Grantee Research Project Results
Final Report: Immunotoxicity of Combined TBT and Co-planar PCB Exposures in Fish
EPA Grant Number: R823881Title: Immunotoxicity of Combined TBT and Co-planar PCB Exposures in Fish
Investigators: Rice, Charles D.
Institution: Mississippi State University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1995 through September 30, 1998
Project Amount: $274,120
RFA: Exploratory Research - Environmental Biology (1995) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Environmental Justice , Human Health , Aquatic Ecosystems
Objective:
Considering the high probability of finding both halogenated aromatic hydrocarbons (HAH) and various organometallic compounds in aquatic environments, the need to investigate the combined toxic effects and mechanisms of action in aquatic organisms is great. Both groups of compounds are lipophilic, and they bioconcentrate and bioaccumulate in the aquatic food chain. The most toxic HAH is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD shows remarkable target specificity for the immune response in rodent models. Other HAHs, especially those that have structural similarity to TCDD, such as the coplanar polychlorinated biphenyls (PCBs), also are immunotoxic to rodent models. Therefore, it is important to establish if they are immunotoxic.
Rice and Schlenk (1995) demonstrated that PCB-126 does not affect the antibody response of channel catfish, Ictalurus punctatus, to Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC). On the other hand, innate immune functions such as phagocyte oxidative burst activity and non-specific cytotoxic cell activity are sensitive. Moreover, as expected, PCB-126 induces hepatic CYP1A activity in catfish. The investigators conclude that innate immune functions are more sensitive to planar HAHs than antigen-specific immunity.
Tributyltin (TBT) also is immunotoxic in mammals. With regard to fish, it was demonstrated that, unlike PCB-126, TBT severely depresses channel catfish antibody responses to Edwardsiella ictaluri. TBT temporarily suppresses catfish phagocyte oxidative burst activity and NCC activity. While PCB-126 has no pronounced effect on leukocyte differentials, it suppresses peripheral blood lymphocytes and elevates neutrophils. Alone, TBT has no effect on channel catfish CYP1A activity in vivo. The mechanisms of toxicity associated with these two classes of xenobiotics are apparently quite different; PCB-126 is an aryl hydrocarbon receptor (Ahr) ligand, while TBT has no known specific cellular ligand. There are even reports of antagonistic effects of TBT on the cytochrome P4501A system. Therefore, the work described herein investigates the effects of combined exposure on various measures of immunity and cytochrome P450 related activities in channel catfish (Ictalurus punctatus). The channel catfish is both ecologically and commercially important. Although considered to be a freshwater species, the channel catfish migrates to brackish waters of many estuaries and is an indicator species in the Louisiana Province EMAP.
The primary hypothesis tested in this current study is that exposures to mixtures of a coplanar PCB and TBT will be synergistic as to immune suppression at doses that are relatively nontoxic when administered alone. Because some of the immunosuppressive effects of PCB-126 may be due to P450 associated end products, an additional hypothesis to be addressed is that by inhibiting P450 related activities, TBT exposure is antagonistic to the immunotoxic actions of PCB-126.
The need to examine toxicological interactions between TBT and planar HAHs is logical. TBT and coplanar PCBs are potent immunotoxic agents and have severe effects on reproduction and development in animal models studied to date. Exposure to coplanar HAHs results in several classical toxic responses including thymic atrophy and immunotoxicity, body weight loss, and reproductive abnormalities (Safe, 1990). Other than the reported effects on CYP1A activity, TBT acts as a calcium ionophore and disrupts both plasma and mitochondrial membrane integrity (Chow, et al., 1992). These effects are the primary mechanisms behind the potent immunotoxic properties of TBT, including thymic atrophy (Raffray, et al., 1993). Immune dysfunction in mouse B-lymphocytes following exposure to HAHs also involves altered calcium homeostasis (Karrass, et al. 1996).
Summary/Accomplishments (Outputs/Outcomes):
Because planar HAHs are active through the Ahr, it was important to validate that the chosen doses of PCB-126 and TBT were physiologically active without overt toxicity. Hepatic CYP1A content and EROD activity as well as body condition factors were chosen. The latter is an index of overt toxicity. As a prerequisite to the study, fish CYP1A-specific monoclonal antibodies were developed (Rice, et al., 1998). Three mAbs, FA-1, C10-7, and B-2-7, were developed using a peptide fragment (Myers, et al., 1993) mapping to the ligand binding domain of the P4501A protein. Methods for assessing CYP1A enzymatic activities (EROD activity) were already developed and applied to channel catfish (Rice and Schlenk, 1995). Therefore, we tested the hypothesis that TBT will inhibit CYP1A activities in vivo.
