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Thyroid hormone status of Atlantic croaker exposed to Aroclor 1254 and selected PCB congeners
Comparative Biochemistry and Physiology, Part C 144 (2006) 263 – 271 www.elsevier.com/locate/cbpc
Thyroid hormone status of Atlantic croaker exposed to Aroclor 1254 and selected PCB congeners Kimberly D. LeRoy, Peter Thomas, Izhar A. Khan ⁎ The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, USA Received 25 May 2006; received in revised form 25 September 2006; accepted 25 September 2006 Available online 6 October 2006
Abstract Atlantic croaker (Micropogonias undulatus) were exposed to the polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or one of three individual congeners (planar PCB 77 or ortho-substituted PCB 47 and PCB 153) in the diet for 30 days to investigate the effects of PCBs on circulating thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) decreased plasma T3 levels consistently, but the effects on T4 levels were inconsistent from year to year. Exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) significantly lowered both T4 and T3, while PCB 47 at the same doses had no effect on thyroid hormone levels. The lower doses of PCB 77 (0.004, 0.01 and 0.02 mg/kg body mass/day) had no effect on T4 or T3, whereas the highest dose (0.1 mg/kg body mass/day) increased T4 levels significantly. The results of the present study demonstrate that exposure to PCBs at environmentally realistic concentrations can have profound effects on the thyroid status of Atlantic croaker. The ortho-substituted PCB 153 appears to contribute at least partially to the deleterious effects of Aroclor 1254 on thyroid status, whereas the planar PCB 77 at concentrations present in the mixture is unlikely to alter thyroid hormone levels. © 2006 Elsevier Inc. All rights reserved. Keywords: Atlantic croaker; Aroclor 1254; PCB congeners; Polychlorinated biphenyls; Thyroid hormones; Fish
1. Introduction Chemical pollutants present in the aquatic environment can disrupt vital physiological processes in fish, such as growth, reproduction, osmoregulation, and thyroid and immune functions (Folmar et al., 1982; McCarthy et al., 2003; Thomas and Khan, 2005). Recent attention has focused on the ability of these synthetic chemicals to interfere with the normal function of the endocrine system. Polychlorinated biphenyls (PCBs) are some of the most widely studied endocrine disrupting chemicals (EDCs) (Van den Berg et al., 1998). These persistent organic pollutants can be found in aquatic environments across the world (Slooff et al., 1983; Van der Oost et al., 1996; Kannan et al., 1998; Mazet et al., 2005). PCBs are able to bioaccumulate in the lipid-rich tissues of many aquatic organisms and cause numerous adverse health effects (Safe, 1994; Van den Berg et al., 1998).
⁎ Corresponding author. Tel.: +1 361 749 6810; fax: +1 361 749 6749. E-mail address: [email protected] (I.A. Khan). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.09.005
Based on their chemical structure, PCB congeners can be divided into two main groups, the planar (non-orthosubstituted) and non-planar (ortho-substituted). Considered the most toxic, planar PCBs (PCB 77, 126, 169) are laterally substituted with no Cl− substitution on ortho-positions. The absence of Cl− atoms from the ortho-position on the rings allows a smaller hydrogen atom to become incorporated into the compound, creating a planar structure that is similar to dioxin (TCDD) and is more resistant to degradation (Walker and Peterson, 1991; Maier et al., 1994; Van den Berg et al., 1998). Planar PCB congeners and their metabolites share a common mechanism of action in vertebrates and are able to bind a cytosolic protein, the aryl hydrocarbon receptor (AhR), induce hepatic enzymes (CYP1A), and cause a variety of toxic responses (Burgin et al., 2001). Reported effects of exposure to planar PCBs include weight loss, enzyme induction, teratogenicity, carcinogenicity, and endocrine disruption (Safe, 1994). The di- to tetra-ortho-substituted PCB congeners have two or more chlorine atoms on the ortho-positions and are not planar due to steric hindrance. The ortho-substituted congeners are most abundant in Aroclor mixtures and can act via different
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mechanisms and elicit a different pattern of toxicity (Kodavanti et al., 1998; Brouwer et al., 1999; Khan et al., 2002). Exposure to these congeners can induce CYP2B and 3A enzymes and elicit neurotoxicity, carcinogenicity, and endocrine disruption (Zoeller et al., 2000; Khan et al., 2002). Thyroid hormones (TH) are known to play important roles in the early development and metamorphosis in different vertebrate groups (reviewed in Power et al., 2001). In particular, there is considerable evidence for thyroidal control of amphibian metamorphosis (reviewed in Tata, 2006). In Japanese flounder, exogenous administration of TH or elevation of endogenous T4 levels by thyroid stimulating hormone induces precocious metamorphosis by increasing the rate of transformation (Miwa and Inui, 1987; Inui et al., 1989). The relevance of TH to metamorphosis has also been demonstrated in fishes exhibiting a relatively less dramatic transformation, such as conger eel (Conger myriaster), telescopic-eye black goldfish (Carassius auratus), zebrafish (Danio rerio) grouper (Epinephelus coioides) and tarpon (Megalops cyprinodes) (Yamano et al., 1991; Reddy and Lam, 1992; Brown, 1997; de Jesus et al., 1998; Shiao and Hwang, 2006). Moreover, TH treatments improve larval survival in several fish species (Brown et al., 1989; Ayson and Lam, 1993; Inui et al., 1994). Thyroid hormones also influence a variety of other functions in fish including growth, smoltification, osmoregulation, reproduction and locomotor activity (Eales and Brown, 1993; Rousseau et al., 2002; Kulczykpwska et al., 2004; Edeline et al., 2005; Swapna et al., 2006). Therefore, the ability of PCBs to disrupt the thyroid system in fish has the potential to influence normal physiology of the adults and/or long term survival of their offspring. Previous studies in Atlantic croaker (Micropogonias undulatus) using Aroclor 1254 have established this species as an excellent vertebrate model for investigations into the mechanisms of PCB neuroendocrine toxicity (Khan and Thomas, 1997, 2001, 2004; Thomas and Khan, 2005). Exposure of croaker to Aroclor 1254 (1 mg/kg body mass/day for 30 days) during the early-recrudescence phase of the gonadal cycle disrupts gonadotropin releasing hormone (GnRH) neuroendocrine function resulting in the impairment of LH secretion and gonadal growth (Khan and Thomas, 2001). This neuroendocrine disruption is caused by inhibition of hypothalamic tryptophan hydroxylase, the rate limiting enzyme in 5-hydroxytryptamine (5-HT) synthesis. Interestingly, the planar PCB 77, but not the orthosubstituted PCB 47 or PCB 153, impairs the hypothalamic 5-HT system in croaker (Khan and Thomas, 2006). Considering the ability of thyroid hormones to influence the development of the 5-HT system in mammals (Schantz et al., 1991), it is worthwhile to determine the effects of the PCB mixture and individual PCB congeners on the thyroidal status of Atlantic croaker. The reported effects of PCB exposure on thyroid function in fish are highly variable. An earlier study in yearling coho salmon (Oncorhynchus kisutch) exposed to a mixture of PCBs in the diet (1:4 mixture of Aroclor 1242 and Aroclor 1254) reported depressed triiodothyronine (T3) levels and slower growth with no effect on T4 levels (Leatherland and Sonstegard, 1978, 1980). In contrast, exposure of coho salmon to a combination of Aroclor 1254 and fuel oil increased levels of
circulating T3 and delayed the plasma T4 surge commonly associated with smoltification (Folmar et al., 1982). European flounder (Platichthys flesus) show increased levels of both T4 and T3 following exposure to the PCB mixture Clophen A50 (Besselink et al., 1996). These variable effects on TH levels reported in many laboratory studies may be due to the variable composition of PCB mixtures in biological systems and the different effects of predominant planar and ortho-substituted congeners. Studies in experimental animals suggest that the selective metabolism and complex behavior of each individual PCB congener or metabolite is important when studying these compounds (Safe, 1994; Van den Berg et al., 1998; Khan et al., 2002). However, only a few studies have examined the effects of individual PCB congeners on circulating TH levels in fish (Adams et al., 2000; Brown et al., 2004). In addition, previous studies in fish have not examined the effects of PCB mixtures and individual PCB congeners in the same species. The present study examined the effects of dietary exposure to the Aroclor 1254 mixture and three individual PCB congeners on circulating TH levels in Atlantic croaker in order to better understand the contributions of the selected planar and nonplanar congeners to the overall effects of the PCB mixture on the thyroid status in fish. A planar (PCB 77) and two orthosubstituted congeners with different degrees of chlorination (tetra-chlorinated PCB 47 and hexa-chlorinated PCB 153) were selected for the present study. Moreover, analyses of Aroclor 1254 and the liver samples of croaker exposed to the mixture were performed to determine individual congener profiles and their bioaccumulation in this species. 2. Materials and methods 2.1. Chemicals and reagents Aroclor 1254 (Lot # K040) was purchased from Foxboro Company (North Haven, CT). PCB 47 (2,2′,4,4′-tetrachlorobiphenyl), PCB 77 (3,3′,4,4′-tetrachlorobiphenyl), and PCB 153 (2,2′,4,4′,5,5′-hexaclorobiphenyl) were purchased from Chemservice (West Chester, PA). Total T4 and T3 ELISA kits were purchased from DSL Laboratories, Inc. (Webster, TX). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2. Experimental animals Young-of-the-year Atlantic croaker were captured by shrimp trawls from Redfish Bay, Texas, during the early fall and maintained in large, recirculating, seawater tanks (salinity 30– 35 ppt) equipped with biofilters. Fish were maintained in a simulated natural fall photoperiod (light/dark cycle, 11:13 h) and temperature conditions (22 ± 1 °C), and fed a mixed diet of chopped shrimp and commercial trout pellets (95:5 w/w). Either male or female fish were used for the experiments in order to avoid variable responses, if any, due to the sex of the individuals. Fish were handled according to the National Institute of Health Guidelines for handling and care of experimental animals. The animal utilization protocol was approved by the
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Institutional Animal Care and Use Committee of the University of Texas at Austin.
