Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs

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Environmental Research 98 (2005) 64–72 www.elsevier.com/locate/envres

Dietary exposure to Aroclor 1254 alters gene expression in Xenopus laevis frogs Anna M. Jelaso, Cari DeLong, Jay Means, and Charles F. Ide Environmental Institute and the Great Lakes Center for Environmental and Molecular Sciences, Western Michigan University, Room 3924 Wood Hall, Kalamazoo, MI 49008, USA Received 17 February 2004; received in revised form 19 May 2004; accepted 21 May 2004 Available online 6 July 2004

Abstract Polychlorinated biphenyls (PCBs) are persistent environmental pollutants that contribute to worldwide health problems. Despite data associating PCBs with adverse health effects, decisions to clean up contaminated sites remain controversial. Cleanup decisions are typically based on risk assessment methods that are not sensitive enough to detect subtle changes in health. We have recently shown that gene expression signatures can serve as sensitive molecular biomarkers of exposure and related health effects. Our initial studies were carried out with developing Xenopus laevis tadpoles that were exposed to the PCB mixture Aroclor 1254 (A1254) for 2 days. A1254 was dissolved in dimethyl sulfoxide and added to the aquarium water for rapid loading of PCBs into the tadpole tissue. These studies showed that increases in the expression of specific genes occurred independent of adverse health effects, and decreases in specific genes correlated with the appearance of observable health effects, including decreased survival and gross morphological and behavioral abnormalities. In this report, we extend our previous work to test the use of gene expression signatures as biomarkers in frogs exposed to PCBs through the diet from early tadpole stages through metamorphosis. This work showed that chronic lowdose exposure to A1254 (24 ppm) in food produced tissue levels of 17 ppm and increased gene expression of nerve growth factor and proopiomelanocortin independent of adverse health effects. Exposure to higher doses of A1254 (200 ppm) produced tissue levels of 80 ppm and increased expression of p450 1A1, also, independent of adverse health effects. This work provides further evidence for the use of gene expression changes as biomarkers of exposure to PCBs. r 2004 Elsevier Inc. All rights reserved. Keywords: PCBs; Gene expression; Biomarker; Metamorphosis; Frog

1. Introduction Polychlorinated biphenyls (PCBs) are ubiquitous pollutants that pose a global environmental health problem (Safe, 1994; Mayes et al., 1998). They were manufactured in the United States in the 1950s for use in electrical transformers and capacitors, plasticizers, and carbonless paper products. Although their manufacture was banned in the 1970s they are highly resistant to degradation and persist in the environment (Danse et al., 1997; Pruitt et al., 1999). PCBs are lipophilic and concentrate in biological tissues, placenta, and blood. Corresponding author. Fax: +269-387-2272.

E-mail address: [email protected] (A.M. Jelaso). 0013-9351/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2004.05.014

PCBs bioaccumulate along successive levels of the food chain (Peakall, 1992; Safe, 1994; Weisglas–Kuperus, 1998). The most common route of exposure is through the consumption of contaminated fish (MDEQ, 1998; Faroon et al., 2001; Schantz et al., 2001). Numerous studies have shown that PCBs cause adverse health effects in wildlife and humans. For example, PCBs cause reproductive system dysfunction (Hornshaw et al., 1983; Heaton et al., 1995; Mendola et al., 1997; Raychoudhury et al., 2000; Fernie et al., 2001), endocrine disruption (Colborn et al., 1993; Fox, 2001; French et al., 2001; Gore, 2001; Kimbrough and Krouskas, 2001), immunological effects (Tryphonas, 1995; Weisglas-Kuperus, 1998; Grasman and Fox, 2001; Belles–Isles et al., 2002), and neurodevelopmental and

