Environmental Toxicology and Chemistry, Vol. 26, No. 6, pp. 1179–1185, 2007 䉷 2007 SETAC Printed in the USA 0730-7268/07 $12.00 ⫹ .00
DIETARY EXPOSURE TO LOW PESTICIDE DOSES CAUSES LONG-TERM IMMUNOSUPPRESSION IN THE LEOPARD FROG (RANA PIPIENS) ANATHEA ALBERT,† KEN DROUILLARD,‡ G. DOUGLAS HAFFNER,‡ and BRIAN DIXON*†‡ †Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada ‡Great Lakes Institute for Environmental Research, University of Windsor, Windsor, Ontario N9B 3P4, Canada ( Received 4 December 2005; Accepted 8 December 2006) Abstract—This study examines the relationship between dietary exposure of pesticides, DDT, and dieldrin and immunosuppression in the northern leopard frog (Rana pipiens). Immune function was measured before, during, and after a 10-week exposure period with the use of both adaptive and innate immunity responses. Exposure to low doses (75 ng/g body wt DDT or 2.1 ng/g dieldrin total dose over the 10 weeks) resulted in significant suppressive effects on antibody production and secondary delayed-type hypersensitivity (DTH). The high doses (750 ng/g DDT and 21 ng/g dieldrin), however, did not affect antibody production, DTH, or oxidative burst in a predictable dose–response manner. The differences in magnitude and direction of the effects of the two dosing regimes were likely due to differences in chemical exposure on the basis of feeding and effectiveness of chemical uptake. The low dose results demonstrated that moderate concentrations of pesticides, frequently observed in the environment, are able to weaken the immune response of R. pipiens. Keywords—Frog
Immunosuppression
Pesticide
DDT
Dieldrin
prey, and in the soil, but none in the water [14]. Unfortunately, most studies on chemical effects in amphibians have been based on intraperitoneal injections, dermal uptake, and water exposure. Yet, evidence that chemical uptake from food can cause immunotoxicological responses is sparse. The purpose of this study was to evaluate the immune effects of chronic dietary exposure to low doses of the organochlorine pesticides DDT and dieldrin in R. pipiens. Three components of the immune response were quantified: specific antibody production to keyhole limpet hemocyanin linked to dinitrophenol (KLH-DNP) as a measure of humoral immunity, oxidative burst as a measure of innate immunity, and delayed-type hypersensitivity (DTH), a T cell–mediated inflammatory response. Immune responses in unexposed R. pipiens were also determined and used to establish a baseline against which chemically induced changes in immune effects could be distinguished from natural changes.
INTRODUCTION
Declining populations and local extinctions of amphibians are of global concern [1–3]. Several pathogens (Bactrachochytrium dendrobatidis and Ranavirus) have frequently been associated with amphibian declines [1,4]. Similarly, exposure to ultraviolet-B (UV-B) radiation has also been related to increased susceptibility of frog embryos to fungal infection [5]. Recent research has demonstrated that selected pesticides have immunosuppressive effects in amphibians [6–9]. Reduced competence to defend against pathogens was observed in Woodhouse’s toads (Bufo woodhousei) after dermal exposure to malathion; in Rana sylvatica after water exposure to malathion, atrazine, and esfenvalerate [6,10]; and in Rana pipiens after water exposure to pesticide mixtures [8]. However, other studies have shown that pesticide exposure reduced virus infection in Ambystoma macrodactylum [11] and Rana clamitans [12]. Reduced antibody production and oxidative burst were observed in R. pipiens when injected with small doses of either malathion, DDT, or dieldrin [7]. A field study revealed that frogs with moderate tissue levels of DDT/dicholorodiphenyldichloroethylene (DDE) and dieldrin exhibited immunosuppressed responses [7]. Other field studies have also reported a strong relationship between limb deformities and parasite cysts in frogs with exposure to agricultural runoff [6]. Christin et al. [9] have shown that pesticides inhibit the proliferation of R. pipiens lymphocytes. Such studies demonstrate that pesticides are capable of causing immunomodulation in amphibians, and there is a need to quantify the relative importance of pesticide exposure as a possible contributing factor to declines and disease outbreaks. Diet is an important exposure route for hydrophobic pesticides [13]. A field study in Bermuda showed DDE in tissues of Bufo marinus and Eleutherodactylus johnstonei, in their
MATERIALS AND METHODS
Study species Adult R. pipiens were collected from five rural sites in Grey and Bruce counties in southern Ontario, Canada, in July 2001. The sites were all chosen in areas that had not been sprayed or used for agriculture recently in an attempt to reduce the possibility of obtaining frogs with high tissue levels of pesticides. Frogs were kept in accordance with Animal Care Protocols for the University of Waterloo and were acclimated in the lab for three months before experimentation. Frogs were divided by weight into six exposure groups of 10 individuals such that that each group contained a similar weight distribution.
