., 2000). The inhibition of COX serves as the rate limiting step in the formation of prostoglandins which are found at the site of inflammation. The COX-1 enzyme is expressed in many tissue types including those in the gastrointestinal tract, and it is responsible for producing prosanoids that function to form cytoprotection in the gastric system. Inhibition of the COX-1 enzyme may lead to the formation of ulcers in the gastrointestinal tract. Meanwhile, COX-2 is present in fewer cell types but is a key mediator in the inflammation cascade and the pain and fever response (Cryer and Kimmey, 1998; Simmons et al., 2004). COX-1 activity is known to play an important role in development in zebrafish (Cha et al., 2005) and some mammals (Cappon et al., 2003a,b). As well, COX mediated production of prostaglandins is important for ovulation in mammals (Gaytan et al., 2006)
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and fishes (Mercure and Van Der Kraak, 1996; Sorbera et al., 2001). Wastewater processing studies in Europe show that between 20 and 90% of various drugs are removed from wastewater during treatment, but the percentages for individual drugs dramatically decrease if disturbances take place during the sludge activation process (Fent et al., 2006). Ibuprofen is biodegraded to carboxylated and hydroxylated metabolites, and between 52 and 90% of ibuprofen is removed depending on treatment conditions (Paxeus, 2004). Studies have identified environmental ibuprofen, along with other NSAIDS, diclofenac, acetylsalicylic acid, and ketoprofen in quantities exceeding l g/L (Fent et al., 2006). Untreated sewage was shown to contain up to75 g/L of ibuprofen; a mean of 4 g/L and maximum of 24.6 g/L was detected in treated effluents (Metcalfe et al., 2003). Treated and untreated wastewater both enter aquatic ecosystems and carry these pharmaceutical products into the environment (Fent et al., 2006). Ibuprofen literature indicates that changes in function of several physiological systems are possible with long-term exposure. Exposure has resulted in histopathological changes in the gills and kidney of fish (Schwaiger et al., 2004; Triebskorn et al., 2004). In mammals, NSAIDS alter ovulation and development (Cappon et al., 2003a,b; Dawood, 1993). Furthermore, longterm treatment in humans frequently produces inflammatory lesions in the gastrointestinal tract (Bjorkman, 1998), and have been associated with in increased risk of heart disease in women (Egan et al., 2004; Grosser et al., 2006). The purpose of this study is to begin to determine the reproductive and physiological effects of chronic, low-level ibuprofen exposure in Oryzias latipes, Japanese medaka. Years of characterization and development of biomarkers make medaka an ideal model species of the assessment of the reproductive and developmental consequences of exposure (Metcalfe et al., 1999). 2. Materials and methods 2.1. Medaka culture Juvenile medaka aged approximately 2 months post-hatch were obtained from laboratory culture, originally stocked with eggs from the University of Mississippi Environmental Toxicology Research Program. Culture and test animals were maintained in balanced salt solution (BSS; Yamamoto, 1975) made with Nanopure® filtered water. Water quality parameters for the culture average pH 6.5, 22 ◦ C, >5.0 mg/L dissolved oxygen, 6.0 mg/L general hardness, and 1.5 g/L salinity. Ammonia and nitrite were generally undetectable. Culture and experimental animals were maintained on a 16:8 light:dark cycle. All fish were fed twice daily with freshly hatched brine shrimp and once daily with commercial flake food (TetraMin© ).