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neurological deficits (Huisman et al., 1995; Jacobson and Jacobson, 1996, 1997; Patandin et al., 1998; Tilson et al., 1998; Faroon et al., 2001). Health effects most commonly occur in developing organisms; however, reproductive and neurological impairments have also been shown in adult laboratory animals (Altmann et al., 1995; Nishida, 1997; Stewart et al., 2000) and humans exposed to PCBs through occupational exposure or through consumption of Great Lakes fish (Kilburn and Warshaw, 1995; Schantz et al., 2001). Decisions with regard to the necessity and extent of cleanup of contaminated sites are often contentious due to political, economic, and scientific concerns. Cleanup decisions rely on environmental risk assessments that draw upon various factors, including health risk data (Fontenot et al., 2000). Determining the health risks of exposure to PCBs is complicated by the fact that environmental levels are frequently associated with health effects that are nonlethal and occur through subtle changes in biochemical and molecular mechanisms that are difficult to measure. To improve environmental health risk assessment, it is necessary to establish sensitive methods that reliably detect effects of low-dose exposures and subtle changes in physiology that produce nonlethal but significant health effects (Glowa, 1991). Biomarkers are commonly used risk assessment tools that indicate not only whether an exposure to a specific contaminant has occurred but also the biological effects related to that exposure (Silbergeld, 1990, 1993). Sensitive biomarkers that detect molecular-level changes may improve our ability to detect sublethal effects of exposure and elucidate underlying mechanisms of contaminant-induced health effects. Using gene expression signatures as potential biomarkers is a relatively new approach. Our intention was to conduct laboratory-based studies on aquatic organisms and then follow up with field testing in related organisms. The current work in this report is the third in a series of laboratory-based studies that deal with patterns of gene expression in Xenopus laevis frogs treated with Aroclor 1254 (A1254). We have previously shown that changes in gene expression can serve as biomarkers of exposure and related health effects to the PCB mixture A1254 in developing X. laevis tadpoles (Jelaso et al., 2002, 2003). Aroclor 1254, commonly found in the environment consists of both ortho and non-ortho substituted congeners. Changes in gene expression were measured after acute exposure to A1254 (Jelaso et al., 2002, 2003). In our previous studies, A1254 was dissolved in dimethyl sulfoxide (DMSO) and added to the tadpole aquarium water. In Jelaso et al. (2003), we showed that exposure to low doses of Aroclor 1254 (up to 50 ppb) increased expression of nerve growth factor (NGF), proopiomelanocortin (POMC), interleukin-1 converting

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enzyme (ICE), b-actin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in 18-day post-fertilization tadpoles after 2 days of exposure. PCB levels in the tadpole tissue were less than 20 ppm. Increased gene expression occurred independent of observable health effects or long-term changes in survival, demonstrating the use of these gene expression changes as biomarkers of exposure. Higher doses of A1254 (400–700 ppb), after 2 days of exposure, produced tissue levels of 80–160 ppm and decreased gene expression of NGF and b-actin that occurred concomitantly with decreased survival and the appearance of gross morphological and behavioral abnormalities. This study showed that increases in expression of specific genes can serve as biomarkers of exposure to A1254 and that decreases in expression of specific genes can serve as biomarkers of exposure and related health effects. In this study, X. laevis tadpoles were exposed to A1254 through the diet from early development through metamorphosis. This study was designed to validate our previous work but in this case using an exposure method that reflects a natural route of exposure. Taqman reverse transcriptase (RT)-PCR was used to measure changes in gene expression for six genes that play important roles in normal development and physiology (NGF, dopamine type 2 receptor (D2 Rt), ICE, POMC, GAPDH, and bactin) and for p450 1A1, a metabolic enzyme that is frequently upregulated by PCBs. Survival, gross morphology, and behavior were monitored and tissue levels of PCBs were measured using GC/MS.

2. Materials and methods 2.1. Animals X. laevis tadpoles were obtained by injecting male and female adult frogs with human chorionic gonadotropin (Sigma, St. Louis, MO). Tadpoles were reared in spring water (Absopure, Plymouth, MI) for the first 14 days to increase survival and then transferred into deionized tap water. Tadpoles were staged according to Nieuwkoop and Faber (1994) and were treated in accordance with an animal use protocol approved by the Institutional Animal Care and Use Committee of Western Michigan University. 2.2. Dietary exposure of tadpoles Dietary exposure to A1254 was started at the time that X. laevis tadpoles begin feeding, at approximately 5 days post-fertilization (approx. stage 44/45). Tadpoles were sorted without bias into the following treatment groups: control, 24, 50, 100, or 200 ppm. Twelve tadpoles were assigned to each treatment group.