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heparinized syringes and kept on ice. Plasma samples were obtained after centrifugation at 3000 ×g for 6 min and were stored at − 80 °C until assayed for T4 and T3.
2.3. Experimental protocols 2.4. Experiment 1: effects of Aroclor 1254 on TH levels PCBs were dissolved in a small volume of ethanol, which was then mixed with vegetable oil and the ethanol subsequently evaporated at 40 °C. The PCBs in vegetable oil were then thoroughly mixed into the fish diet. Fish were fed the prepared food laced with sub-lethal levels of PCBs or mixed with vegetable oil alone at the rate of 3% of their body weight per day for 30 days, between 08:00 and 10:00 h. In the fall, young-of-the-year fish (40–60 g body weight each) were randomly sorted into groups of 25 and maintained in 1500-l recirculating seawater tanks equipped with biofilters. They were allowed to acclimate for a minimum of 1 month prior to initiating PCB feeding experiments. All fish used in fall experiments were at early stages of gametogenesis at the beginning of each experiment and the control fish were fully mature by the end of the experiment, as evidenced by the presence of readily expressible milt in males and late vitellogenic oocytes in females. Due to the limited number of experiments that could be carried out within the short reproductive period of croaker (October–November), some fish were maintained on a shortened seasonal photoperiod–temperature cycle in order to simulate their natural fall breeding season in the spring. In our laboratory, croaker can be moved through a shortened seasonal photoperiod–temperature cycle and can be induced to become reproductively mature off-season (unpublished observation). Using this established protocol, fish were cycled through a fourand-a-half month shortened seasonal photoperiod–temperature cycle corresponding to winter, summer and fall conditions. Fish used in spring experiments were 6 months older and gonadally mature when the PCB exposures were initiated in April–May, unlike those in the fall experiments in which PCB exposure occurred during the gonadal growth phase of the reproductive cycle. At the end of the experiments, all fish were sampled between 0900 and 1100 h to avoid possible diurnal fluctuations in hormone levels (e.g., Leiner et al., 2000) from influencing the results. Fish from each tank were divided into two subgroups at the time of sampling. One subgroup of 8–10 fish was sampled to collect blood for TH measurements, while the remaining fish were used to collect brain samples without bleeding in order to evaluate neuroendocrine parameters. Blood sampling time for each tank was b15 min and the sampling time for each experiment (3–4 tanks) was b1 h. Body, liver, and gonad weights were recorded in order to determine hepato-somatic indices (HSIs; liver weight expressed as percent body weight) and gonado-somatic indices (GSIs; gonad weight expressed as percent body weight). Livers were carefully removed, collected on dry ice, and stored at − 80 °C for subsequent PCB analysis. Studies have shown that measuring total plasma T4 and T3 levels provides relative measurement of the systemic availability of thyroid hormones in fish (Eales and Brown, 1993). For all experiments, blood was collected from the caudal vessels using
In the fall of 2003 and 2005, 25 croaker with testes at early stages of spermatogenesis were fed a control diet of chopped shrimp and commercial trout pellets, while two other groups of fish were exposed to Aroclor 1254 mixed in the diet (0.2 and 1.0 mg/kg body mass/day) for 30 days. In the spring of 2005, gonadally mature female croaker cycled through compressed photoperiod–temperature regimes were exposed to Aroclor 1254. One group of 25 fish was fed a control diet each morning, while two other groups were fed Aroclor 1254 mixed in the diet (0.2 and 1.0 mg/kg body mass/ day) for 30 days. This experiment was repeated in the fall of 2005 with gonadally recrudescing female croaker. 2.5. Experiment 2: effects of selected PCB congeners on TH levels In the fall of 2003 and 2004, one tank of 25 male croaker with testes at early stages of spermatogenesis was fed a control diet, while two other groups of fish were exposed to the orthoPCB 153 (0.1 and 1.0 mg/kg body mass/day) in the diet for 30 days. Two similar experiments were carried out in the fall of 2003 and 2004 with PCB 47 using the same doses and duration of exposure as the PCB 153 experiments. In the fall of 2004, one tank of 25 male fish was fed a control diet, while two other groups were exposed to the planar PCB 77 (0.1 and 1.0 mg/kg body mass/day). Due to high mortality, the experiment was terminated early and dosage levels for future experiments using PCB 77 were reduced more than 10 fold. In the spring of 2005, one group of 25 mature males was fed a control diet, while three other groups were exposed to sub-lethal doses of the planar PCB 77 mixed in the diet (0.004, 0.02 and 0.1 mg/kg body mass/day) for 30 days. Another experiment with PCB 77 (0.01 and 0.1 mg/kg body mass/day) exposure in males was conducted in the fall of 2005 to confirm the results. 2.6. Thyroid hormone assays Thyroid hormones were extracted from plasma samples using a mixture of chloroform and methanol, following the method described by Greenblatt et al. (1989) with minor modifications. Preliminary experiments showed that the extraction of both T4 and T3 from plasma was best accomplished using a mixture of chloroform and methanol (data not shown). Extracted samples were stored at − 80 °C and were resuspended in enzyme immunoassay (EIA) buffer in one half the original sample volumes before starting the enzyme-linked immunosorbent assays (ELISAs). Total T4 and total T3 levels were measured in extracted samples with commercially available ELISA kits following the protocols provided with the kits (DSL Laboratories Inc., Webster, TX). Standards and extracted samples were added to the 96-well plates in duplicate.
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Absorbance was measured at 450 nm (Spectra Max 190, Molecular devices Corp., Sunnyvale, CA) immediately following the final step in the ELISA protocol. Concentrations of T4 and T3 were subsequently determined using SOFTmaxPRO 4.0 software. Intra-assay variation ranged from 8.12% to 4.37%, while interassay variation was 10.62% and 8.95% for T4 and T3, respectively. 2.7. Liver PCB analyses Liver samples and Aroclor 1254 standard were analyzed by CRG Marine Laboratories, Inc., Torrance, CA for total PCB concentrations and individual congener profiles. Samples were analyzed by GCMS using EPA method 8270Cm. 2.8. Statistical analyses All statistical tests were conducted using SYSTAT Version 10.2. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple range test for the comparison of group means. Differences between groups were considered significant at a p-value b 0.05.
Fig. 2. Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in female Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01, ⁎⁎ = P b 0.001).
3. Results No mortality was observed during any experiments except in the first trial using the highest dose of PCB 77 (1 mg/kg body mass/day). This trial experiment with PCB 77 was terminated early and TH levels were not examined in this experiment. 3.1. Experiment 1: effects of Aroclor 1254 on TH levels There was no significant difference in body weights of fish from control and Aroclor 1254 treatment groups (data not
Fig. 1. Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01; ⁎⁎ = P b 0.001).
included), suggesting that these doses of the PCB mixture do not adversely affect growth. There was also no significant correlation between gonadal stage and thyroid status when examined by a comparison between the GSI and TH levels in control fish (data not included). Plasma concentrations of T4 and T3 were significantly altered in male fish exposed to both the low and high doses of Aroclor 1254 (Fig. 1). In 2003, the T4 response showed a Ushaped curve, with the low dose producing a 30% decrease in T4, and the high dose producing a 48% increase in T4 levels when compared to controls. When the experiment was repeated in 2005, no statistically significant difference was observed in T4 levels between the control and treatment groups, indicating that the effects of Aroclor 1254 on T4 levels were not consistent from year to year. However, T3 levels were reduced consistently in all groups of male croaker exposed to Aroclor 1254 (Fig. 1). In 2003, the significant decrease in T3 levels was not dosedependent. However, when the experiment was repeated in 2005, T3 levels decreased in a dose-related manner, with a 41%
Fig. 3. Total PCB accumulation in the livers of croaker exposed to the high dose of Aroclor 1254 (1.0 mg/kg body mass/day) showed a positive correlation with the hepato-somatic indices (HSIs) of individual fish.