control “stock” was 1 L water. Other ibuprofen stock solutions were made by adding 80, 800, or 8000 g of the drug to water. Stocks were stored in the dark at 4 ◦ C. New stocks were made every 2 weeks. Thirty-two quart jars were filled with 790 mL of BSS and randomly placed in a room temperature water bath maintained at 26 ± 1 ◦ C; five fish were distributed to each container. Each water bath was lit with overhead fluorescent lights and a single 18 in. fluorescent fixture (510 lumens) mounted on the wall approximately 60 cm above the jars. Each jar was then dosed with 10 mL of ibuprofen solutions (at 4 ◦ C) to obtain final water concentrations of 0, 1, 10 and 100 g/L. These concentrations were chosen to overlap mean and maximal reported ibuprofen concentrations in sewage and effluent (Metcalfe et al., 2003). Each day, 100% of water was changed and a fresh dose of ibuprofen solution was administered to each jar. Nominal concentrations were not confirmed analytically. However, current Material Safety Data Sheets indicate that ibuprofen is stable and extremely soluable in water (>2 mg/mL at 25 ◦ C). Huggett et al. (2004) measured concentrations of ibuprofen from a 96 h flow through experiment at 900 g/L recovered 920 g/L. In this experiment, recoveries of fortified water samples at 700 and 900 g/L were 100%. Research has shown that photolysis of ibuprofen is undetectable in sunlight (Packer et al., 2003). We expect nominal concentrations of ibuprofen to be representative of the actual exposure. After 4 weeks, O. latipes in the same exposure group were separated into 80 jars with single sex pairs and acclimated to 28 ± 1 ◦ C. We have observed that housing medaka in same sex groups prior to reproductive assessment increases the consistency of egg production and fertilization once the animals are switched to male–female pairs. Reproductive adult pairs were switched within treatments to male–female pairs after 1 week and allowed to breed. Over the course of 7 days, eggs were collected from reproductively active pairs every 24 h during water renewal and ibuprofen exposure. The frequency of reproduction, total clutch size, number of fertilized eggs, and average egg size were recorded. Animals were sacrificed following 1 week of reproductive monitoring. All animals were anesthetized with MS-222 (Sigma, 3-aminobenzoic acid ethyl ester, methanesulfonate salt), weighed and measured. Livers were removed from 20 males and 20 females in each treatment for mass determination, hepatosomatic index (HSI) calculation, and later used in a cyclooxygenase activity assay. Animals were decapitated and livers removed and weighed. Livers were placed in a microcentrifuge tube with 5 L of PMSF solution and stored at −80 ◦ C. The remaining animals were fixed in 10% neutral buffered formalin (VWR Scientific, West Chester, PA) for histological analysis. 2.3. COX enzyme activity
2.2. Exposure Stock solutions for the exposure were prepared by dissolving ibuprofen (Sigma Chemical, St. Louis, MO) into 1 L of Nanopure® water. One stock was made for each treatment. The
COX activity assays were performed on whole liver tissue homogenate using the Cayman Chemical (Ann Arbor, MI) COX activity assay. Livers were thawed on ice, homogenized and centrifuged at 4 ◦ C with 10,000 rpm (11,180 × g)
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for 30 min. The supernatant was removed and protein content measured using Bio-Rad protein assay protein dye and standards of bovine serum albumin (BSA; Sigma, cold alcohol precipitated, frac V; 0.0625–0.5 mg/mL). Liver supernatant was assayed in a 96-well plate with appropriate COX activity reagents. This assay measures the peroxidase activity of both COX enzymes, and the COX-1 and COX-2 specific activity was not determined. Samples were incubated with assay buffer and a colorometric substrate and the appearance of tetramethylp-phenylenediene (TMPD) was compared to an ovine COX-1 standard. Background absorbance was measured from tissue after enzyme activity was destroyed by boiling, and this background value was subtracted from each sample. The plate was allowed to develop for 15 min at room temperature and COX activity was determined with absorbance at 595 nm for each sample. The specificity of the assay was determined with the addition of inhibitors for both COX-1 and COX-2 enzymes (SC-560 and DuP-697, respectively). No COX activity was detected in these samples. Reported COX activities are normalized to the protein content (mg) in the homogenized liver supernatant. 2.4. Data analysis Data for reproduction and COX activity were analyzed by nonparametric statistical tests (Mann–Whitney U tests, Kruskal–Wallis tests, and Kendall rank correlations) unless samples met requirements for normality. Many of the endpoints exhibited unequal variance between treatments. All analyses were conducted using StatView (nonparametric tests) or SAS 9.0 (regressions) with statistical significance defined at an α-level of p ≤ 0.05.