Blood sampling A maximum 500 l of blood was collected with a 27G, 1.25-cm needle (Becton Dickinson, Franklin Lakes, NJ, USA)
* To whom correspondence may be addressed (
[email protected]). 1179
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via cardiac puncture. Two microliters were removed for the chemiluminescence reaction and the remainder was stored overnight at 4⬚C and allowed to clot. The serum was collected and stored at ⫺20⬚C.
Antibody detection Specific Immunoglobulin M (IgM) production was measured by an enzyme-linked immunosorbent assay (ELISA) as described in Gilbertson et al. [7]. Only IgM was measured as a result of a lack of anti-frog antibodies for other immunoglobulin isotypes such as IgY. Plates (96-well) were coated with KLH-DNP (Sigma-Aldrich, St Louis, MO, USA). The plates were blocked with a 1% bovine serum albumin solution, and 100 l of frog serum was added to four replicate wells for each individual. The primary antibody was called 6-16, a mouse anti-Xenopus IgM antibody, provided by M. Flanjik, which was able to cross-react with IgM of R. pipiens. The secondary antibody was goat anti-mouse alkaline phosphatase conjugate (Sigma, St. Louis, MO, USA) diluted 1:1,000. The plate was washed between each step with Tris-buffered saline containing Tween 20 (polyoxyethylene sorbitan monolaurate). The substrate for the conjugate was the Sigmafast p-nitrophenyl phosphate tablet set (Sigma) dissolved in distilled water. Absorbance was measured on a Versimax plate reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm. The four replicate wells for each individual were used to obtain an average absorbance value for that individual. Because all groups received the vehicle control, individual absorbance values were normalized to the vehicle control absorbance values. Each plate contained three negative controls: one lacking primary antibody, one lacking secondary antibody, and one lacking both antibodies. The positive controls, a commercial mouse anti-KLH antibody (Sigma), and a goat antimouse conjugate secondary antibody yielded strong color reaction.
Oxidative burst Whole blood was used in a zymosan A–induced chemiluminescence reaction to measure oxidative burst levels as described by Marnila et al. [15]. Whole frog blood was mixed with luminol (5-amino-2,3-dihydro 1,4-phthalazinedione; Sigma) and Frog Ringer’s solution in a 96-well plate. Just before measurement, zymosan A (Sigma) was added to each well to stimulate oxidative burst. Chemiluminescence was measured with a 1450 Wallac Microbeta Trilux 2 luminescence counter (Wallac Oy, Turku, Finland). Results were represented as luminescence counts per second. Over the course of the reaction, a curve of luminescence counts per second was generated for each sample, and the peak value of the curve was taken to represent oxidative burst. Peak values for each group were normalized to the vehicle control in a bar graph by SigmaPlot 2000 (Systat Software, San Jose, CA, USA). Controls contained all reagents except blood.
Delayed type hypersensitivity Delayed-type hypersensitivity tests were carried out at three time points. The first was completed during the pre-exposure period after frogs had been sensitized with chicken egg lysozyme (CEL). The second was completed after five weeks of pesticide exposure and two weeks after sensitization with KLH-DNP. The third, generating a secondary DTH response, was carried out after 10 weeks of pesticide exposure and two weeks after a boost injection of KLH-DNP. Toe thickness was
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measured with electronic calipers (Digimatic Outside Micrometer 0–2.5 cm, Mitutoyo, Morgan Precision Tools, Aurora, IL, USA). Nine days after sensitization, the middle toe on one foot was injected with KLH-DNP to induce an inflammatory reaction, and the middle toe on the other foot was injected with phosphate-buffered saline as a control. Toe thickness was again measured at 24, 48, and 72 h postinjection. All measurements were completed a minimum of three times, and an average of the measurements was used as the final value. The maximum swelling occurred between 24 and 48 h.