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Tadpoles were reared in 20
20-cm2 glass dishes; four tadpoles per dish. A1254 was purchased from AccuStandard (New Haven, CT). Tadpole food was prepared by making a stock solution of A1254 (50 mg) dissolved in hexane (100 mL) for a final concentration of 500 ppm. An aliquot of stock solution was dissolved in 95% ethanol and added to commercially available tadpole food (Nasco) to obtain doses of either 24, 50, 100, or 200 ppm A1254. The food was incubated at room temperature in a fume hood until dry. PCB levels were measured using GC/MS. Control food was prepared in a similar manner, but by adding hexane with no A1254 and 95% ethanol. Tadpoles (one dish) were fed a scoop (0.02 g) of frog food containing A1254 (or control food) every day and changed into fresh deionized tap water every 3 days. Each bowl received the same amount of food per feeding. Tadpoles were exposed from 5 days post-fertilization (approx. stage 44/45) through metamorphosis (stage 64/65). Gross morphology, behavior, and development were observed throughout the course of the exposure period. The experiment was repeated twice with tadpoles from two independent spawnings. Animals from the first experiment were used to optimize RT-PCR conditions and to measure PCB tissue levels using GC/MS. Animals from the second experiment were used for RT-PCR analysis of gene expression. Frogs were collected at stage 64/65. Frogs were staged using gross morphological characteristics described in Nieuwkoop and Faber (1994); almost complete resorption of the tail was the most prevalent staging characteristic. We chose to harvest the animals at a specific stage and record the time to that stage in order to assess changes in metamorphic rate in the different treatment groups. Stage 64/65 was chosen because mortality occurs between stages 64/65 and complete metamorphosis at stage 66. Therefore, we chose to assay animals that had clearly reached a metamorphic state, but were a few days short of complete metamorphosis. The number of days that it took the frogs to reach stage 64/65 was recorded. The frogs were anesthetized in tricaine methanesulfonate (MS-222; diluted 1:2000), rinsed in deionized tap water, weighed, and dissected to collect tissue samples for RNA isolation and GC/MS analysis. The limbs were removed and stored at 20 1C until extraction for GC/MS analysis. The head and body region was snap frozen in liquid nitrogen and stored at 80 1C until RNA isolation. 2.3. RNA extraction Total RNA was isolated using Qiazol reagent and Qiagen RNEasy Maxi kits following the manufacturer’s instructions (Qiagen, Valencia, CA). Following lysis, 50 ng of yeast mRNA (Clontech, Palo Alto, CA) was