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3. Results 3.1. Reproduction Long-term ibuprofen exposure had a concentration dependent effect on the pattern of reproduction in exposure pairs. With increasing exposure, pairs spawned less frequently during the week and produced more eggs when they did spawn. The number of days that O. latipes produced eggs during 1 week of reproduction depended on treatment concentration. The total number of eggs produced by pairs over the week of assessment did not differ with exposure (Kruskal–Wallis test, H = 1.792, p = 0.62). As ibuprofen treatment increased, the frequency of egg production decreased (Kendall rank correlation, τ = −0.138, p = 0.03; Fig. 1). Increasing ibuprofen exposure also had a significant effect on the number of eggs produced by a pair per spawning event (Kendall rank correlation, τ = 0.248, p = 0.01). The greatest number of eggs per day was produced by breeding pairs exposed to the highest concentration of ibuprofen. On days when they do reproduce, pairs exposed to 100 g/L of ibuprofen produced nearly twice as many eggs as those in the control group (Fig. 1). The rate of fertilization was generally greater than 90% for all treatment groups; no difference was observed in the rate of fertilization between treatment groups (Kruskal–Wallis test, H = 2.278, p = 0.52). Mean egg diameter was 1.25 mm, and did not differ with ibuprofen treatment (Kruskal–Wallis test, H = 1.402, p = 0.71).
2.5. Histology For histological analysis, medaka were anesthetized until opercular movement stopped and no response was observed to a pinch of the caudal peduncle. Ten control animals (5 male and 5 female) and 10 animals from the highest exposure group were analyzed. Fish were immersion fixed in 10% neutral buffered formalin for at least 1 week. Prior to slicing, each fish was decalcified in 50% Cal-Ex (Fisher Scientific, Fair Lawn, NJ) for 24 h and rinsed in phosphate buffered saline for 24 h. Each animal was placed at 4 ◦ C in 30% sucrose made in phosphate buffer and allowed to absorb the sucrose overnight or until the animal sank to the bottom of the vial. Whole medaka were embedded in Cryo-M-Bed (VWR) and frozen using dry ice. Embedded frozen tissue was mounted in a Leica cryostat and sliced into 10-m thick sagital sections which were melted onto glass slides. Sections were washed in phosphate buffer and stained in hemotoxylin and eosin. Slides were then dehydrated through a series of ethanol washes (50–100%), soaked twice in xylene, and coverslipped under Permount (VWR). Each section was viewed at 100× magnification for visual damage. Tissues (gills, liver, gonads, and gastrointestinal track) from each animal were viewed at 400×. No quantitative histological measurements were made.
Fig. 1. Changes in the pattern of reproduction for pairs of Japanese medaka exposed to ibuprofen for 6 weeks. The number of days that pairs produced eggs declined with increasing ibuprofen exposure concentrations (p = 0.03). On days that pairs reproduced, the number of eggs released (clutch size) increased with exposure concentration (p = 0.01). Bars represent mean values for the group ± standard error.
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Fig. 2. Hepatosomatic index (HSI ± standard error), the relative weight of the liver, is shown for females and males following ibuprofen exposure. Although no significant differences were detected from controls, female HSI (gray bars) tended to increase while male HSI (white bars) tended to decrease. Female HSI are significantly higher than males for each exposure concentration (all p > 0.04).
Over the week that reproduction was monitored, the average number of eggs produced was related to the weight of the female (regression ANOVA, F = 7.573, R2 = 0.18, p = 0.01). The residual difference in the relationship between size (weight) and egg production was not significantly different between treatment groups (Kruskal–Wallis test, H = 6.643, p = 0.08). Although the variability in residuals is greatest in the 10 and 100 g/L ibuprofen treatment groups. 3.2. Hepatosomatic index The relative liver weight of ibuprofen exposed animals was not different than controls for males or females (Kruskal–Wallis tests, both p > 0.48). However, the HSI of females tended to increase with exposure to ibuprofen, while male HSI seemed to decrease (Fig. 2). When compared directly, the HSI of males and females differs significantly for each exposure concentration (1 g/L: Mann–Whitney U, Z = −2.03, p = 0.04; 10 g/L: Mann–Whitney U, Z = −3.55, p > 0.01; 100 g/L: Mann–Whitney U, Z = −2.50, p = 0.01) but not controls (Mann–Whitney U, Z = −0.68, p = 0.49). The pattern of HSI changes indicates that males and females may respond differently to ibuprofen exposure. 3.3. Hepatic COX activity The COX activity in homogenized liver tissue was highly variable in control groups, and a decrease in this variability was the primary effect of ibuprofen exposure. Females in the control treatment exhibited the most variability in individual COX activity (Fig. 3). Females showed a significant decline in this variability following ibuprofen exposure at 10 and 100 g/L (F0,10 = 8.43, p0,10 = 0.02; F0,100 = 21.54, p0,100 > 0.001; F1,100 = 8.65, p1,100 = 0.02). Therefore, ibuprofen exposure changed the range of activity but did not significantly alter the mean value for COX activity. COX activity in male liver samples was the most variable in controls. However, variability in male COX activity was significantly dif-
Fig. 3. Cyclooxygenase (COX) activity normalized to the amount of protein (mg) in liver homogenates (±standard error) is shown for males and females following ibuprofen exposure. In both sexes, variability of COX activity was highest in control samples. For females, variability in enzyme activity declines following 10 and 100g/L exposure (all p > 0.02). A change in variability was detected in males between tissues from controls and those exposed to 1.0 g/L (p = 0.01).