K.D. LeRoy et al. / Comparative Biochemistry and Physiology, Part C 144 (2006) 263–271 Table 1 Percent of individual PCB congeners detected in Aroclor 1254 and liver samples from Atlantic croaker exposed to the mixture in the diet (1 mg/kg body mass/ day) Congener
IUPAC # Aroclor 1254 Liver (% of total PCBs) (% of total PCBs)
2,2′,5 2,4,4′ 2,4′,5 2′,3,4 3,4,4′ 2,2′,3,5′ 2,2′,4,5′ 2,2′5,5′ 2,3′4,4′ 2,3′,4′,5 2,4,4′,5 3,3′,4,4′ 3,4,4′,5 2,2′,3,4,5′ 2,2′,3,5′,6 2,2′,3′,4,5 2,2′,4,4′,5 2,2′,4,5,5′ 2,3,3′,4,4′ 2,3,3′,4′,6 2,3,4,4′,5 2,3′,4,4′,5 2,3′,4,4′,6 2′,3,4,4′,5 3,3′,4,4′,5 2,2′,3,3′,4,4′/2,3′,4,4′,5,5′ 2,2′,3,4,4′,5′ 2,2′,3,4,5,5′ 2,2′,3,4′,5′,6 2,2′,3,5,5′,6 2,2′,4,4′,5,5′ 2,3,3′,4,4′,5 2,3,3′,4,4′,5′ 2,3,3′,4,4′,6 2,3′,4,4′,5′,6/2,2′3,3′,4,6′ 3,3′,4,4′,5,5′ 2,2′,3,3′,4,4′,5 2,2′,3,3′,4′,5,6 2,2′,3,4,4′,5,5′ 2,2′,3,4,4′,5′,6 2,2′,3,4′,5,5′,6 2,3,3′,4,4′,5,5′ 2,2′,3,3′,4,4′,5,5′ 2,2′,3,3′,4,5,6,6′ 2,2′,3,3′,4,5′,6,6′ 2,2′,3,3′,4,4′,5,5′,6
18 28 31 33 37 44 49 52 66 70 74 77 81 87 95 97 99 101 105 110 114 118 119 123 126 128/167 138 141 149 151 153 156 157 158 168/132 169 170 177 180 183 187 189 194 200 201 206
0.06 0.07 0.04 0.02 0.02 0.82 0.39 1.11 4.06 7.85 2.75 0.31 0.13 3.43 2.62 4.95 5.35 6.72 7.57 9.05 0.56 13.47 0.19 0.38 6.60 0.10 6.60 0.86 2.18 0.27 4.00 1.78 1.13 0.91 1.51 0.21 0.40 0.11 ND 0.04 0.91 0.38 ND 0.09 ND 0.02
0.02 0.06 0.04 0.01 0.00 0.67 0.39 1.13 4.62 5.97 2.26 0.15 0.14 3.71 2.33 5.36 6.04 7.52 9.40 8.78 0.68 16.29 0.23 0.58 0.03 2.29 7.60 0.98 2.42 0.28 4.62 1.51 0.51 1.11 0.87
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total PCB accumulation in the livers of croaker exposed to the high dose of Aroclor 1254 showed a positive correlation with the HSIs of individual fish (Fig. 3), despite high individual variation (20–30% standard error; N = 7). GSIs in fish exposed to Aroclor 1254 were not affected when considering only the fish in which TH levels were measured; however, when considering the whole tank of fish (N = 25), GSIs were significantly lower in Aroclor 1254-exposed males and females (data not shown). Aroclor 1254 and seven liver samples from fish exposed to the high dose of the PCB mixture were analyzed for 46 individual PCB congeners and total PCB levels (Table 1). The predominant congeners found in Aroclor 1254, in the order of decreasing concentration were PCB 118, 110, 70, 105, 101, 126, 138, 99, 97, 66, and 153, which accounted for 75% of the total detectable PCBs. Homolog groups contributing the most to the PCB content in liver samples were (in order of contribution to the total) the penta-, hexa-, tetra-, and heptachlorinated groups. Predominant congeners found in liver tissue, in the order of decreasing concentration were PCB 118, 105, 110, 138, 101, 99, 70, 97, and 153, which accounted for 77% of the total detectable PCBs. Congeners present in Aroclor 1254 but not detected in any liver samples were PCB 37, 169, 200, and 206. In terms of the planar PCBs, congener 77 was present at the highest concentration in livers, followed by PCB 126 and PCB 169. This was not the same pattern found in the Aroclor 1254 however, in which PCB 126 was found at the highest concentration, followed by PCB 77 and PCB 169. Plasma T4 or T3 levels did not show a strong correlation with total PCB accumulation in liver samples of fish exposed to the high dose of Aroclor 1254 as the TH levels were in the normal range of
0.49 0.08 0.55 0.14 0.12 0.02 0.01 ND ND ND
Numbers in bold indicate the ten most predominant congeners. ND = nondetectable. (N = 7).
decrease seen at the highest dose of Aroclor 1254. Similar to males, T3 levels in Aroclor 1254-exposed female croaker decreased significantly in the low and high dose groups (Fig. 2). A dose-related decrease in average T3 levels was seen in both the fall and spring experiments, while average T4 levels were not significantly affected in any treatment group. The HSIs in fish exposed to Aroclor 1254 increased in a dose-related manner: Males, 0.86 ± 0.08, 1.26 ± 0.14 and 1.38 ± 0.21%; Females, 0.93 ± 0.07, 1.28 ± 0.08 and 2.05 ± 0.28%, in the control, low and high dose groups, respectively. In addition,
Fig. 4. Effects of exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01; ⁎⁎ = P b 0.001).
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individual variability within the treatment group (data not shown). 3.2. Experiment 2: effects of selected PCB congeners on TH levels Both plasma T4 and T3 levels decreased in all groups of male croaker exposed to the ortho-substituted PCB 153 (0.1 and 1.0 mg/kg body mass/day) when compared to control fish, although there was no effect on T3 levels in the fall of 2004 (Fig. 4). In all experiments during the fall of 2004, average levels of both T4 and T3 were only 30% of average levels observed in the previous year and might be responsible for the lack of significant decrease in T3 levels in PCB 153-treated fish in this experiment. The amount of PCB 153 represented 4% of the total PCB congeners in Aroclor 1254 and 4.62% of the total PCBs detected in liver samples from male croaker exposed to the highest dose of the PCB mixture (Table 1). Dietary exposure to the ortho-substituted PCB 47 (0.1 and 1.0 mg/kg body mass/day) for 30 days resulted in no significant alterations in plasma T4 or T3 levels (data not included). Fully mature and gonadally recrudescing male croaker exposed to the highest dose of the planar PCB 77 (0.1 mg/kg body mass/day) had significantly higher levels of plasma T4 than control fish (Fig. 5). In both the spring and fall experiments, average T4 levels more than doubled in fish fed the highest dose of PCB 77. However, there was no significant change in average T3 levels following any exposure to PCB 77. The highest dose of PCB 77 also resulted in decreased appetite towards the end of the exposure period. PCB 77 was 0.31% of the total PCBs in the Aroclor 1254 and 0.15% of the total PCBs detected in liver samples from male croaker exposed to the highest dose of the PCB mixture (Table 1).
Fig. 5. Effects of exposure to PCB 77 on circulating levels of T4 and T3 in Atlantic croaker (A) 0.004, 0.02 and 0.1 mg/kg body mass/day (B), 0.01 and 0.1 mg/kg body mass/day. Bars represent average TH levels of 9 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎⁎ = P b 0.001).
4. Discussion The results presented in this paper demonstrate the effects of an environmentally relevant PCB mixture, along with those of individual ortho-substituted and planar congeners on the thyroid status of an estuarine fish, Atlantic croaker. Interestingly, both Aroclor 1254 and PCB 153 significantly decreased circulating TH levels in croaker, while PCB 77 and PCB 47 failed to decrease TH levels. The results of this study suggest that di-ortho-substituted congeners with higher chlorination such as the hexa-chlorinated PCB 153 may contribute to the depression of TH levels observed in croaker exposed to Aroclor 1254, whereas planar congeners such as PCB 77 present at low concentrations in the PCB mixture are unlikely to alter thyroidal homeostasis in this species. Aroclor 1254 is thought to generally depress both T4 and T3 levels in mammals. The finding that Aroclor 1254 decreases plasma T3 levels in croaker is significant because T3 is considered to be the biologically active thyroid hormone (Eales and Brown, 1993). Unlike its effects on T3 levels, the effects of Aroclor 1254 on T4 levels in this study were inconsistent, with no significant effect observed in subsequent experiments with males or females. Similar to the results in croaker, Aroclor 1254 decreases circulating T3 levels without significantly altering T4 levels in salmonids (Leatherland and Sonstegard, 1978). Overall, the average TH levels in control fish were quite variable from year to year, which probably reflects differences in the underlying TH status of the experimental fish that may account for the variable degree of responses to various PCB exposures. Interestingly, one of the most prevalent congeners found in the environment, di-ortho-PCB 153 (Jordan and Feeley, 1999) was also the most effective in reducing circulating TH levels in croaker. In view of the fact that PCB 153 is one of the major congeners found in Aroclor 1254 (4% of the total), and that it bioaccumulates in the livers of croaker (4.6% of total PCBs accumulated), PCB 153 appears to contribute at least partially to the observed decrease in TH levels in croaker exposed to the PCB mixture. In contrast to the hexa-chlorinated PCB 153, the tetra-chlorinated di-ortho-substituted PCB 47 had no significant effect on TH levels in the present study. This suggests that the degree of chlorination may be an important factor contributing to the effects of individual PCB congeners on TH levels. The PCB 153-induced decrease in T4 levels observed in croaker is consistent with similar findings in mammals (Van Birgelen et al., 1992; Ness et al., 1993). PCB 77, considered one of the most toxic congeners, is thought to generally depress circulating levels of T4 in mammals (Brouwer et al., 1999). However, American plaice injected with PCB 77 show no effect on plasma T4, but have lower T3 levels (Adams et al., 2000). In the present study, there was no significant effect of the lower doses of PCB 77 on T4 or T3 levels, while T4 levels increased with the highest dose employed. Based on the findings that PCB 77 constitutes only 0.31% of the Aroclor 1254 mixture used in the present study and less than 0.15% bioaccumulates in croaker liver tissues, it is highly unlikely that this PCB congener contributes significantly