added to the homogenate for each treatment group to obtain an exogenous control for Taqman RT-PCR. mRNA was isolated from the total RNA from each treatment group using Oligotex mini kits according to the manufacturer’s instructions (Qiagen). The mRNA concentration of each treatment group was determined by measuring the absorbance at A260 using a GeneQuant Pro spectrophotometer (Amersham Pharmacia Biotech, Piscataway, NJ). 2.4. Real-time PCR Gene sequences used for primer and probe design were obtained from GenBank. Primers and dual-labeled fluorescent probes were designed with Primer Express software (version 1.5; Applied Biosystems, Foster City, CA). All probes were synthesized with a FAM (6carboxy-fluorescein) reporter molecule attached to the 50 end and a TAMRA (6-carboxy-tetramethyl-rhodamine) quencher linked at the 30 end. Taqman RT-PCRs were carried out for the following seven genes: NGF, D2 Rt, ICE, POMC, p450 1A1, GAPDH, and b-actin. RTPCRs using primers and probe for yeast actin were run in each plate to serve as an exogenous control. Primer and probe sequences and the physiological functions of each gene were previously published in Jelaso et al. (2002, 2003). Each RT-PCR was performed in a 25-mL final volume containing 1.25 U/mL of MultiScribe RT, 300 nM forward and reverse primer, 200 nM Taqman probe, 2
Master Mix without uracil N-glycoslyase, and an equivalent concentration of mRNA template for each treatment group. All RT-PCR reagents were purchased from Applied Biosystems. For each gene, all RT-PCR runs contained control reactions without template and without RT and with a four-point standard curve. All reactions were run in duplicate in 96-well plates. An Applied Biosystems Prism 7700 Sequence Detection System was used for amplification and fluorescence detection. Thermal cycling parameters were 48 1C, 30 min, 10 min at 95 1C, and then 40 cycles of 95 1C for 15 s, 60 1C for 1 min. The relative standard curve method was used to quantitate mRNA levels for each target gene (Jelaso et al., 2002, 2003). Standard curves were generated using serial dilutions of mRNA (1, 2, 4, and 8 ng) from untreated control frogs. These concentrations were used to generate the standard curve as they produced curves with correlation coefficients of 0.99 and slopes closest to –3.3, indicators of efficient RT-PCR conditions; 4 ng of mRNA from each treatment group was used as a template for each RT-PCR. An equivalent amount of yeast mRNA (50 ng) was added to each sample during the total RNA isolation as an exogenous control for the RT-PCR. For each RTPCR run, reactions using mRNA template from each

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2.5. PCBs GC/MS analysis Reagents: PCB standards and internal standards were purchased from AccuStandard and Ultra Scientific (North Kingstown, RI). An internal standard, 4,40 dibromo-octafluoro biphenyl (DBOFP) was purchased from Ultra Scientific. Solvents utilized in the extraction process were pesticide-grade hexane and dichloromethane obtained from Baker Scientific (Phillipsburg, NJ). Solid-phase C-18 used for matrix solid-phase dispersion (MSPD) extraction was purchased from Varian Scientific (Cary, NC). Extraction: frog tissue samples were extracted for PCBs using a MSPD method (Barker et al., 1993). Briefly, the tissue was weighed and combined with 5 g of dry, precleaned C-18 solid-phase material in a glass mortar, spiked with 25 mL of a solution containing the five deuterated surrogate standards (40 mg/mL each), and then thoroughly ground into a homogeneous powder with a glass pestle. The C18 was then transferred quantitatively into the barrel of a disposable 10-mL plastic syringe plugged at the end with a 4-mm glass fiber filter and a small amount of inactivated glass wool. The MSPD columns were then eluted as follows: the column bed was first saturated with approximately 2 mL of dichloromethane and allowed to stand for 20 min. An additional 8 mL of dichloromethane was passed through each MSPD column and allowed to elute by gravity into a 40-mL glass vial. In some cases, the solvent was slowly forced through the column using a syringe. The solvent eluent was evaporated to approximately 2 mL under a stream of pure nitrogen, transferred quantitatively to a 4-mL amber glass vial, and further evaporated under nitrogen to a final volume of 200 mL. Samples were stored at –20 1C until gas chromatograph/ mass spectrometer analysis. GC/MS analysis of PCB congeners: the PCB congeners were quantified by the multiple selected ion monitoring (SIM) GC/MS method adapted from Means (1998) and McMillin and Means (1996). Analysis was performed on 2-mL samples of the extracts using an Agilent Technologies (Palo Alto, CA) (AG) 6890A gas chromatograph equipped with the capillary column (AG DB-5MS) (30 m
0.025 mm i.d.), which was directly interfaced to an AG 5973N mass selective detector,

equipped with a 7683 autosampler. A temperature program for the GC oven using a series of linear temperature ramps from 50 to 300 1C to optimally separate the analytes was developed. The mass spectrometer was tuned daily and/or after each 16 h of analysis using perfluorotributylamine. A multiple SIM method was developed for monitoring the total amounts of CL1–CL10 congener groups in the appropriate retention window(s). The congener groups were quantified using a calibration standard containing a representative congener from each chlorination group (AccuStandard). Individual congeners were quantified using authentic individual standards or mixtures. Determination of method detection limits: detection limits for each analyte in the sample matrix type were estimated from statistical information derived from standard calibration curves (Taylor, 1987). For the frog tissue samples, this limit was typically 5–12 ng/g wet wt (ppb) with a mean value of 9 ng/g. Samples were spiked immediately before injection with 10 mL of a 100mg/mL solution of DBOFP as an internal standard.