ferent only between controls and the 1.0 g/L exposure group (F0,10 = 49.09, p0,10 = 0.01). Measurements of COX activity, along with HSI, indicate that male and female medaka respond differently to ibuprofen exposure. 3.4. Histology Serial sections from the gills, head kidneys, and livers of exposed animals resembled those of controls. Gills filaments were normal in length and distribution, without degradation or fusion. Head kidneys appeared to have normal tubule shape and numbers. Livers exhibited no evidence of hepatocyte degradation. The liver of one female exposed to 100 g/L ibuprofen contained acellular solid deposits, although deposits were not detected in any other animal. 4. Discussion Six weeks of exposure to aqueous ibuprofen produced a change in the pattern of reproduction. With increasing concentration, pairs of Japanese medaka reproduced less frequently and produced a greater number of fertilized eggs when they did spawn. NSAID-related changes in reproduction are not unprecedented in the literature. NSAIDS, specifically COX-2 inhibitors, are known to inhibit ovulation in mammals (Gaytan et al., 2006). Human reproductive studies show that ibuprofen may increase the time interval between the luteinizing hormone surge and follicular collapse (Uhler et al., 2001). In teleosts, prostaglandins have been reported to be produced in the ovaries (Goetz et al., 1989; Knight et al., 1995), and are known to play a role in oocyte maturation in sea bass (Sorbera et al., 2001). Indomethacin, which is a general inhibitor of COX-1 and -2, repressed human gonadotropin-induced maturation of harvested oocytes but not spontaneous maturation of oocytes (Sorbera et al., 2001). Therefore, limiting the induction of COX activity in the ovary may slow the rate of oocyte maturation or ovulation in medaka.
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HSI changes with exposure were divergent for males and females. Females exhibited hepatic growth relative to males with exposure. The observed sexually dimorphic response may be related to changes in reproduction. Since the liver is site of production of vitellogenin, a glycoprotein precursor to yolk (Lazier and MacKay, 1993), the size of the liver may increase with the number of eggs maturing or spawned in a single event. HSI has been shown to increase with measures of vitellogenin production (Pereira et al., 1993) and decrease with ovarian development (Yoneda et al., 2001). The liver is also the site of a major portion of metabolism and detoxification of contaminants (Treinen-Moslen, 2001). Eicosanoids can alter the production of steroid hormones and steroid metabolizing enzymes (Gravel and Vijayan, 2006; Mercure and Van Der Kraak, 1996). Exposure of rainbow trout to diclofenac caused a visual depletion of glycogen from liver hepatocytes (Triebskorn et al., 2004). Thus, growth of hepatocytes may be altered by their change in function with NSAID exposure. Hepatic COX activity was found to be less variable among males than females. Neither males nor females showed a significant inhibition of COX activity following 6 weeks of exposure. One explanation is that ibuprofen is ineffective in its inhibition of teleost COX activity, as evidenced by a study of sunfish (Cavallaro and Burnside, 1988). Another possibility is that 6 weeks of exposure allowed recovery of COX activity, either through an increase in the synthesis of COX enzymes or through increased metabolism of ibuprofen. However, we found no evidence in the literature of changing effective concentrations of ibuprofen with long-term use or exposure. Another possibility is that the natural variability of hepatic COX activity in female medaka precludes the detection of inhibition of activity; measurements of hepatic COX activity from exposed females are similar to lower values measured from female controls. In this study, we did not observe histological changes in the gill or head kidney following 6 weeks of ibuprofen exposure. One animal displayed solid, acelluar deposits in the liver. Fish exposure to environmentally relevant levels of another NSAID, diclofenac, has been shown to change organ structure (Schwaiger et al., 2004; Triebskorn et al., 2004). Studies involving