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to the effects of Aroclor 1254 on TH levels observed in this species. A recent study by Khan and Thomas (2006), along with data presented here, suggest that the reproductive neuroendocrine impairment seen previously in this species after Aroclor 1254 exposure may not be due to the deleterious effects of PCB exposure on T4 or T3. The planar PCB 77, but not orthosubstituted PCB 47 or PCB 153, inhibits hypothalamic TPH activity and reproductive development, suggesting that planar PCBs in the Aroclor mixture are most likely responsible for the reproductive neuroendocrine disruption in croaker. In contrast, PCB 77 had no effect on circulating levels of the biologically active T3 and reductions in circulating TH levels following Aroclor 1254 exposure appear to be primarily influenced by PCB 153 and other similar non-planar congeners. Circulating TH levels can be reduced by increasing TH excretion, which is accomplished by increasing the activity of glucuronidation enzymes and by displacing THs from transport proteins, such as the thyroid hormone-binding protein transthyretin. Due to their structural similarity, some hydroxylated PCB congeners are able to serve as binding ligands for T4binding proteins in mammals (Darnerud et al., 1996). PCBs can also alter plasma TH levels by directly affecting TH synthesis and/or inhibiting the proteolysis of thyroglobulin, a T4 precursor (Van Birgelen et al., 1995). The contribution of one or more of these mechanisms to the reduced TH levels after PCB exposures needs further evaluation. The consequences of reduced TH levels in PCB-exposed females on egg quality and larval health also warrant investigation because THs are known to impact early larval development in a variety of fish species (Brown et al., 1989; Ayson and Lam, 1993; Inui et al., 1994; Brown, 1997). In fact, exposure of Atlantic croaker females to Aroclor 1254, at a dose similar to that used in the present study has been shown to impair growth and survival skills of their offspring (McCarthy et al., 2003). Since a variety of physiological functions are influenced by TH in fish (Eales and Brown, 1993; Rousseau et al., 2002; Kulczykpwska et al., 2004; Edeline et al., 2005; Swapna et al., 2006), it is reasonable to assume that the impaired thyroidal status of PCB-exposed croaker may have some physiological consequences for adult animals as well. Uptake from food is thought to be the major pathway by which hydrophobic organic chemicals accumulate in fish (Connolly and Pederson, 1988; Gobas et al., 1988). After exposure, PCBs are cleared from the blood stream within minutes and quickly accumulate in the liver where they are metabolized by microsomal cytochrome P-450 to less lipophilic metabolites that can undergo conjugation and excretion (Van den Berg et al., 1998). The increase in HSIs of croaker exposed to the Aroclor 1254 mixture is a common response usually associated with the induction or stimulation of hepatic enzyme activities (Slooff et al., 1983). Jordan and Feeley (1999) have reported that PCBs 110, 118, 138, and 153 are the predominant PCB congeners in chinook salmon (Oncorhynchus tshawytscha) collected from the Great Lakes. Similarly, PCB 153 and PCB 138 have been shown to be abundant congeners in fish from the Drome River (Mazet et al.,
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2005). In the present study, similar congeners were found to be abundant in Atlantic croaker exposed to the highest concentration of Aroclor 1254, thereby demonstrating the environmental relevance of the congeners employed in the present study. Aroclor mixtures are defined by the weight percentage of chlorine atoms, represented by the last two numbers in the name. Aroclor 1254 should therefore be 54% chlorine by weight. However, Burgin et al. (2001) recently reported that the congener composition of Aroclor mixtures could vary significantly from lot to lot and found different effects on T4 levels in rats after using two different lots of Aroclor 1254. Similarly, the percent of individual PCB congeners in the Aroclor 1254 mixture used in the present study differs greatly from those of several other studies reported in the literature (Kodavanti et al., 1998). The inconsistent composition of Aroclor mixtures further complicates the risk assessment for these chemicals. In conclusion, this study demonstrates the ability of a PCB mixture, Aroclor 1254, and selected congeners to alter thyroidal status in Atlantic croaker by changing circulating levels of T4 and/or T3. The results suggest that di-orthosubstituted PCB 153 and similar non-planar congeners in the Aroclor 1254 mixture contribute, at least partially, to the deleterious effects of the PCB mixture on TH levels observed in the present study. Acknowledgements This work was supported by a Public Health Service Grant (ES07672). The authors would like to thank Dr. Hakan Berg and Ms. Susan Lawson for their help with the in vivo exposure experiments. This article is contribution number 972 of The University of Texas Marine Science Institute. References Adams, B.A., Cyr, D.G., Eales, J.G., 2000. Thyroid hormone deiodination in tissues of the American plaice, Hippoglosssoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126. Comp. Biochem. Physiol. C 127, 367–378. Ayson, F.G., Lam, T.J., 1993. Thyroxine injection of female rabbitfish (Siganus guttatus) broodstock: changes in thyroid hormone levels in plasma, yolk, and yolk–sac larvae, and its effect on larval growth and survival. Aquaculture 109, 83–93. Besselink, H.T., Van Beusekom, S., Roex, R., Vethaak, A.D., Koeman, J.H., Brouwer, A., 1996. Low hepatic 7-ethoxyresorufin-O-dethylase (EROD) activity and minor alterations in retinoid and thyroid hormone levels in flounder (Platichthys flesus) exposed to the polychlorinated biphenyl mixture Clophen A50. Environ. Pollut. 92, 267–274. Brouwer, A., Longnecker, M.P., Birnbaum, L.S., Cogliano, J., Kostyniak, P., Moore, J., Schantz, S., Winneke, G., 1999. Characterization of potential endocrine-related health effects at low dose levels of exposure to PCBs. Environ. Health Perspect. 107, 639–649. Brown, D.D., 1997. The role of the thyroid hormone in zebrafish and axolotl development. Proc. Natl. Acad. Sci. U. S. A. 94, 13011–13016. Brown, C.L., Cochran, M., Doroshov, S., Bern, H.A., 1989. Enhanced survival in striped bass fingerlings after maternal triiodothyronine treatment. Fish Physiol. Biochem. 7, 295–299. Brown, S.B., Evans, R.E., Vandenbyllardt, L., Finnson, K.W., Palace, V.P., Kane, A.S., Yarechewski, A.Y., Muir, D.C.G., 2004. Altered thyroid status in lake trout (Salvelinus namaycush) exposed to co-planar 3,3′,3,4′,5pentachlorobiphenyl. Aquat. Toxicol. 67, 75–85.
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Burgin, D.E., Diliberto, J.J., Derr-Yellin, E.C., Kannan, N., Kodavanti, P.R.S., Birnbaum, L.S., 2001. Differential effects of two lots of Aroclor 1254 on enzyme induction, thyroid hormones, and oxidative stress. Environ. Health Perspect. 109, 1163–1168. Connolly, J.P., Pederson, C.J., 1988. Accumulation of hydrophobic organic chemicals in fish. Environ. Sci. Technol. 22, 99–103. Darnerud, P.O., Morse, D., Klasson-Wehler, E., Brouwer, A., 1996. Binding of a 3,3′,4,4′-tetrachlorobiphenyl (CB-77) metabolite to fetal transthyretin and effects on fetal thyroid hormone levels in mice. Toxicology 106, 105–114. de Jesus, E.G., Toledo, J.D., Simpas, M.S., 1998. Thyroid hormones promote early metamorphosis in grouper (Epinephelus coioides) larvae. Gen. Comp. Endocrinol. 112, 10–16. Eales, J.G., Brown, S.B., 1993. Measurement and regulation of thyroidal status in teleost fish. Rev. Fish Biol. 3, 299–347. Edeline, E., Bolliet, V., Dufour, S., Elie, P., 2005. Endocrine control of Anguilla anguilla glass eel dispersal: effect of thyroid hormones on locomotor activity and rheotactic behavior. Horm. Behav. 48, 53–63. Folmar, L.C., Dickhoff, W.W., Zaugg, W.S., Hodgins, H.O., 1982. The effects of Aroclor 1254 and No. 2 fuel oil on smoltification and sea-water adaptation of coho salmon (Oncorhynchus kisutch). Aquat. Toxicol. 2, 291–299. Gobas, F.A.P.C., Muir, D.C.G., Mackay, D., 1988. Dynamics of dietary bioaccumulation and faecal elimination of hydrophobic organic chemicals in fish. Chemosphere 17, 943–962. Greenblatt, M., Brown, C.L., Lee, M., Dauder, S., Bern, H.A., 1989. Changes in thyroid hormone levels in eggs and larvae and in iodide uptake by eggs of coho and chinook salmon, Oncorhynchus kisutsch and O. tschawytscha. Fish Physiol. Biochem. 6, 261–278. Inui, Y., Miwa, S., Hirano, T., 1989. Effects of bovine TSH on the tissue thyroxine level and metamorphosis in prometamorphic flounder larvae. Gen. Comp. Endocrinol. 74, 406–410. Inui, Y., Tagawa, M., Miwa, S., Yamano, K., Hirano, T., 1994. Hormonal control of flounder metamorphosis. In: Davey, K.G., Peter, R.E., Tobe, S.S. (Eds.), Perspectives in Comparative Endocrinology. National Research Council Canada, pp. 408–411. Jordan, S.A., Feeley, M.M., 1999. PCB congener patterns in rats consuming diets containing Great Lakes salmon: analysis of fish, diets, and adipose tissue. Environ. Res. 80, S207–S212. Kannan, K., Nakata, H., Stafford, R., Masson, G.R., Tanabe, S., Giesy, J.P., 1998. Bioaccumulation and toxic potential of extremely hydrophobic polychlorinated biphenyl congeners in biota collected from a superfund site contaminated with Aroclor 1268. Environ. Sci. Technol. 32, 1214–1221. Khan, I.A., Thomas, P., 1997. Aroclor 1254-induced alterations in hypothalamic monoamine metabolism in the Atlantic croaker (Micropogonias undulatus): correlation with pituitary gonadotropin release. Neurotoxicology 18, 553–560. Khan, I.A., Thomas, P., 2001. Disruption of neuroendocrine control of luteinizing hormone secretion by Aroclor 1254 involves inhibition of hypothalamic tryptophan hydroxylase activity. Biol. Reprod. 64, 955–964. Khan, I.A., Thomas, P., 2004. Vitamin E reduces Aroclor 1254-induced impairment of reproductive neuroendocrine function in Atlantic croaker. Mar. Environ. Res. 58, 333–336. Khan, I.A., Thomas, P., 2006. PCB congener specific distribution of reproductive neuroendocrine function in Atlantic croaker. Mar. Environ. Res. 62, 25–28. Khan, M.A., Lichtensteiger, C.A., Faroon, O., Mumtaz, M., Schaeffer, D.J., Hansen, L.G., 2002. The hypothalamo–pituitary–thyroid (HPT) axis: a target of nonpersistent ortho-substituted PCB congeners. Toxicol. Sci. 65, 52–61. Kodavanti, P.R.S., Ward, T.R., Derr-Yellin, E.C., Mundy, W.R., Casey, A.C., Bush, B., Tilson, H.A., 1998. Conger-specific distribution of polychlorinated biphenyls in brain regions, blood, liver, and fat of adult rats following repeated exposure to Aroclor 1254. Toxicol. Appl. Pharmacol. 153, 199–210. Kulczykpwska, E., Sokolowska, E., Takvam, B., Stefansson, S., Ebbesson, L., 2004. Influence of exogenous thyroxin on plasma melatonin in juvenile Atlantic salmon (Salmo salar). Comp. Biochem. Physiol. B 137, 43–47. Leatherland, J.F., Sonstegard, R.A., 1978. Lowering of serum thyroxine and triiodothyronine levels in yearling coho salmon (Oncorhynchus kisutch), by dietary mirex and PCBs. J. Fish. Res. Board Can. 35, 1285–1289.