Tissue analysis To determine pesticide accumulation in the frogs during the study, whole-body contaminant analysis was performed at six, 10, and 26 weeks for all control groups. Preparation of samples was completed following the procedure of Lazar et al. [16]. Frogs were homogenized then dried with Na2SO4, and pesticides were extracted with a glass 24-cm liquid chromatography column containing hexane and dichloromethane (50%, v/v). Fully activated Florisil was used to clean up the sample, and three fractions (fraction 1, 50 ml hexanes; fraction 2, 50 ml dichloromethane/hexanes 85%:15% [v/v]; fraction 3, 130 ml dichloromethane/hexanes 60%:40% [v/v]) were collected, with DDT/DDE being in the first two fractions and dieldrin in the third fraction. A Hewlett-Packard HP-5890 gas chromatograph (GC; Avondale, PA, USA) with electron-capture detector and autosampler (HP-7673) was used for analysis. Reference materials, blanks, and standards were run with every set of samples.
Data analysis Differences in immune responses over time were compared with by-groups repeated measures analysis of variance (RMANOVA) with SPSS威 Version 10 (SPSS, Chicago, IL, USA). The Greenhouse–Geisser (G-G) value of significance was used to correct for the time correlation. Post hoc comparisons were done with Tukey testing. Pre-exposure values for antibody production and chemiluminescence, although obtained with a different antigen, were included in the statistical analysis because all values were normalized to the vehicle control. Differences between primary and secondary DTH response were determined with a paired t test for each group. Differences in the DTH response between groups at 10 weeks were determined by one-way ANOVA, followed by Tukey’s post hoc testing.
Study outline One of the six groups of 10 frogs were fed a net quantity of 750 and 75 g (or e L⫺1) DDT, representing high-dose (HDDT) and low-dose (LDDT) exposure regimes. Similarly, 21 g L⫺1 and 2.1 g L⫺1 of dieldrin were used for the high (HDI) and low (LDI) dosing regimes. Cyclophosphamide (CY) 9,000 g L⫺1 was used as a positive control. Because the pesticides had low water solubility (log KOW ⬎ 5.5), they were dissolved in dimethyl sulfoxide (DMSO) and injected into live crickets. A food control group of crickets received just DMSO. To ensure that frogs received the correct dose, they were isolated during feeding. Additional unexposed crickets were offered to the frogs afterwards. The pesticides were injected into crickets that were fed to the frogs over a 10-week period, representing chronic dietary exposure. The dose given at each feeding was 21.4 ng/g body weight p,p⬘-DDT (Aldrich, Milwaukee, WI, USA), 2.14
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ng/g DDT, 0.60 ng/g dieldrin, 0.06 ng/g dieldrin (Sigma-Aldrich), or 257 ng/g cyclophosphamide (Sigma-Aldrich). Frogs were fed three times weekly and were watched to ensure they ate enough crickets to obtain the full dose. Two weeks before exposure, frogs were assessed for initial immune system functioning by immunizing with horse muscle myoglobin to initiate an antibody response and with CEL to generate a DTH response. This challenge was not expected to influence results because previous research indicated that no changes in antibody levels were observed if the original immunization was done before exposure [7]. After exposure, frogs were immunized with KLH-DNP to test for both antibody and DTH responses. After three weeks of pesticide exposure, frogs were immunized intramuscularly with KLH-DNP (Sigma), dimethyldioctadecylammonium bromide (Fluka Chemika, Ronkonkoma, NY, USA), and Titer max adjuvant (Sigma) in a ratio of 1:1:1. Blood samples were obtained at five, seven, and 10 weeks from the start of the exposure studies. Two weeks before obtaining blood samples, frogs were boosted with KLH-DNP. At five and 10 weeks, DTH tests were completed. After 10 weeks of exposure, the frogs were fed untreated crickets, and blood sampling occurred at 12, 14, 18, 22, 26, 30, 34, and 38 weeks to determine how long immunotoxicological responses would be maintained in the frogs.