rainbow trout, Oncorhynchus mykiss, found 28 days of exposure produced kidney damage, specifically damage to tubular epithelial cells (Schwaiger et al., 2004). In one study, gill damage was evident in the secondary lamellae following exposure (Schwaiger et al., 2004). Ultrastructual analysis using electron microscopy revealed further damage to hepatocytes and glycogen depletion in the liver (Triebskorn et al., 2004). These investigations determined the lowest observed effect concentration (LOEC) for “cytological alterations in the liver, kidney, and gills” to be l g/L (Triebskorn et al., 2004). By comparison, histological damage was not observed in Japanese medaka following 6 weeks of ibuprofen exposure. This disparity may be due to differences in the COX inhibition between ibuprofen and diclofenac, differences in species sensitivity, or the use of more sensitive ultrastructral comparisons by Triebskorn et al. (2004). In vitro COX potencies of diclofenac and ibuprofen differ significantly. For instance, the COX-2 IC50 for diclofenac is 0.06 M,
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while the IC50 for ibuprofen is 19 M (Blain et al., 2002). These data suggest that diclofenac at equivalent concentrations would be more portent than ibuporofen in in vivo systems. Further studies should look at the relationship between the specificity of inhibition of COX-1 and -2 and histopathological damage from environmental exposure. Disruption of COX-1 expression disrupts development in zebrafish embryos but not mammalian embryos (Cappon et al., 2003a,b; Cha et al., 2005; Grosser et al., 2002). Although the different patterns of COX expression in fish and mammals have important implications for risk assessment for fishes, we saw no evidence that exposure during sexual maturation would inhibit either reproductive output or hepatic COX activity. However, studies have reported developmental and reproductive consequences of exposure in fish (Cha et al., 2005; Sorbera et al., 2001) and mammals (Cappon et al., 2003a,b; Gaytan et al., 2006). Developmental or reproductive impairment mediated by COX inhibitors could have a significant impact on fish populations. More research is needed to completely characterize the risk of environmental NSAID exposure. 5. Conclusion Environmental pharmaceuticals are frequently detected in wastewater and surface water. These contaminants, although expected to be found in relatively low concentrations, may be biologically potent. In this study we examined the long-term reproductive and physiological consequences of ibuprofen exposure. As expected, no acute toxicity was observed. Six weeks of exposure did produce a change in the temporal pattern of spawning in pairs of Japanese medaka. With increasing concentrations, pairs were more likely to spawn less frequently and produce more eggs when they did spawn. Timing of spawning is not likely to impair population growth in a laboratory species. However, if a similar delay is possible in species with highly coordinated spawning, such as cod (Scott et al., 2006) and salmon (de Gaudemar and Beall, 1998), fertilization and offspring survival may be compromised. Females and males responded differently to long-term ibuprofen exposure. Females exhibited more variable COX activity, and that variability decreased with exposure concentration. The relative liver weight of exposed females also tended to increase with exposure, potentially related to the change in spawning and vitellogenin production. Sex differences in the response to NSAIDS have been reported in the clinical and mammalian literature (Egan et al., 2004; Grosser et al., 2006; Reese et al., 2001). However, we present the first evidence of sexspecific changes in a teleost. Although the mammalian studies indicate estrogen upregulates COX-2, the potential for steroids to regulate expression of these genes in fish is unknown. Acknowledgments The authors would like to thank E. Anderson, Dr. J.R. Ripley, and T. Steuckle for their assistance, and Dr. K. Willett for providing O. latipes fry. This research was funded in part by West Virginia University’s Eberly College of Arts and Sciences.
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