Leatherland, J.F., Sonstegard, R.A., 1980. Effects of dietary Mirex and PCBs in combination with food deprivation and testosterone administration on thyroid activity and bioaccumulation of organochlorines in rainbow trout Salmon gairdneri Richardson. J. Fish Dis. 3, 115–124. Leiner, K.A., Han, G.S., MacKenzie, D.S., 2000. The effects of photoperiod and feeding on the diurnal rhythm of circulating thyroid hormones in the red drum, Sciaenops ocellatus. Gen. Comp. Endocrinol. 120, 88–98. Maier, W.E., Kodavanti, P.R., Harry, G.J., Tilson, H.A., 1994. Sensitivity of adenosine triphosphatases in different brain regions to polychlorinated biphenyl congeners. J. Appl. Toxicol. 14, 225–229. Mazet, A., Keck, G., Berny, P., 2005. Concentrations of PCBs, organochlorine pesticides and heavy metals (lead, cadmium, and copper) in fish from the Drome river: potential effects on otters (Lutra lutra). Chemosphere 61, 810–816. McCarthy, I.D., Fuiman, L.A., Alvarez, M.C., 2003. Aroclor 1254 affects growth and survival skills of Atlantic croaker Micropogonias undulatus larvae. Mar. Ecol., Prog. Ser. 252, 295–301. Miwa, S., Inui, Y., 1987. Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceous). Gen. Comp. Endocrinol. 67, 356–363. Ness, D.K., Schantz, S.L., Moshtaghian, J., Hansen, L.G., 1993. Effects of perinatal exposure to specific PCB congeners on thyroid hormone concentrations and thyroid histology in the rat. Toxicol. Lett. 68, 311–323. Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Björnsson, B.Th., Einarsdottir, I.E., Canario, A.V.M., Sweeney, G.E., 2001. Thyroidal hormones in growth and development of fish. Comp. Biochem. Physiol. C 130, 447–459. Reddy, P.K., Lam, T.J., 1992. Effects of thyroid hormones on morphogenesis and growth of larvae and fry of telescopic-eye black goldfish, Carassius auratus. Aquaculture 107, 383–394. Rousseau, K., Le Belle, N., Sbaihi, M., Marchelidon, J., Schmitz, M., Dufour, S., 2002. Evidence for a negative feedback in the control of eel growth hormone by thyroid hormones. J. Endocrinol. 175, 605–613. Safe, S., 1994. Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit. Rev. Toxicol. 24, 87–149. Schantz, S.L., Levin, E.D., Bowman, R.E., 1991. Long-term neurobehavioral effects of perinatal polychlorinated biphenyl (PCB) exposure in monkeys. Environ. Toxicol. Chem. 10, 747–756. Shiao, J.-C., Hwang, P.-P., 2006. Thyroid hormones are necessary for the metamorphosis of tarpon Megalop cyprinoides leptocephali. J. Exp. Mar. Biol. Ecol. 331, 121–132. Slooff, W., Van Kreijl, C.F., Baars, A.J., 1983. Relative liver weights and xenobiotic-metabolizing enzymes of fish from polluted surface waters in the Netherlands. Aquat. Toxicol. 4, 1–14. Swapna, I., Rajasekhar, M., Supriya, A., Raghuveer, K., Srenivasulu, G., Rasheeda, M.K., Majumdar, K.C., Kagawa, H., Tanaka, H., Dutta-Gupta, A., Senthilkumaran, B., 2006. Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in air-breathing catfish, Clarias gariepinus. Comp. Biochem. Physiol. A 144, 1–10. Tata, J.R., 2006. Amphibian metamorphosis as a model for the developmental actions of thyroid hormones. Mol. Cell. Endocrinol. 246, 10–20. Thomas, P., Khan, I.A., 2005. Disruption of nongenomic steroid actions on gametes and serotonergic pathways controlling reproductive neuroendocrine function by environmental chemicals. In: Naz, R.K. (Ed.), Endocrine Disruptors: Effects on Male and Female Reproductive Systems. CRC Press, Boca Raton, FL, pp. 3–45. Van Birgelen, A.P.J.M., Van der Kolk, J., Poiger, H., Van den Berg, M., Brouwer, A., 1992. Interactive effects of 2,2′,4,4′,5,5′-hexachlorobiphenyl and 2,3,7,8-tetra-chlorodibenzo-P-dioxin on thyroid hormone, vitamin A, and vitamin K metabolism in the rat. Chemosphere 25, 1239–1244. Van Birgelen, A.P.J.M., Smit, E.A., Kampen, I.M., Groenveld, C.N., Fase, K.M., Van der Kolk, J., Poiger, H., Van den Berg, M., Koeman, J.H., Brouwer, A., 1995. Subchronic effects of 2,3,7,8-TCDD or PCBs on thyroid hormone metabolism: use in risk assessment. Eur. J. Pharmacol. 293, 77–85. Van den Berg, M., Birnbaum, L., Bosveld, A.T.C., Brunstrom, B., Cook, P., Feeley, M., Giesy, J.P., Hanberg, A., Hasegawa, R., Kennedy, S.W., Kubiak, T., Larsen, J.C., Van Leeuween, F.X., Leim, A.K., Nolt, C., Peterson, R.E.,
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Thyroid hormone status of Atlantic croaker exposed to Aroclor 1254 and selected PCB congeners Kimberly D. LeRoy, Peter Thomas, Izhar A. Khan ⁎ The University of Texas at Austin, Marine Science Institute, 750 Channel View Drive, Port Aransas, Texas 78373, USA Received 25 May 2006; received in revised form 25 September 2006; accepted 25 September 2006 Available online 6 October 2006
Abstract Atlantic croaker (Micropogonias undulatus) were exposed to the polychlorinated biphenyl (PCB) mixture (Aroclor 1254) or one of three individual congeners (planar PCB 77 or ortho-substituted PCB 47 and PCB 153) in the diet for 30 days to investigate the effects of PCBs on circulating thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) decreased plasma T3 levels consistently, but the effects on T4 levels were inconsistent from year to year. Exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) significantly lowered both T4 and T3, while PCB 47 at the same doses had no effect on thyroid hormone levels. The lower doses of PCB 77 (0.004, 0.01 and 0.02 mg/kg body mass/day) had no effect on T4 or T3, whereas the highest dose (0.1 mg/kg body mass/day) increased T4 levels significantly. The results of the present study demonstrate that exposure to PCBs at environmentally realistic concentrations can have profound effects on the thyroid status of Atlantic croaker. The ortho-substituted PCB 153 appears to contribute at least partially to the deleterious effects of Aroclor 1254 on thyroid status, whereas the planar PCB 77 at concentrations present in the mixture is unlikely to alter thyroid hormone levels. © 2006 Elsevier Inc. All rights reserved. Keywords: Atlantic croaker; Aroclor 1254; PCB congeners; Polychlorinated biphenyls; Thyroid hormones; Fish
1. Introduction Chemical pollutants present in the aquatic environment can disrupt vital physiological processes in fish, such as growth, reproduction, osmoregulation, and thyroid and immune functions (Folmar et al., 1982; McCarthy et al., 2003; Thomas and Khan, 2005). Recent attention has focused on the ability of these synthetic chemicals to interfere with the normal function of the endocrine system. Polychlorinated biphenyls (PCBs) are some of the most widely studied endocrine disrupting chemicals (EDCs) (Van den Berg et al., 1998). These persistent organic pollutants can be found in aquatic environments across the world (Slooff et al., 1983; Van der Oost et al., 1996; Kannan et al., 1998; Mazet et al., 2005). PCBs are able to bioaccumulate in the lipid-rich tissues of many aquatic organisms and cause numerous adverse health effects (Safe, 1994; Van den Berg et al., 1998).