Channel catfish were exposed to PCB-126, TBT, or both in combination, with corn oil serving as the carrier control. CYP1A protein was determined by ELISA and EROD activity was measured after: (1) a single injected (i.p.) dose of 0.01, 0.1, or 1 mg/kg of each or both in combination; and (2) six injections (i.p.) of 0.017, 1.7, or 17 µg/kg of each (or both in combination) given at 3-day intervals over a 16-day period to yield a cumulative dose of 0.01, 0.1, or 1 mg/kg. The results have been published (Rice and Roszell, 1998).
As expected, 3 and 7 days following both an acute exposure and six fractionated doses of PCB-126 administered over a 16-day period elevated hepatic CYP1A activity. However, the magnitude of response after repeated exposures was higher. CYP1A induction was almost twice that of acute exposure 3 and 7 days after the last injection. Ethoxyresorufin O-deethylase activity did not follow these responses, although it was elevated in all PCB-126 treated fish. Tributyltin is not a ligand for the Ahr, therefore any induction of CYP1A activity by this compound alone was unexpected. Yet, all three levels of TBT slightly elevated EROD activity 3 days after the last injection of repeatedly exposed fish. The biological significance of this observation, if any, is unclear because EROD activity returned to that of corn oil treated fish 4 days later.
The interactive effects of TBT and PCB-126 on hepatic CYP1A protein and enzyme activity were unpredictable and unexpected, in this and a previous study (DeLong and Rice, 1997). CYP1A protein data from our acute exposure studies affirmed the EROD data of Fent and Stegeman (1991) and Bruschweiler, et al., (1996) in that TBT inhibited PCB-126 induced responses (Day 3 of our study). However, this effect was not evident 4 days later. This is not surprising considering that fish rapidly metabolize and eliminate TBT (Lee, 1985). Furthermore, TBT modulated CYP1A protein induction by PCB-126 in the repeated exposure studies involving mid- and high-dose ranges. Based on our current understanding of Ahr-mediated pathways leading to induction, this is difficult to explain.
Enzyme (EROD) activity associated with CYP1A induction conflicts with the protein data. High levels of TBT enhance low-level PCB-126 related EROD induction, and all three levels of TBT potentiate high-level PCB-126 related activity. This effect is greater in the repeated, fractionated dose study, although the effects are eliminated by day 7 at the higher levels of PCB-126. Such conflicting data between CYP1A protein and EROD activity have been reported for 3,3'4,4'-tetrachlorobiphenyl (Hahn and Stegeman, 1994) and may be a result of substrate saturation. Again, it is not clear at this point how low levels of PCB-126 (0.01 to 1 mg/kg), administered as repeated fractionated doses, lead to higher EROD induction patterns than does a single dose. It may be that the Ahr and other cytosolic and nuclear components associated with transcription activation are maximally induced over the 2-week period of repeated dosing. This would then lead to greater than expected responses from basal, or newly induced levels of Ahr (and other factors) after a single dose.
Considering these experiments, any explanations for the enhancing effects of TBT on this system are only speculative and appropriately cautious. TBT-related potentiation of PCB-126-induced CYP1A may come from our basic understanding of leukocyte biology. Elevated intracellular calcium is a critical factor in leukocyte activation leading to reactive oxygen intermediate production by phagocytes and the differentiation and proliferation of lymphocytes. There are, however, both calcium-dependent and calcium-independent kinases involved in each of these examples. Moreover, elevated intracellular calcium and subsequent endonuclease activity are related to apoptotic events in the thymus and other lymphoid organs. Nonetheless, elevated calcium leads to the up-regulation of many cellular signal transduction pathways. One of these may enhance the transcription of Ahr, aryl receptor nuclear transferases (ARNT), or both. It may even facilitate interactions of the Ahr and the ligand or heat shock chaperones. Tributyltin is a calcium ionophore (Rice and Weeks, 1989; Raffray, et al., 1993, Girard, et al., 1997) and, as such, may elevate hepatic and endothelial intracellular calcium levels. This potential mechanism needs to be explored, possibly through the use the fish hepatoma cell line PLHC-1 and the rat hepatoma cell line 4HIIE. Both cell lines have inducible CYP1A activities (Hahn, et al., 1993; Tillit, et al., 1991).