Unexposed frogs To observe natural variation in antibody and oxidative burst levels over time, four frogs that were not exposed to pesticide or the carrier DMSO were immunized with KLH-DNP. Blood samples were obtained at two-week intervals at the start of the experiment, but later in the study when it became clear that recovery was not occurring in the treatment groups, they were taken at four-week intervals to match the schedule of sampling in the treatment groups. These samples were used in chemiluminescence and ELISA assays. Frogs were boosted two weeks before each blood sampling. The immune responses of these frogs unexposed to pesticide and DMSO were also compared with the immune responses of the control group from the feeding study to determine whether there were differences between DMSO-exposed individuals and pesticide and DMSO individuals.
Fig. 1. Levels of specific immunoglobulin M (IgM) antibodies generated by immunization with keyhole limpet hemocyanin coupled to dinitrophenyl (KLH-DNP). Of the 12 time points, one occurred before exposure (pre-exp), three occurred during exposure (5, 7, 10 weeks), and eight occurred during the postexposure period (12, 14, 18, 22, 26, 30, 34, 38 weeks). Anti–KLH-DNP IgM levels were measured by an enzyme-linked immunosorbent assay (ELISA), and absorbance readings were expressed as a percentage of the control group, dimethyl sulfoxide (DMSO). Bars represent mean ⫾ 1 SE anti-KLH IgM levels as a percentage of the control group levels. Letters represent statistically significant differences, bars with the same letters are not different, and bars with different letters are different from each other. (A) Low-dose dieldrin (LDI) group, exposed to a total dose of 2.1 ng/g dieldrin, n ⫽ 6. (B) High-dose dieldrin (HDI) group received a total dose of 21 ng/g dieldrin, n ⫽ 5. (C) Low-dose DDT (LDDT) group received a total dose of 75 ng/g DDT, n ⫽ 4. (D) High-dose DDT (HDDT) group received a total dose of 750 ng/g DDT, n ⫽ 5. (E) Cyclophosphamide (Cy)-exposed positive control group received a total dose of 9,000 ng/g cyclophosphamide, n ⫽ 4.
RESULTS
Health of organisms No mortalities occurred while the frogs were held before the start of the study; however, two deaths occurred during the pre-exposure period and 16 occurred during the posttreatment period. Most tanks lost two or three individuals to skin damage–related infections before a treatment could be found. Because mortalities occurred after the frogs were treated, a high sensitivity in these organisms to laboratory-induced stress is indicated.
Antibody response after pesticide exposure Antibody levels in the treatment and control groups preexposure were not significantly different (ANOVA, p ⬎ 0.05), although it was observed that antibody levels in the LDI, HDI, LDDT, and CY groups (Fig. 1A to C and E) were somewhat higher than those observed in the DMSO and HDDT groups (Fig. 1D). The pre-exposure antibody levels showed large variability in several groups, including HDI and HDDT (Fig. 1B
Fig. 2. Specific antibody production, in response to keyhole limpet hemocyanin conjugated to dinitrophenyl (KLH-DNP) immunization, of unexposed frogs and dimethyl sulfoxide (DMSO)–exposed frogs to observe the variation in this response over time. (A) Levels of specific, anti-KLH, immunoglobulin M antibodies in the serum of immunized but unexposed frogs, n ⫽ 4. Specific antibodies were measured with an enzyme-linked immunosorbent assay (ELISA) reaction. Times correspond to the number of weeks after initial immunization. (B) Antibody response to KLH immunization of DMSOexposed frogs from the feeding study, n ⫽ 5. Times correspond to the number of weeks after initiation of DMSO exposure. Frogs were immunized at three weeks.