⁎ Corresponding author. Tel.: +1 361 749 6810; fax: +1 361 749 6749. E-mail address: [email protected] (I.A. Khan). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.09.005
Based on their chemical structure, PCB congeners can be divided into two main groups, the planar (non-orthosubstituted) and non-planar (ortho-substituted). Considered the most toxic, planar PCBs (PCB 77, 126, 169) are laterally substituted with no Cl− substitution on ortho-positions. The absence of Cl− atoms from the ortho-position on the rings allows a smaller hydrogen atom to become incorporated into the compound, creating a planar structure that is similar to dioxin (TCDD) and is more resistant to degradation (Walker and Peterson, 1991; Maier et al., 1994; Van den Berg et al., 1998). Planar PCB congeners and their metabolites share a common mechanism of action in vertebrates and are able to bind a cytosolic protein, the aryl hydrocarbon receptor (AhR), induce hepatic enzymes (CYP1A), and cause a variety of toxic responses (Burgin et al., 2001). Reported effects of exposure to planar PCBs include weight loss, enzyme induction, teratogenicity, carcinogenicity, and endocrine disruption (Safe, 1994). The di- to tetra-ortho-substituted PCB congeners have two or more chlorine atoms on the ortho-positions and are not planar due to steric hindrance. The ortho-substituted congeners are most abundant in Aroclor mixtures and can act via different
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mechanisms and elicit a different pattern of toxicity (Kodavanti et al., 1998; Brouwer et al., 1999; Khan et al., 2002). Exposure to these congeners can induce CYP2B and 3A enzymes and elicit neurotoxicity, carcinogenicity, and endocrine disruption (Zoeller et al., 2000; Khan et al., 2002). Thyroid hormones (TH) are known to play important roles in the early development and metamorphosis in different vertebrate groups (reviewed in Power et al., 2001). In particular, there is considerable evidence for thyroidal control of amphibian metamorphosis (reviewed in Tata, 2006). In Japanese flounder, exogenous administration of TH or elevation of endogenous T4 levels by thyroid stimulating hormone induces precocious metamorphosis by increasing the rate of transformation (Miwa and Inui, 1987; Inui et al., 1989). The relevance of TH to metamorphosis has also been demonstrated in fishes exhibiting a relatively less dramatic transformation, such as conger eel (Conger myriaster), telescopic-eye black goldfish (Carassius auratus), zebrafish (Danio rerio) grouper (Epinephelus coioides) and tarpon (Megalops cyprinodes) (Yamano et al., 1991; Reddy and Lam, 1992; Brown, 1997; de Jesus et al., 1998; Shiao and Hwang, 2006). Moreover, TH treatments improve larval survival in several fish species (Brown et al., 1989; Ayson and Lam, 1993; Inui et al., 1994). Thyroid hormones also influence a variety of other functions in fish including growth, smoltification, osmoregulation, reproduction and locomotor activity (Eales and Brown, 1993; Rousseau et al., 2002; Kulczykpwska et al., 2004; Edeline et al., 2005; Swapna et al., 2006). Therefore, the ability of PCBs to disrupt the thyroid system in fish has the potential to influence normal physiology of the adults and/or long term survival of their offspring. Previous studies in Atlantic croaker (Micropogonias undulatus) using Aroclor 1254 have established this species as an excellent vertebrate model for investigations into the mechanisms of PCB neuroendocrine toxicity (Khan and Thomas, 1997, 2001, 2004; Thomas and Khan, 2005). Exposure of croaker to Aroclor 1254 (1 mg/kg body mass/day for 30 days) during the early-recrudescence phase of the gonadal cycle disrupts gonadotropin releasing hormone (GnRH) neuroendocrine function resulting in the impairment of LH secretion and gonadal growth (Khan and Thomas, 2001). This neuroendocrine disruption is caused by inhibition of hypothalamic tryptophan hydroxylase, the rate limiting enzyme in 5-hydroxytryptamine (5-HT) synthesis. Interestingly, the planar PCB 77, but not the orthosubstituted PCB 47 or PCB 153, impairs the hypothalamic 5-HT system in croaker (Khan and Thomas, 2006). Considering the ability of thyroid hormones to influence the development of the 5-HT system in mammals (Schantz et al., 1991), it is worthwhile to determine the effects of the PCB mixture and individual PCB congeners on the thyroidal status of Atlantic croaker. The reported effects of PCB exposure on thyroid function in fish are highly variable. An earlier study in yearling coho salmon (Oncorhynchus kisutch) exposed to a mixture of PCBs in the diet (1:4 mixture of Aroclor 1242 and Aroclor 1254) reported depressed triiodothyronine (T3) levels and slower growth with no effect on T4 levels (Leatherland and Sonstegard, 1978, 1980). In contrast, exposure of coho salmon to a combination of Aroclor 1254 and fuel oil increased levels of
circulating T3 and delayed the plasma T4 surge commonly associated with smoltification (Folmar et al., 1982). European flounder (Platichthys flesus) show increased levels of both T4 and T3 following exposure to the PCB mixture Clophen A50 (Besselink et al., 1996). These variable effects on TH levels reported in many laboratory studies may be due to the variable composition of PCB mixtures in biological systems and the different effects of predominant planar and ortho-substituted congeners. Studies in experimental animals suggest that the selective metabolism and complex behavior of each individual PCB congener or metabolite is important when studying these compounds (Safe, 1994; Van den Berg et al., 1998; Khan et al., 2002). However, only a few studies have examined the effects of individual PCB congeners on circulating TH levels in fish (Adams et al., 2000; Brown et al., 2004). In addition, previous studies in fish have not examined the effects of PCB mixtures and individual PCB congeners in the same species. The present study examined the effects of dietary exposure to the Aroclor 1254 mixture and three individual PCB congeners on circulating TH levels in Atlantic croaker in order to better understand the contributions of the selected planar and nonplanar congeners to the overall effects of the PCB mixture on the thyroid status in fish. A planar (PCB 77) and two orthosubstituted congeners with different degrees of chlorination (tetra-chlorinated PCB 47 and hexa-chlorinated PCB 153) were selected for the present study. Moreover, analyses of Aroclor 1254 and the liver samples of croaker exposed to the mixture were performed to determine individual congener profiles and their bioaccumulation in this species. 2. Materials and methods 2.1. Chemicals and reagents Aroclor 1254 (Lot # K040) was purchased from Foxboro Company (North Haven, CT). PCB 47 (2,2′,4,4′-tetrachlorobiphenyl), PCB 77 (3,3′,4,4′-tetrachlorobiphenyl), and PCB 153 (2,2′,4,4′,5,5′-hexaclorobiphenyl) were purchased from Chemservice (West Chester, PA). Total T4 and T3 ELISA kits were purchased from DSL Laboratories, Inc. (Webster, TX). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 2.2. Experimental animals Young-of-the-year Atlantic croaker were captured by shrimp trawls from Redfish Bay, Texas, during the early fall and maintained in large, recirculating, seawater tanks (salinity 30– 35 ppt) equipped with biofilters. Fish were maintained in a simulated natural fall photoperiod (light/dark cycle, 11:13 h) and temperature conditions (22 ± 1 °C), and fed a mixed diet of chopped shrimp and commercial trout pellets (95:5 w/w). Either male or female fish were used for the experiments in order to avoid variable responses, if any, due to the sex of the individuals. Fish were handled according to the National Institute of Health Guidelines for handling and care of experimental animals. The animal utilization protocol was approved by the
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Institutional Animal Care and Use Committee of the University of Texas at Austin.
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heparinized syringes and kept on ice. Plasma samples were obtained after centrifugation at 3000 ×g for 6 min and were stored at − 80 °C until assayed for T4 and T3.
2.3. Experimental protocols 2.4. Experiment 1: effects of Aroclor 1254 on TH levels PCBs were dissolved in a small volume of ethanol, which was then mixed with vegetable oil and the ethanol subsequently evaporated at 40 °C. The PCBs in vegetable oil were then thoroughly mixed into the fish diet. Fish were fed the prepared food laced with sub-lethal levels of PCBs or mixed with vegetable oil alone at the rate of 3% of their body weight per day for 30 days, between 08:00 and 10:00 h. In the fall, young-of-the-year fish (40–60 g body weight each) were randomly sorted into groups of 25 and maintained in 1500-l recirculating seawater tanks equipped with biofilters. They were allowed to acclimate for a minimum of 1 month prior to initiating PCB feeding experiments. All fish used in fall experiments were at early stages of gametogenesis at the beginning of each experiment and the control fish were fully mature by the end of the experiment, as evidenced by the presence of readily expressible milt in males and late vitellogenic oocytes in females. Due to the limited number of experiments that could be carried out within the short reproductive period of croaker (October–November), some fish were maintained on a shortened seasonal photoperiod–temperature cycle in order to simulate their natural fall breeding season in the spring. In our laboratory, croaker can be moved through a shortened seasonal photoperiod–temperature cycle and can be induced to become reproductively mature off-season (unpublished observation). Using this established protocol, fish were cycled through a fourand-a-half month shortened seasonal photoperiod–temperature cycle corresponding to winter, summer and fall conditions. Fish used in spring experiments were 6 months older and gonadally mature when the PCB exposures were initiated in April–May, unlike those in the fall experiments in which PCB exposure occurred during the gonadal growth phase of the reproductive cycle. At the end of the experiments, all fish were sampled between 0900 and 1100 h to avoid possible diurnal fluctuations in hormone levels (e.g., Leiner et al., 2000) from influencing the results. Fish from each tank were divided into two subgroups at the time of sampling. One subgroup of 8–10 fish was sampled to collect blood for TH measurements, while the remaining fish were used to collect brain samples without bleeding in order to evaluate neuroendocrine parameters. Blood sampling time for each tank was b15 min and the sampling time for each experiment (3–4 tanks) was b1 h. Body, liver, and gonad weights were recorded in order to determine hepato-somatic indices (HSIs; liver weight expressed as percent body weight) and gonado-somatic indices (GSIs; gonad weight expressed as percent body weight). Livers were carefully removed, collected on dry ice, and stored at − 80 °C for subsequent PCB analysis. Studies have shown that measuring total plasma T4 and T3 levels provides relative measurement of the systemic availability of thyroid hormones in fish (Eales and Brown, 1993). For all experiments, blood was collected from the caudal vessels using
In the fall of 2003 and 2005, 25 croaker with testes at early stages of spermatogenesis were fed a control diet of chopped shrimp and commercial trout pellets, while two other groups of fish were exposed to Aroclor 1254 mixed in the diet (0.2 and 1.0 mg/kg body mass/day) for 30 days. In the spring of 2005, gonadally mature female croaker cycled through compressed photoperiod–temperature regimes were exposed to Aroclor 1254. One group of 25 fish was fed a control diet each morning, while two other groups were fed Aroclor 1254 mixed in the diet (0.2 and 1.0 mg/kg body mass/ day) for 30 days. This experiment was repeated in the fall of 2005 with gonadally recrudescing female croaker. 2.5. Experiment 2: effects of selected PCB congeners on TH levels In the fall of 2003 and 2004, one tank of 25 male croaker with testes at early stages of spermatogenesis was fed a control diet, while two other groups of fish were exposed to the orthoPCB 153 (0.1 and 1.0 mg/kg body mass/day) in the diet for 30 days. Two similar experiments were carried out in the fall of 2003 and 2004 with PCB 47 using the same doses and duration of exposure as the PCB 153 experiments. In the fall of 2004, one tank of 25 male fish was fed a control diet, while two other groups were exposed to the planar PCB 77 (0.1 and 1.0 mg/kg body mass/day). Due to high mortality, the experiment was terminated early and dosage levels for future experiments using PCB 77 were reduced more than 10 fold. In the spring of 2005, one group of 25 mature males was fed a control diet, while three other groups were exposed to sub-lethal doses of the planar PCB 77 mixed in the diet (0.004, 0.02 and 0.1 mg/kg body mass/day) for 30 days. Another experiment with PCB 77 (0.01 and 0.1 mg/kg body mass/day) exposure in males was conducted in the fall of 2005 to confirm the results. 2.6. Thyroid hormone assays Thyroid hormones were extracted from plasma samples using a mixture of chloroform and methanol, following the method described by Greenblatt et al. (1989) with minor modifications. Preliminary experiments showed that the extraction of both T4 and T3 from plasma was best accomplished using a mixture of chloroform and methanol (data not shown). Extracted samples were stored at − 80 °C and were resuspended in enzyme immunoassay (EIA) buffer in one half the original sample volumes before starting the enzyme-linked immunosorbent assays (ELISAs). Total T4 and total T3 levels were measured in extracted samples with commercially available ELISA kits following the protocols provided with the kits (DSL Laboratories Inc., Webster, TX). Standards and extracted samples were added to the 96-well plates in duplicate.