3. Results 3.1. Aroclor 1254 alters metamorphic rate in developing frogs Fig. 1 shows the average number of days that it took for the animals in each treatment group to reach stage 64/65 for both experiments. In experiment 1, the tadpoles exposed to higher doses (100–200 ppm) reached stage 64/65 later than the controls. This delay was 70 Expt 1 Expt 2

65 Age (days)

treatment group were performed with primers and a Taqman probe for yeast actin. The relative expression level for each target gene was calculated by dividing the target gene value by the yeast actin (exogenous control) value (Jelaso et al., 2002, 2003; Smith et al., 2003). The mRNA values for each treatment group were expressed as a percentage of the untreated control group expression value. Statistical comparisons were performed using ANOVA and Fisher’s post-hoc test (Statview; SAS Institute, Cary, NC).

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*

*

100 ppm

200 ppm

60

55

50

45 Control

24 ppm

50 ppm

Treatment Group Fig. 1. Number of days that the frogs took to reach stage 64/65 of development. Growth rate was recorded in two independent experiments. In Experiment 1 (triangles) there was a significant delay in growth rate in frogs treated with 100 and 200 ppm compared to controls ( N ¼ 8, Po 0:05). In Experiment 2 (squares) there was a similar trend, but the results were not statistically significant.

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Table 1 Statistical values of gene expression changes

350 300

*

NGF POMC

P value 0.032 0.004 F value 3.282 5.436

0.411 1.040

GAPDH b-actin P450 1A1

0.069 0.408 2.577 1.063

0.643 0.638

0.004 8.024

250 % Control

NGF POMC D2 Rt ICE

One-way ANOVA was calculated using Statview software. P and F values for each gene are presented. Fisher’s post-hoc test was calculated for genes with P values o0.05. P values for Fisher’s posthoc tests are described in the legend to Fig. 2.

200

*

150 100 50 0 Control

24 ppm

(A)

50 ppm

100 ppm

200 ppm

Treatment Group 350 300

ICE D2 Rt

250 % Control

statistically significant (N ¼ 8, Po 0:05). In the second experiment, frogs exposed to higher doses of A1254 (100–200 ppm) also reached stage 64/65 later than the controls. However, this result was not statistically significant. There were no significant changes in survival or any observable changes in gross morphology or behavior in any of the treatment groups.

GAPDH b-actin

200 150 100

3.2. Aroclor 1254 alters gene expression in newly metamorphosed frogs

50 0 Control

24 ppm

(B)

800

100 ppm

200 ppm

*

p450

700 600 500 400 300 200 100 0 Control

3.3. PCB accumulation in frog tissues

50 ppm Treatment Group

900

% Control

ANOVA P and F values for each gene are listed in Table 1. Fig. 2A shows that dietary exposure to 24 ppm A1254 produced a statistically significant increase in expression of POMC and NGF (N ¼ 5). Fig. 2B shows that exposure to 24 ppm A1254 also increased expression of the D2 Rt, ICE, GAPDH, and b-actin (D2 Rt, ICE: N ¼ 5; GAPDH, b-actin: N ¼ 4), although this effect was not statistically significant. Exposure to higher doses (50–200 ppm) had no significant effect on expression of these genes. Exposure to 200 ppm A1254 significantly increased expression of p450 1A1 (N ¼ 4; Fig. 2C).

(C)

24 ppm

50 ppm

100 ppm

200 ppm

Treatment Group

PCB levels were measured in the hindlimbs and forelimbs of individual frogs from each treatment group from the first experiment using GC/MS. PCB accumulation in frog limbs ranged from 16.4371.61 to 79.81+5.59 ppm (24 and 200 ppm in food, respectively). The mean PCB tissue levels for each treatment group are shown in Table 2. There were no detectable levels of PCBs present in the control frog tissues.