A more likely explanation is that endogenous factors are released as a direct result of either handling stress, TBT exposure, or both. CYP1A1, glutathione S-transferases, and UDP-glucuronosyl transferase are potentiated by dexamethasone in rats (Prough, et al., 1996); a response that is inhibited by RU486. Fish CYP1A1 induction also is potentiated by glucocorticoids (Celander, et al., 1997). These effects are not restricted to glucocorticoids because androgens and growth hormone have a role in CYP2A2 and CYP3A2 expression, respectively (Prough, et al., 1996). To our knowledge, no one has reported that TBT elevates glucocorticoids or other neuroendocrine secretions directly. However, in the case of molluscs, TBT blocks P-450 associated aromatases leading to elevated androgens. However, hormone profiles will be measured in future studies. To minimize circadian variations in physiological responses, all sampling in this study occurred in the mid-morning. Interestingly, this is a time of day associated with elevated cortisol (Levi, et al., 1991), and lowered growth hormone levels (Leatherland, et al., 1974) as well as prolactin levels (Nevid and Meier, 1995) in several species of fish.
It is important to realize that the effects of TBT on PCB-126-induced CYP1A activity appear to be only additive, and not synergistic. Even if only additive, this may still be an environmentally significant interaction because induction of this system is associated with enhanced metabolism of several environmental pro-carcinogens to ultimate mutagens and carcinogens (e.g., benzo-(a)-pyrene) that also are potent immunotoxic agents (White and Holsapple, 1984). It is likely that most environmental mixtures of xenobiotics have similar interactive effects on other biological systems including reproduction, development, and immune function. In fact, most of our concerns about xenobiotics over the last few decades are related to Ahr-active structures because of their potent pathobiological effects.
Intraperitoneal injections of test agents are not environmentally relevant. However, the animals were exposed to a desired concentration and hepatic CYP1A activity was not modulated by similar activity in other organs, especially the gill, skin, and gut. Extrahepatic CYP1A and other P450 isozymes may dramatically modulate the properties that this study ascribes to TBT. Whether TBT affects PCB-126-induced CYP1A activity following dietary or sediment-borne exposures obviously needs to be determined. Furthermore, it is important to realize that CYP1A induction is only a physiological marker of exposure and not necessarily one of toxicological effect. Beyond induction of xenobiotic metabolizing enzymes, other responses such as immune function, reproduction, and behavior need to be examined to fully evaluate the effects of TBT not only on HAH-related toxicity, but other classes of xenobiotics as well.
Additional Studies and Results. Because the above studies involved intraperitoneal injections as the route of exposure, one can question the effects of exposure to TBT, PCB-126, and both in combination. To that end, channel catfish were treated with dietary TBT, PCB-126, and combinations of both for 21 days. Hepatic CYP1A protein and EROD activity were determined in these fish exposed continually for 3, 14, and 21 days. As expected, TBT (10 ppb and 1 ppm) had no effect, while PCB-126 (1 ppm) induced both CYP1A protein and EROD activity by day 21. As with intraperitoneal exposures, low levels of TBT potentiated PCB-126-induced CYP1A protein, but it suppressed EROD activity. Although high levels of TBT had no effect on PCB-126-induced CYP1A protein, it suppressed EROD activity.
Summary of Effects on CYP1A. At reasonably low levels, TBT enhances PCB-126-induced CYP1A activity in channel catfish as it does in B6C3F1 mice. Only at high levels (1 mg/kg) does TBT inhibit CYP1A enzymatic activity (EROD), and then only in the presence of an inducer like PCB-126. The channel catfish is an appropriate model to investigate the toxicity, and mechanisms of action, following co-exposure to TBT and PCB-126 on a variety of systems.
Immune Functions. The immunotoxicity of PCB-126, TBT, and combinations of both were determined using the above experimental design for acute and repeated exposures. Because the low and high dose of each resulted in a full range of effects on CYP1A, this study focused on low (0.01 mg/kg) and high (1 mg/kg). Fish were immunized at the time of the last injection. Phagocyte oxidative burst activity (Rice, et al., 1995) was measured 14 and 21 days following the last dose for each of the acute and repeated exposure studies. These time points complimented earlier studies (Rice, et al., 1995; Rice and Schlenk, 1995) that measured innate immune responses 3 and 7 days after exposure. Antibody responses were evaluated as plaque assays 14 days after exposure in the earlier studies.