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Absorbance was measured at 450 nm (Spectra Max 190, Molecular devices Corp., Sunnyvale, CA) immediately following the final step in the ELISA protocol. Concentrations of T4 and T3 were subsequently determined using SOFTmaxPRO 4.0 software. Intra-assay variation ranged from 8.12% to 4.37%, while interassay variation was 10.62% and 8.95% for T4 and T3, respectively. 2.7. Liver PCB analyses Liver samples and Aroclor 1254 standard were analyzed by CRG Marine Laboratories, Inc., Torrance, CA for total PCB concentrations and individual congener profiles. Samples were analyzed by GCMS using EPA method 8270Cm. 2.8. Statistical analyses All statistical tests were conducted using SYSTAT Version 10.2. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's multiple range test for the comparison of group means. Differences between groups were considered significant at a p-value b 0.05.
Fig. 2. Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in female Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01, ⁎⁎ = P b 0.001).
3. Results No mortality was observed during any experiments except in the first trial using the highest dose of PCB 77 (1 mg/kg body mass/day). This trial experiment with PCB 77 was terminated early and TH levels were not examined in this experiment. 3.1. Experiment 1: effects of Aroclor 1254 on TH levels There was no significant difference in body weights of fish from control and Aroclor 1254 treatment groups (data not
Fig. 1. Effects of exposure to Aroclor 1254 (0.2 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01; ⁎⁎ = P b 0.001).
included), suggesting that these doses of the PCB mixture do not adversely affect growth. There was also no significant correlation between gonadal stage and thyroid status when examined by a comparison between the GSI and TH levels in control fish (data not included). Plasma concentrations of T4 and T3 were significantly altered in male fish exposed to both the low and high doses of Aroclor 1254 (Fig. 1). In 2003, the T4 response showed a Ushaped curve, with the low dose producing a 30% decrease in T4, and the high dose producing a 48% increase in T4 levels when compared to controls. When the experiment was repeated in 2005, no statistically significant difference was observed in T4 levels between the control and treatment groups, indicating that the effects of Aroclor 1254 on T4 levels were not consistent from year to year. However, T3 levels were reduced consistently in all groups of male croaker exposed to Aroclor 1254 (Fig. 1). In 2003, the significant decrease in T3 levels was not dosedependent. However, when the experiment was repeated in 2005, T3 levels decreased in a dose-related manner, with a 41%
Fig. 3. Total PCB accumulation in the livers of croaker exposed to the high dose of Aroclor 1254 (1.0 mg/kg body mass/day) showed a positive correlation with the hepato-somatic indices (HSIs) of individual fish.
K.D. LeRoy et al. / Comparative Biochemistry and Physiology, Part C 144 (2006) 263–271 Table 1 Percent of individual PCB congeners detected in Aroclor 1254 and liver samples from Atlantic croaker exposed to the mixture in the diet (1 mg/kg body mass/ day) Congener
IUPAC # Aroclor 1254 Liver (% of total PCBs) (% of total PCBs)
2,2′,5 2,4,4′ 2,4′,5 2′,3,4 3,4,4′ 2,2′,3,5′ 2,2′,4,5′ 2,2′5,5′ 2,3′4,4′ 2,3′,4′,5 2,4,4′,5 3,3′,4,4′ 3,4,4′,5 2,2′,3,4,5′ 2,2′,3,5′,6 2,2′,3′,4,5 2,2′,4,4′,5 2,2′,4,5,5′ 2,3,3′,4,4′ 2,3,3′,4′,6 2,3,4,4′,5 2,3′,4,4′,5 2,3′,4,4′,6 2′,3,4,4′,5 3,3′,4,4′,5 2,2′,3,3′,4,4′/2,3′,4,4′,5,5′ 2,2′,3,4,4′,5′ 2,2′,3,4,5,5′ 2,2′,3,4′,5′,6 2,2′,3,5,5′,6 2,2′,4,4′,5,5′ 2,3,3′,4,4′,5 2,3,3′,4,4′,5′ 2,3,3′,4,4′,6 2,3′,4,4′,5′,6/2,2′3,3′,4,6′ 3,3′,4,4′,5,5′ 2,2′,3,3′,4,4′,5 2,2′,3,3′,4′,5,6 2,2′,3,4,4′,5,5′ 2,2′,3,4,4′,5′,6 2,2′,3,4′,5,5′,6 2,3,3′,4,4′,5,5′ 2,2′,3,3′,4,4′,5,5′ 2,2′,3,3′,4,5,6,6′ 2,2′,3,3′,4,5′,6,6′ 2,2′,3,3′,4,4′,5,5′,6
18 28 31 33 37 44 49 52 66 70 74 77 81 87 95 97 99 101 105 110 114 118 119 123 126 128/167 138 141 149 151 153 156 157 158 168/132 169 170 177 180 183 187 189 194 200 201 206
0.06 0.07 0.04 0.02 0.02 0.82 0.39 1.11 4.06 7.85 2.75 0.31 0.13 3.43 2.62 4.95 5.35 6.72 7.57 9.05 0.56 13.47 0.19 0.38 6.60 0.10 6.60 0.86 2.18 0.27 4.00 1.78 1.13 0.91 1.51 0.21 0.40 0.11 ND 0.04 0.91 0.38 ND 0.09 ND 0.02
0.02 0.06 0.04 0.01 0.00 0.67 0.39 1.13 4.62 5.97 2.26 0.15 0.14 3.71 2.33 5.36 6.04 7.52 9.40 8.78 0.68 16.29 0.23 0.58 0.03 2.29 7.60 0.98 2.42 0.28 4.62 1.51 0.51 1.11 0.87
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total PCB accumulation in the livers of croaker exposed to the high dose of Aroclor 1254 showed a positive correlation with the HSIs of individual fish (Fig. 3), despite high individual variation (20–30% standard error; N = 7). GSIs in fish exposed to Aroclor 1254 were not affected when considering only the fish in which TH levels were measured; however, when considering the whole tank of fish (N = 25), GSIs were significantly lower in Aroclor 1254-exposed males and females (data not shown). Aroclor 1254 and seven liver samples from fish exposed to the high dose of the PCB mixture were analyzed for 46 individual PCB congeners and total PCB levels (Table 1). The predominant congeners found in Aroclor 1254, in the order of decreasing concentration were PCB 118, 110, 70, 105, 101, 126, 138, 99, 97, 66, and 153, which accounted for 75% of the total detectable PCBs. Homolog groups contributing the most to the PCB content in liver samples were (in order of contribution to the total) the penta-, hexa-, tetra-, and heptachlorinated groups. Predominant congeners found in liver tissue, in the order of decreasing concentration were PCB 118, 105, 110, 138, 101, 99, 70, 97, and 153, which accounted for 77% of the total detectable PCBs. Congeners present in Aroclor 1254 but not detected in any liver samples were PCB 37, 169, 200, and 206. In terms of the planar PCBs, congener 77 was present at the highest concentration in livers, followed by PCB 126 and PCB 169. This was not the same pattern found in the Aroclor 1254 however, in which PCB 126 was found at the highest concentration, followed by PCB 77 and PCB 169. Plasma T4 or T3 levels did not show a strong correlation with total PCB accumulation in liver samples of fish exposed to the high dose of Aroclor 1254 as the TH levels were in the normal range of
0.49 0.08 0.55 0.14 0.12 0.02 0.01 ND ND ND
Numbers in bold indicate the ten most predominant congeners. ND = nondetectable. (N = 7).
decrease seen at the highest dose of Aroclor 1254. Similar to males, T3 levels in Aroclor 1254-exposed female croaker decreased significantly in the low and high dose groups (Fig. 2). A dose-related decrease in average T3 levels was seen in both the fall and spring experiments, while average T4 levels were not significantly affected in any treatment group. The HSIs in fish exposed to Aroclor 1254 increased in a dose-related manner: Males, 0.86 ± 0.08, 1.26 ± 0.14 and 1.38 ± 0.21%; Females, 0.93 ± 0.07, 1.28 ± 0.08 and 2.05 ± 0.28%, in the control, low and high dose groups, respectively. In addition,
Fig. 4. Effects of exposure to PCB 153 (0.1 and 1.0 mg/kg body mass/day) on circulating levels of T4 and T3 in Atlantic croaker. Bars represent average TH levels of 6–8 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎ = P b 0.01; ⁎⁎ = P b 0.001).
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individual variability within the treatment group (data not shown). 3.2. Experiment 2: effects of selected PCB congeners on TH levels Both plasma T4 and T3 levels decreased in all groups of male croaker exposed to the ortho-substituted PCB 153 (0.1 and 1.0 mg/kg body mass/day) when compared to control fish, although there was no effect on T3 levels in the fall of 2004 (Fig. 4). In all experiments during the fall of 2004, average levels of both T4 and T3 were only 30% of average levels observed in the previous year and might be responsible for the lack of significant decrease in T3 levels in PCB 153-treated fish in this experiment. The amount of PCB 153 represented 4% of the total PCB congeners in Aroclor 1254 and 4.62% of the total PCBs detected in liver samples from male croaker exposed to the highest dose of the PCB mixture (Table 1). Dietary exposure to the ortho-substituted PCB 47 (0.1 and 1.0 mg/kg body mass/day) for 30 days resulted in no significant alterations in plasma T4 or T3 levels (data not included). Fully mature and gonadally recrudescing male croaker exposed to the highest dose of the planar PCB 77 (0.1 mg/kg body mass/day) had significantly higher levels of plasma T4 than control fish (Fig. 5). In both the spring and fall experiments, average T4 levels more than doubled in fish fed the highest dose of PCB 77. However, there was no significant change in average T3 levels following any exposure to PCB 77. The highest dose of PCB 77 also resulted in decreased appetite towards the end of the exposure period. PCB 77 was 0.31% of the total PCBs in the Aroclor 1254 and 0.15% of the total PCBs detected in liver samples from male croaker exposed to the highest dose of the PCB mixture (Table 1).