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Fig. 3. Zymosan-induced oxidative burst reaction before (pre-exp), during (5–10 weeks), and after (12–38 weeks) a 10-week dietary exposure to pesticides or immunosuppressive drugs. Oxidative burst levels were measured with a luminol-enhanced chemiluminescence reaction. Results were expressed as the mean ⫾ 1 SE peak chemiluminescence as a percentage of the peak chemiluminescence of the control group (n ⫽ 6). Letters represent statistically significant differences, bars with the same letters are not different, bars with different letters are different from each other. (A) Exposed to a low dieldrin dose, the net dose was 2.1 ng/g dieldrin (n ⫽ 6). (B) Exposed to a high dieldrin dose, a net dose of 21 ng/g dieldrin (n ⫽ 5). (C) Exposed to a net dose of 75 ng/g DDT (n ⫽ 4). (D) Exposed to a net dose of 750 ng/g DDT (n ⫽ 5). (E) Exposed to 9,000 ng/g cyclophosphamide (Cy) (n ⫽ 4).
and D). Because the groups were not compared with each other, such differences were not of toxicological significance. Ten weeks after exposure, the LDI and LDDT groups had antibody (IgM) levels that were significantly lower (RMANOVA: LDI group, G-G ⫽ 0.009; LDDT group, G-G ⫽ 0.043) than their respective pre-exposure levels (Fig. 1A and C). For the LDDT group, this trend continued for 38 weeks after exposure (Fig. 1C). For the LDI group, antibody levels at 10, 18, and 22 weeks were all significantly lower than the preexposure level (Fig. 1A), although they seemed to recover to normal levels with a lot of variation by 38 weeks after exposure. The HDI and HDDT groups, however, did not have significant reductions in antibody levels when compared with pre-exposure levels, although the HDDT group did have a low level at week 18 that was significantly lower than those seen at weeks 5, 14, 26, and 34.
Antibody levels in unexposed frogs Natural variations of antibody levels and oxidative burst were quantified in unexposed frogs and compared with the responses of the DMSO-exposed frogs from the feeding study. Some interindividual variability was observed in both the unexposed (Fig. 2A) and DMSO-exposed (Fig. 2B) frogs, and it was interesting to note that some individuals consistently had
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Fig. 4. Oxidative burst response of unexposed and dimethyl sulfoxide (DMSO)–exposed frogs over time, measured with a zymosan A-induced, luminol-enhanced chemiluminescence reaction. Values are shown as the mean peak chemiluminescence for individual frogs at multiple times. (A) Levels of oxidative burst in whole blood of unexposed frogs. The responses of individual frogs are shown over a 34 week period, n ⫽ 4. (B) Levels of oxidative burst in whole blood of DMSO-exposed frogs from the feeding study. Oxidative burst levels over time are shown for individual frogs over a 340-week period n ⫽ 5. Times correspond to the number of weeks after initiation of DMSO exposure.
high or low antibody levels. Antibody levels in unexposed frogs remained constant for the first 10 weeks after immunization, then doubled by 14 weeks and remained high until 26 weeks (Fig. 2A). The DMSO-exposed frogs also experienced this doubling of antibody levels at around the same time (Fig. 2B). Variation in antibody response over time was similar in unexposed and DMSO-exposed groups.
Oxidative burst The oxidative burst for all of the groups was generally constant over time, but specific isolated peaks in oxidative burst activity were observed (Fig. 3). Statistically, only the LDI (Fig. 3A), HDDT (Fig. 3D), and CY (Fig. 3E) groups had significant enhancement of oxidative burst (RMANOVA by group: LDI p ⫽ 0.02, HDDT p ⫽ 0.005, CY p ⫽ 0.002) compared with the pre-exposure levels. At 10 weeks, the LDI, HDDT, and CY groups revealed significant increases in oxidative burst, but this was not observed in the LDDT and HDI groups, suggesting that oxidative burst might well be regulated by factors other than pesticide dose. Oxidative burst in the unexposed frogs remained low over time and demonstrated little variation among individuals (Fig. 4A). Oxidative burst of the DMSO-exposed group from the feeding study fluctuated more than that observed in the unexposed frogs (Fig. 4B), but no statistically significant difference in peak chemiluminescence was observed between the two groups.