After acute (single) exposures, PCB-126 and TBT suppressed antibody responses only at high doses. No interactive effects of TBT/PCB-126 combinations were observed. Combinations of high TBT and low PCB-126, as well as high TBT and high PCB-126 also suppressed antibody responses, but this suppression was comparable to high PCB-126 and high TBT when they were administered alone.
For repeated exposures resulting in a cumulative exposure that was the same as a single exposure in the acute studies, only a high dose of TBT suppressed the antibody response. There were no interactive effects of TBT/PCB-126 combinations. In summary, TBT and PCB-126 suppress the antibody response to V. anguillarum, but only at the highest exposure level of each. In contrast, Rice and Schlenk (1995) demonstrated that a single dose of 1 mg/kg PCB-126 had no effect on the antibody response to Edwardsiella ictaluri measured using a plaque assay. This difference is probably due to the antigen used, the assay for determining antibody responses, or both. Serum antibodies to V. anguillarum seem to be more sensitive.
Phagocyte oxidative burst activity (NBT assay) is sensitive to TBT and PCB-126 after a single exposure (injection). Phagocyte activity was suppressed 14 days postexposure by both levels of TBT and PCB-126. This response was still suppressed 21 days postexposure in fish treated with high and low TBT and PCB-126, respectively. The only interactive effect of TBT and PCB-126 combinations was in the low doses of each. Oxidative burst activity was suppressed three-fold in this group 21 days after exposure. Combinations of high and low, and high and high doses of TBT and PCB-126, respectively, also suppressed this response, but activities were comparable to each compound given alone.
In the repeated exposure study, both levels of TBT enhanced oxidative burst activity 14 days postexposure; however, this response was suppressed 7 days later. PCB-126 had no effect when given alone. In contrast to the study of Rice and Schlenk (1995), this study showed that a single dose of 1 mg/kg PCB-126 lowered this endpoint 7 days after exposure.
In summary, there are no synergistic effects of TBT and PCB-126 on immune function. Of particular note, the innate immune system of channel catfish is more sensitive than the humoral (antibody) response to both PCB-126 and TBT. As with CYP1A activity, there is an interaction between the two compounds in terms of oxidative burst activity at low levels of both compounds in mixture. The mechanisms behind potentiation of effects in both CYP1A activity and phagocyte oxidative burst activity are unclear. However, both systems involve an enzyme system for full activity. The former is classically referred to as EROD activity, while the latter involves NADPH oxidase activity. Enhanced PCB-126-induced CYP1A and suppressed oxidative burst activity by TBT may be related to cortisol secretion because cortisol enhances CYP1A activity (Celander, et al., 1997), but suppresses phagocyte activity (Rice, 2000). However, we found no effect of exposure on cortisol production. The mechanisms remain unclear.
Conclusions. Only in rare cases do Ahr-binding compounds exist alone in the environment. Most exist in mixtures with other, non-related compounds, especially in industrialized harbor estuaries and their tributaries. To date, the interactive effects of xenobiotic mixtures containing both Ahr active and non-Ahr active structures remain unclear, if not controversial. In this regard, TBT and PCB-126 mixtures have potent effects on hepatic CYP1A and innate immune functions in fish. Because fish rely more on innate immune functions than specific immune functions (Rice, 2000), these findings question current approaches to assessing the risk associated with planar HAHs and organometals. Clearly, we cannot accurately assign a risk associated with environmental exposure to these compounds as single agents because they do not occur alone in the environment except for unusual circumstances such as accidental spills and experimentation. Future studies should focus on the mechanism(s) behind the interactive effects of TBT and PCB-126 on CYP1A and phagocyte oxidative burst activity; two systems affected by Ahr-ligands.
References:
White Jr., KL, Holsapple MP. Direct suppression of in vitro antibody production by mouse spleen cells by the carcinogen benzo-(a)-pyrene but not by the noncarcinogenic congener benzo-(e)-pyrene. Cancer Research 1984;44(8):3388-3393.
Leatherland JF, Mckeown BA, John TM. Circadian rhythm of plasma prolactin, growth hormone, glucose and free fatty acid in juvenile kokanee salmon, Oncorhynchus nerka. Comparative Biochemistry and Physiology Part A: Physiology 1974;47(3):821-828.
Fent K, Stegeman JJ. Effects of tributyltin chloride in vitro on the hepatic microsomal mono-oxygenase system in the fish Stenotomus chrysops. Aquatic Toxicology 1991;20(3):159-168.