Fig. 5. Effects of exposure to PCB 77 on circulating levels of T4 and T3 in Atlantic croaker (A) 0.004, 0.02 and 0.1 mg/kg body mass/day (B), 0.01 and 0.1 mg/kg body mass/day. Bars represent average TH levels of 9 individuals. Data are presented as the mean ± SEM. Asterisks denote values significantly different from the respective control group (⁎⁎ = P b 0.001).
4. Discussion The results presented in this paper demonstrate the effects of an environmentally relevant PCB mixture, along with those of individual ortho-substituted and planar congeners on the thyroid status of an estuarine fish, Atlantic croaker. Interestingly, both Aroclor 1254 and PCB 153 significantly decreased circulating TH levels in croaker, while PCB 77 and PCB 47 failed to decrease TH levels. The results of this study suggest that di-ortho-substituted congeners with higher chlorination such as the hexa-chlorinated PCB 153 may contribute to the depression of TH levels observed in croaker exposed to Aroclor 1254, whereas planar congeners such as PCB 77 present at low concentrations in the PCB mixture are unlikely to alter thyroidal homeostasis in this species. Aroclor 1254 is thought to generally depress both T4 and T3 levels in mammals. The finding that Aroclor 1254 decreases plasma T3 levels in croaker is significant because T3 is considered to be the biologically active thyroid hormone (Eales and Brown, 1993). Unlike its effects on T3 levels, the effects of Aroclor 1254 on T4 levels in this study were inconsistent, with no significant effect observed in subsequent experiments with males or females. Similar to the results in croaker, Aroclor 1254 decreases circulating T3 levels without significantly altering T4 levels in salmonids (Leatherland and Sonstegard, 1978). Overall, the average TH levels in control fish were quite variable from year to year, which probably reflects differences in the underlying TH status of the experimental fish that may account for the variable degree of responses to various PCB exposures. Interestingly, one of the most prevalent congeners found in the environment, di-ortho-PCB 153 (Jordan and Feeley, 1999) was also the most effective in reducing circulating TH levels in croaker. In view of the fact that PCB 153 is one of the major congeners found in Aroclor 1254 (4% of the total), and that it bioaccumulates in the livers of croaker (4.6% of total PCBs accumulated), PCB 153 appears to contribute at least partially to the observed decrease in TH levels in croaker exposed to the PCB mixture. In contrast to the hexa-chlorinated PCB 153, the tetra-chlorinated di-ortho-substituted PCB 47 had no significant effect on TH levels in the present study. This suggests that the degree of chlorination may be an important factor contributing to the effects of individual PCB congeners on TH levels. The PCB 153-induced decrease in T4 levels observed in croaker is consistent with similar findings in mammals (Van Birgelen et al., 1992; Ness et al., 1993). PCB 77, considered one of the most toxic congeners, is thought to generally depress circulating levels of T4 in mammals (Brouwer et al., 1999). However, American plaice injected with PCB 77 show no effect on plasma T4, but have lower T3 levels (Adams et al., 2000). In the present study, there was no significant effect of the lower doses of PCB 77 on T4 or T3 levels, while T4 levels increased with the highest dose employed. Based on the findings that PCB 77 constitutes only 0.31% of the Aroclor 1254 mixture used in the present study and less than 0.15% bioaccumulates in croaker liver tissues, it is highly unlikely that this PCB congener contributes significantly
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to the effects of Aroclor 1254 on TH levels observed in this species. A recent study by Khan and Thomas (2006), along with data presented here, suggest that the reproductive neuroendocrine impairment seen previously in this species after Aroclor 1254 exposure may not be due to the deleterious effects of PCB exposure on T4 or T3. The planar PCB 77, but not orthosubstituted PCB 47 or PCB 153, inhibits hypothalamic TPH activity and reproductive development, suggesting that planar PCBs in the Aroclor mixture are most likely responsible for the reproductive neuroendocrine disruption in croaker. In contrast, PCB 77 had no effect on circulating levels of the biologically active T3 and reductions in circulating TH levels following Aroclor 1254 exposure appear to be primarily influenced by PCB 153 and other similar non-planar congeners. Circulating TH levels can be reduced by increasing TH excretion, which is accomplished by increasing the activity of glucuronidation enzymes and by displacing THs from transport proteins, such as the thyroid hormone-binding protein transthyretin. Due to their structural similarity, some hydroxylated PCB congeners are able to serve as binding ligands for T4binding proteins in mammals (Darnerud et al., 1996). PCBs can also alter plasma TH levels by directly affecting TH synthesis and/or inhibiting the proteolysis of thyroglobulin, a T4 precursor (Van Birgelen et al., 1995). The contribution of one or more of these mechanisms to the reduced TH levels after PCB exposures needs further evaluation. The consequences of reduced TH levels in PCB-exposed females on egg quality and larval health also warrant investigation because THs are known to impact early larval development in a variety of fish species (Brown et al., 1989; Ayson and Lam, 1993; Inui et al., 1994; Brown, 1997). In fact, exposure of Atlantic croaker females to Aroclor 1254, at a dose similar to that used in the present study has been shown to impair growth and survival skills of their offspring (McCarthy et al., 2003). Since a variety of physiological functions are influenced by TH in fish (Eales and Brown, 1993; Rousseau et al., 2002; Kulczykpwska et al., 2004; Edeline et al., 2005; Swapna et al., 2006), it is reasonable to assume that the impaired thyroidal status of PCB-exposed croaker may have some physiological consequences for adult animals as well. Uptake from food is thought to be the major pathway by which hydrophobic organic chemicals accumulate in fish (Connolly and Pederson, 1988; Gobas et al., 1988). After exposure, PCBs are cleared from the blood stream within minutes and quickly accumulate in the liver where they are metabolized by microsomal cytochrome P-450 to less lipophilic metabolites that can undergo conjugation and excretion (Van den Berg et al., 1998). The increase in HSIs of croaker exposed to the Aroclor 1254 mixture is a common response usually associated with the induction or stimulation of hepatic enzyme activities (Slooff et al., 1983). Jordan and Feeley (1999) have reported that PCBs 110, 118, 138, and 153 are the predominant PCB congeners in chinook salmon (Oncorhynchus tshawytscha) collected from the Great Lakes. Similarly, PCB 153 and PCB 138 have been shown to be abundant congeners in fish from the Drome River (Mazet et al.,
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2005). In the present study, similar congeners were found to be abundant in Atlantic croaker exposed to the highest concentration of Aroclor 1254, thereby demonstrating the environmental relevance of the congeners employed in the present study. Aroclor mixtures are defined by the weight percentage of chlorine atoms, represented by the last two numbers in the name. Aroclor 1254 should therefore be 54% chlorine by weight. However, Burgin et al. (2001) recently reported that the congener composition of Aroclor mixtures could vary significantly from lot to lot and found different effects on T4 levels in rats after using two different lots of Aroclor 1254. Similarly, the percent of individual PCB congeners in the Aroclor 1254 mixture used in the present study differs greatly from those of several other studies reported in the literature (Kodavanti et al., 1998). The inconsistent composition of Aroclor mixtures further complicates the risk assessment for these chemicals. In conclusion, this study demonstrates the ability of a PCB mixture, Aroclor 1254, and selected congeners to alter thyroidal status in Atlantic croaker by changing circulating levels of T4 and/or T3. The results suggest that di-orthosubstituted PCB 153 and similar non-planar congeners in the Aroclor 1254 mixture contribute, at least partially, to the deleterious effects of the PCB mixture on TH levels observed in the present study. Acknowledgements This work was supported by a Public Health Service Grant (ES07672). The authors would like to thank Dr. Hakan Berg and Ms. Susan Lawson for their help with the in vivo exposure experiments. This article is contribution number 972 of The University of Texas Marine Science Institute. References Adams, B.A., Cyr, D.G., Eales, J.G., 2000. Thyroid hormone deiodination in tissues of the American plaice, Hippoglosssoides platessoides: characterization and short-term responses to polychlorinated biphenyls (PCBs) 77 and 126. Comp. Biochem. Physiol. C 127, 367–378. Ayson, F.G., Lam, T.J., 1993. Thyroxine injection of female rabbitfish (Siganus guttatus) broodstock: changes in thyroid hormone levels in plasma, yolk, and yolk–sac larvae, and its effect on larval growth and survival. Aquaculture 109, 83–93. Besselink, H.T., Van Beusekom, S., Roex, R., Vethaak, A.D., Koeman, J.H., Brouwer, A., 1996. Low hepatic 7-ethoxyresorufin-O-dethylase (EROD) activity and minor alterations in retinoid and thyroid hormone levels in flounder (Platichthys flesus) exposed to the polychlorinated biphenyl mixture Clophen A50. Environ. Pollut. 92, 267–274. Brouwer, A., Longnecker, M.P., Birnbaum, L.S., Cogliano, J., Kostyniak, P., Moore, J., Schantz, S., Winneke, G., 1999. Characterization of potential endocrine-related health effects at low dose levels of exposure to PCBs. Environ. Health Perspect. 107, 639–649. Brown, D.D., 1997. The role of the thyroid hormone in zebrafish and axolotl development. Proc. Natl. Acad. Sci. U. S. A. 94, 13011–13016. Brown, C.L., Cochran, M., Doroshov, S., Bern, H.A., 1989. Enhanced survival in striped bass fingerlings after maternal triiodothyronine treatment. Fish Physiol. Biochem. 7, 295–299. Brown, S.B., Evans, R.E., Vandenbyllardt, L., Finnson, K.W., Palace, V.P., Kane, A.S., Yarechewski, A.Y., Muir, D.C.G., 2004. Altered thyroid status in lake trout (Salvelinus namaycush) exposed to co-planar 3,3′,3,4′,5pentachlorobiphenyl. Aquat. Toxicol. 67, 75–85.
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