Delayed type hypersensitivity The LDI, LDDT, HDI, CY, and DMSO groups all had no significant differences between the primary and secondary DTH response to KLH (i.e., between pre-exposure and the five-week measurement). None of the groups had significant differences between five and 10 weeks (paired t test, p ⬎ 0.05),
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Table 1. Whole-body tissue residues of DDT, dichlorodiphenyldichloroethylene (DDE), dichlorodiphenyldichloroethane (DDD), and dieldrin (ng/ g body weight) at three time points from the feeding study. Weights and weight changes are presented for the analyzed individuals (n ⫽ 1 per group per time) and for the corresponding exposure groups at each time n ⫽ 4 to 6
Time (weeks) Cyclophosphamide Cyclophosphamide DMSOa DMSO DMSO Low dieldrin Low dieldrin Low dieldrin High dieldrin High dieldrin High dieldrin Low DDT Low DDT Low DDT High DDT High DDT High DDT Crickets unexposed a
6 10 6 10 26 6 10 26 6 10 26 6 10 26 6 10 26
% Lipid
Dieldrin (ng/g)
DDT (ng/g)
DDE (ng/g)
DDD (ng/g)
2.49 4.9 4.38 3.04 4.78 4.04 4.34 5.47 3.56 3.32 5.19 2.72 4.38 4.13 3.21 3.42 5.02 6.5
ND ND ND ND ND 0.54 0.57 0.35 1.85 1.84 0.89 ND 0.38 ND ND 0.34 ND ND
0.91 2.46 1.83 1.15 3.19 0.95 3.35 0.9 0.62 ND 0.55 0.92 ND 0.84 48.73 39.58 16.06 4.1
0.71 1.33 1.36 3.51 6.06 3.39 2.65 5.08 2.41 1.27 2.21 16.26 6.79 4.91 12.15 11.47 12.33 1.5
0.77 0.72 1.44 ND 16.41 0.47 0.95 0.31 0.14 0.29 0.16 ND 0.41 0.35 12.61 10.81 4.47 4.1
Group average Original Weight weight gain Weight weight (g) change (g) (g) gain SD (g) 26.8 48 26.6 26.9 41.1 22.4 28.7 30.8 25.2 28.8 35.3 37.5 21.8 26.7 33.1 24.5 22.9
⫺1.2 5.5 7.5 5.1 0.5 1.1 0.6 2.1 5.7 1.2 ⫺1.3 ⫺1.2 2.5 11.4 2.2 6.7 4.4
8.45 11.1 1.57 2.75 3.02 3.92 5.43 7.68 6.94 8.5 10.8 3.43 5.6 5.43 5.88 8.18 8.54
4.04 6.2 1.16 1.83 2.61 4.24 6.25 5.76 5.64 6.53 11.91 4.03 4.74 5.84 2.5 3.19 4.65
DMSO ⫽ dimethyl sulfoxide; ND ⫽ not done.
and none of the groups were significantly different from each other at either time point (ANOVA, p ⬎ 0.05).
Tissue analysis and weight Cyclophosphamide- and DMSO-exposed individuals were originally included in the tissue analysis to obtain background levels for residues of DDT or dieldrin that might have accumulated in the wild before collection, although they accumulated pesticides over time. To determine why this was happening, we tested the crickets used in the feeding study. This indicated that DDT was detected in the crickets used to feed the frogs (Table 1) at concentrations higher than the planned low dose. For both DDT and dieldrin, greater tissue concentrations were found in the high-dose groups than in the lowdose groups, although concentrations did not reflect the 10fold differences in the planned feeding regime. In general, the doses of the pesticide being administered within the treated groups tended to remain constant during the feeding period and declined after the food-based dosing stopped. Weight gain varied considerably among individuals and groups as indicated by the relatively large standard deviations (Table 1). The CY, HDDT, and HDI groups had the greatest average weight gain within the first 10 weeks. Individuals exposed to DMSO gained the least weight. Antibody levels correlated weakly but significantly with total animal weight (r ⫽ 0.30, p ⫽ 0.0002), but neither antibody levels nor oxidative burst response correlated significantly with weight change over the course of the experiment. DISCUSSION
Dose and uptake of pesticides The pesticides were taken up in a dose-dependent manner in the sense that frogs fed a low-concentration diet had a lower body burden than those fed a high-concentration diet, as demonstrated by the tissue analysis data (Table 1). However, the frogs accumulated less pesticide than expected on the basis of our injection study [7], and even the highly dosed individuals