Rice CD, Weeks BA. Influence of tributyltin on in vitro activation of oyster toadfish macrophages. Journal of Aquatic Animal Health 1989;1(1):62-68.
Lee RF. Metabolism of tributyltin oxide by crabs, oysters and fish. Marine Environmental Research 1985;17(2-4):145-148.
Safe S. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental and mechanistic considerations which support the development of toxic equivalency factors (TEFs). Critical Reviews in Toxicology 1990;21(1):51-88.
Journal Articles on this Report : 13 Displayed | Download in RIS Format
Other project views: | All 22 publications | 14 publications in selected types | All 13 journal articles |
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Arnold RE, Rice CD. Channel catfish, Ictalurus punctatus, leukocytes secrete immunoreactive adrenal corticotropin hormone (ACTH). Fish Physiology and Biochemistry 2000;22(4):303-310. |
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Bruschweiler BJ, Fent K, Wurgler FE. Inhibition of cytochrome P4501A by organotins in fish hepatoma cells PLHC-1. Environmental Toxicology and Chemistry 1996;15(5):728-735. |
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Burton JE, Dorociak IR, Schwedler TE, Rice CD. Circulating lysozyme and hepatic CYP1A activities during a chronic dietary exposure to tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures in channel catfish, Ictalurus punctatus. Journal of Toxicology and Environmental Health-Part A 2002;65(8):589-602. |
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DeLong GT, Rice CD. Tributyltin potentiates 3,3',4,4',5-pentachlorobiphenyl-induced cytochrome P-4501A related activity. Journal of Toxicology and Environmental Health 1997;51(2):131-148. |
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Girard J-P, Ferrua C, Pesando D. Effects of tributyltin on Ca2+ homeostasis and mechanisms controlling cell cycling in sea urchin eggs. Aquatic Toxicology 1997;38(4):225-239. |
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Karras JG, Morris DL, Matulka RA, Kramer CM, Holsapple MP. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) elevates basal B-cell intracellular calcium concentration and suppresses surface lg- but not CD40-induced antibody secretion. Toxicology and Applied Pharmacology 1996;137(2):275-284. |
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Nevid NJ, Meier AH. Timed daily administrations of hormones and antagonists of neuroendocrine receptors alter day-night rhythms of allograft rejection in the gulf killifish, Fundulus grandis. General and Comparative Endocrinology 1995;97(3):327-339. |
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Prough RA, Linder MW, Pinaire JA, Xiao GH, Falkner KC. Hormonal regulation of hepatic enzymes involved in foreign compound metabolism. FASEB Journal 1996;10(12):1369-1377. |
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Regala RP, Rice CD, Schwedler TE, Dorociak IR. The effects of tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures on antibody responses and phagocyte oxidative burst activity in channel catfish, Ictalurus punctatus. Archives of Environmental Contamination and Toxicology 2001;40(3):386-391. |
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Rice CD, Banes MM, Ardelt TC. Immunotoxicity in channel catfish, Ictalurus punctatus, following acute exposure to tributyltin. Archives of Environmental Contamination and Toxicology 1995;28(4):464-470. |
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Rice CD, Schlenk D. Immune function and cytochrome P4501A activity after acute exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB 126) in channel catfish. Journal of Aquatic Animal Health 1995;7(3):195-204. |
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Rice CD, Schlenk D, Ainsworth J, Goksoyr A. Cross-reactivity of monoclonal antibodies against peptide 277-294 of rainbow trout CYP1A1 with hepatic CYP1A among fish. Marine Environmental Research 1998;46(1-5):87-91. |
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Rice CD, Roszell LE. Tributyltin modulates 3,3',4,4',5-pentachlorobiphenyl (PCB-126)-induced hepatic CYP1A activity in channel catfish, Ictalurus punctatus. Journal of Toxicology and Environmental Health-Part A 1998;55(3):197-212. |
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Supplemental Keywords:
fish, catfish, PCBs, tributyltin, P4501A, CYP1A, immunotoxicity, harbor estuaries, environmental toxicology., Health, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Toxicology, Bioavailability, Environmental Chemistry, Health Risk Assessment, Epidemiology, Risk Assessments, Biology, organometals, immune system effects, epidemelogy, toxic metabolites, immunotoxicology, toxic environmental contaminants, bioaccumulation, cell culture, fish-borne toxicants, co-planar PCB exposuresProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.