Fig. 2. Changes in gene expression in frogs after chronic exposure to A1254. (A) Exposure to 24 ppm A1254 significantly increased expression of NGF (Po 0:05, N ¼ 5) and POMC compared to control (Po 0:01, N ¼ 5). (B) Exposure to 24 ppm A1254 increased expression of ICE (N ¼ 5), D2 Rt (N ¼ 5), GAPDH (N ¼ 4), and b-actin (N ¼ 4). b-actin expression was elevated after exposure to 50 and 100 ppm Aroclor 1254. These results followed the same trend as those of NGF and POMC, but were not statistically significant. (C) Exposure to 200 ppm Aroclor 1254 significantly increased p450 1A1 expression (Po 0:01, N ¼ 4).

4. Discussion

Table 2 PCB tissue levels of frogs exposed to A1254

The concentrations of A1254 (24–200 ppm) added to the frog food reflect levels of PCBs found in fish and wildlife in contaminated watersheds, including the Hudson River and Kalamazoo River Superfund sites. For example, levels of up to 160 ppm in surfical sediment and of 50–250 ppm have been reported in fish tissues in the Kalamazoo River Superfund site (MDEQ,

Treatment group (ppm)

PCB level (ppm)

Control 24 50 100 200

0 16.4371.61 24.6871.83 55.2574.01 79.8175.59

Tissue levels are represented as mean7SD.

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1998; Blasland, 2000). In addition, the concentrations of A1254 used in this study are similar to those in other laboratory-based exposure studies (Gray et al., 1993; Corey et al., 1996; Fowles et al., 1997; Hood and Klaasen, 2000). For example, Corey et al. (1996) exposed adult rats to 125 or 250 ppm A1254 through diet for 60 days and found depressed levels of choline acetyltransferase activity in the brain and memory impairment in exposed animals, and Hood and Klaasen (2000) exposed adult rats to either 25, 50, 100, or 200 ppm A1254 through diet and showed altered levels of thyroid hormone deiodinase activities. Long-term dietary exposure to A1254 produced no significant changes in survival, gross morphology, or behavior. In previous studies, we showed that acute exposure to A1254 significantly reduced survival in 11and 18-day-old tadpoles (25 and 50 ppm in 11-day-old tadpoles; 700 ppb in 18-day-old tadpoles). Reduced survival occurred concomitantly with the appearance of gross morphological and swimming behavior abnormalities (Jelaso et al., 2002). In the previous studies, A1254 was dissolved in DMSO and added to the aquarium water, a method that rapidly loaded the tadpole tissues with PCBs. Adverse health effects appeared in tadpoles with high tissue levels of PCBs (216 ppm in 11-day-old tadpoles exposed to 50 ppm A1254, 171 ppm in 18-day-old tadpoles exposed to 700 ppb A1254). In the present study, frog tissue levels were considerably lower (80 ppm in highest dose) than the levels associated with mortality and observable health effects in our previous studies that exposed tadpoles via A1254 dissolved in DMSO in aquarium water. 4.1. Exposure to A1254 altered metamorphic rate In this study, we showed that exposure to higher doses of A1254 (100 and 200 ppm) delayed metamorphosis. This effect may be related to a PCB-induced alteration of POMC gene expression. Although not statistically significant, POMC gene expression decreased to 50% and 64% in animals treated with 100 and 200 ppm A1254, respectively. Amphibian metamorphosis is regulated by complex interactions among brain-derived neurohormones and regulatory factors and the thyroid hormone system (Eliceiri and Brown, 1994; Tata, 1996; Denver et al., 1997). Corticotropin-releasing hormone, produced in the brain, stimulates production of adrenocorticotropic hormone (ACTH) and thyroid-stimulating hormone in the pituitary (Denver et al., 1997). ACTH is one of three peptides cleaved from the POMC prohormone with well-described physiological effects. Gutleb et al. (1999, 2000) also showed that exposure to PCBs delays metamorphosis in developing X. laevis frogs, although in those studies frogs were exposed to lower doses of a different PCB mixture (2 ppm Clophen

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A50) or a single PCB congener (200 ppb PCB 126). Reduced growth rate has been reported in Dutch infants exposed to PCBs in utero (Patandin et al., 1998) and in children exposed in the Yusho and Yu-Cheng incidents (Yoshimura and Ikeda, 1978).