revealed whole-body concentrations much lower than that observed in contaminated feral populations, in which levels of 630 to 1,000 ng/g DDE and 56 to 199 ng/g dieldrin have been reported [7,17]. Evidence is limited that metabolism could produce the low body concentrations observed in the experimental frogs, and it is possible that gut uptake efficiency might be low or that the frogs would regurgitate contaminated crickets, although this was not observed. Because exposures were well below that anticipated, it was interesting to note that changes in immune responses were still observed in these organisms compared with the negative controls. The groups of frogs that gained the most weight during exposure had fewer detrimental effects on their immune response. It is apparent that the health of an organism, as measured by weight gain, can regulate immune response. Longterm laboratory studies with frogs such as R. pipiens will need to address the sensitivity of these organisms to rearing and handling. Dimethyl sulfoxide did not affect the antibody response in this study, as determined by comparison of DMSOexposed frogs with unexposed frogs (Fig. 2). According to the evidence, antibody response can vary with time, with a doubling of antibody response occurring rather abruptly after 14 weeks in the unexposed and 16 weeks in the DMSO-exposed animals. This is probably a secondary response, but it appears late compared with mammals because, at this point, the animals had already received at least six injections of KLH. Given that the animals have been stressed equally at two-week intervals, this cannot be solely a stress response either.
Immunological effects of dietary pesticides Several of the experimental groups (CY, DMSO, and LDI) contained more DDT than the LDDT group (Table 1) but did not display the reduction in antibody levels as observed in the LDDT group. The LDDT group did have significantly higher levels of DDE and sum DDTs, however, and it is possible that DDE or other DDT-metabolites were initiating the observed toxicological stress. Metabolites can have greater or fewer
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effects than the parent compound, and the elevated DDE concentrations measured in both the LDDT and LDI groups might have contributed to the observed suppression of the antibody response (Fig. 1). Overall, however, the groups subjected to the low doses of both DDT and dieldrin showed a significant reduction in antibody response, indicating that dietary uptake of pesticides can cause immunosuppression. Although the dieldrin-exposed group showed some recovery after 38 weeks, the DDT-exposed group did not. This suggests different magnitudes of effect or perhaps even different mechanisms of immunosuppression by these two compounds. Oxidative burst is the release of reactive oxygen products from cells, and plays an important role in breaking down antigens within these cells. The sudden increase in oxidative burst that occurred in the HDDT, CY, and LDI groups at week 10 indicated that this aspect of innate immunity might be enhanced by dietary pesticide exposure (Fig. 3). Enhancement of oxidative burst by dieldrin has previously been shown in vitro in both humans and rats [18,19]. In frogs, Gilbertson et al. [7] found suppressed oxidative burst compared with vehicle-exposed controls after intramuscular exposure to 50 ng/g dieldrin. The large variation in oxidative burst levels among individuals and over time in this study indicates that multiple factors were regulating the process; therefore, this assay might be unreliable as an indication of toxicological effects on immune responses (Fig. 4). Moreover, previous studies have demonstrated enhanced oxidative burst resulting from in vitro DMSO exposure [20]. The effects of in vivo DMSO exposure on oxidative burst should be investigated directly in the future to rule out questions on the validity of its use as a control for this test. Delayed-type hypersensitivity, DTH, is a measure of T cell function. These cells are important in mediating B cell activation, mediating some inflammatory responses and killing infected cells. Although Gilbertson et al. [7] found increased cellular response after intramuscular exposure to DDT, dieldrin, and malathion, other studies have found reduced cellular response after dietary exposure to cyclophosphamide and PAHs, as well as DDT and dieldrin [21–23]. None of the above studies, however, measured true secondary DTH response because they did not preinject their antigen. During a normal DTH response, the secondary response should be higher than the primary response. None of our groups showed a significant change between the primary and secondary DTH response, indicating that the secondary response was suppressed and that DMSO had an effect on DTH (Fig. 5). Because the effects occurred in the group treated with DMSO alone, it is impossible to determine whether this effect is mostly mediated by DMSO or if it also includes a pesticide-induced effect. This assay showed substantial interindividual variability and is perhaps not a good method for assessing cellular responses in frogs. Humoral immunity is the component of the immune system that is responsible for protection against bacterial and other extracellular pathogens. Antibodies play an important role in binding and facilitating uptake of antigens into cells so that they can be destroyed. Suppression of specific IgM by DDT and dieldrin, as observed in this study, as well as in previous studies [7], can compromise the ability of R. pipiens to overcome infections. Further substantiation of effects on animal health caused by this suppression will require a disease challenge study. Antibody suppression was previously reported in R. pipiens after a single dose of 923 g kg⫺1 p,p⬘-DDT, 50
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Fig. 5. Change in toe thickness because of a delayed-type hypersensitivity (DTH) reaction before (Pre), five weeks after (5 wk), and 10 weeks after (10 wk) pesticide exposure. Each bar represents the mean ⫾ 1 SE change in toe thickness, between 24 and 48 h, due only to the antigen. The pre-exposure antigen was chicken egg lysozyme (CEL), and the five-week and ten-week antigen was keyhole limpet hemocyanin linked to dinitrophenol (KLH-DNP). (A) The DTH response of control, dimethyl sulfoxide (DMSO)–exposed frogs (n ⫽ 6). (B) The DTH response of the cyclophosphamide (Cy)-exposed group. By 10 weeks, the net dose was 9,000 ng/g (n ⫽ 4). (C) The DTH response of frogs exposed to a low dose of dieldrin (LDI); the net dose over 10 weeks was 2.1 ng/g (n ⫽ 6). (D) The DTH response of frogs exposed to a high dose of dieldrin (HDI); the net dose over 10 weeks was 21 ng/g (n ⫽ 5). (E) The DTH response of frogs exposed to a low dose of DDT (LDDT); the net dose over 10 weeks was 75 ng/g (n ⫽ 4). (F) The DTH response of frogs exposed to a high dose of DDT (HDDT); the net dose over 10 weeks was 750 ng/g (n ⫽ 5).
g kg⫺1 dieldrin, or 990 g kg⫺1 malathion [7]. Additionally, mice, rats, and rabbits displayed suppressed antibody levels after chronic dietary or water exposure to DDT [24–26], indicating that DDT has this effect in mammals as well as amphibians. This study revealed, however, that when exposure occurs through diet, antibody suppression was more pronounced at the lower doses than at the higher doses. Suppression of immune function after exposure to very low pesticide doses, but not after higher doses, indicates a possible hormetic effect. Previous evidence of hormetic effects on the immune response was reported by Fan et al. [27]. They found that the DTH response of rats was enhanced and then suppressed with increased exposure to 2,3,7,8-tetrachlorodibenzodioxin [27]. Rana pipiens exposed to food containing DDT and dieldrin did not demonstrate measurable effects on innate immunity or cell-mediated immunity during chronic exposure. This is in contrast to previous studies, and although tissue analysis demonstrated that the pesticides were present in the body, they were present at lower levels because of the different methods of dosing. It is also conceivable that the dose–response relationships might differ when pesticides are administered as an acute intraperitoneal injection or via chronic exposures to contaminated food. During acute exposures, pesticide concentrations in blood would be maximized soon after injection and then decrease as the pesticide becomes distributed throughout body tissues or is lost via metabolic biotransformation or passive diffusion to air, feces, or urine. In contrast, chronic dietary dosing would be expected to produce much lower blood pesticide concentrations after assimilation of ingested food items,
Dietary pesticide–induced immunosuppression in Rana pipiens
but the lower blood pesticide concentration would slowly increase with each feeding event until the animal achieves steady state with its food. Given the long half-lives of DDE and dieldrin in frogs [7], it is unlikely that the animals achieved steady state with their food even after 10 weeks; therefore, the true toxicological significance of this dietary dose could be underestimated. Low levels of pesticide exposure, 75 ng/g DDT and 2.1 ng/g dieldrin, demonstrated obvious negative effects on the immune response of R. pipiens that last for 38 weeks or more, which, in the wild, could possibly compromise the ability of this species to deal with stress and pathogens in its environment. Because pesticides and other chemicals are widely used and released into the environment, their presence could be a contributing factor to the declines in amphibians.
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