4.2. Exposure to low doses of A1254 increased gene expression In our previous studies, we showed that increases in gene expression occurred in developing tadpoles after acute exposure to low doses of A1254 (Jelaso et al., 2003). Eighteen-day-old tadpoles exposed to 5 and 50 ppb A1254 for 2 days had PCB tissue levels of less than 20 ppm and showed significant increases in NGF, POMC, ICE, GAPDH, b-actin, and p53. Increases in gene expression occurred independent of any observable health effects, indicating that they may be useful biomarkers of exposure. In the present article, we show that long-term dietary exposure to low doses of A1254 (24 ppm) in food produced tissue levels (16 ppm) similar to those of 18day-old tadpoles exposed to 50 ppb A1254 (15 ppm) through the aquarium water. In both experiments, gene expression of NGF and POMC increased. Increases in ICE, D2 Rt, GAPDH, and b-actin also occurred but were not statistically significant. Most likely, the changes in ICE, D2 Rt, GAPDH, and b-actin were not statistically significant because there was greater variability in the animals’ response for these genes. In previous work and in this study, increased expression of NGF and POMC occurred in frogs using two very different exposure paradigms, indicating that changes in expression of specific genes are reliable indicators of A1254 exposure in frogs. NGF is a neurotrophic factor molecule that plays an important role in the developing nervous system. It regulates growth, differentiation, and survival of developing neurons and regulates the neural response to injury in both the developing and the adult nervous systems (Deshmukh and Johnson, 1997; Levi-Montalcini and Aloe, 1985). In the present report, NGF gene expression was increased by chronic exposure to 24 ppm A1254. Subtle health effects were not analyzed and whether there are any adverse health effects that result from excess NGF is not known. However, studies with other laboratory animal species have shown that excess NGF is associated with adverse health effects. For example, Heath et al. (1998) showed that overexpression of NGF in the mouse heart decreases the current density of potassium channels, which contributes to ventricular arrhythmias associated with cardiac hypertrophy and failure. Excess NGF is also associated with ovarian dysfunction and the formation of ovarian follicular cysts (Dissen et al., 2000; Lara et al., 2000).

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POMC is a gene that encodes for three individual proteins involved in the regulation of endocrine system functions: ACTH, b-endorphin, and melanocyte-stimulating hormone. In this report, exposure to low levels of A1254 (24 ppm) increased expression of POMC. This result is similar to that of our previous study which showed that acute exposure to low doses of A1254 increased expression of POMC in 18-day-old tadpoles (Jelaso et al., 2003) and that of Bestervelt et al. (1998) who showed an increase in circulating levels of ACTH in mice after exposure to TCDD. 4.3. Exposure to high doses of A1254 increased expression of p450 1A1 Cytochrome p450 enzymes play important roles in steroid metabolism and the detoxification of xenobiotics (Denison and Whitlock Jr., 1995; Troisi and Mason, 2000). Cytochrome p450 1A1 is an inducible enzyme that mediates the partial degradation and detoxification of PCBs through activation of the aryl hydrocarbon (Ah) receptor. Non-ortho substituted PCB congeners can bind to the Ah receptor. A1254 is a mixture that contains both ortho and non-ortho substituted congeners. Numerous studies have shown that p450 1A1 is induced after exposure to PCBs (Dragnev et al., 1995; Bandiera et al., 1997; Drahushuk et al., 1997; Fowles et al., 1997; Yeung et al., 2003). P450 1A1 is a wellestablished biomarker of exposure to PCBs in many different organisms. In this study, frogs exposed to the highest dose (200 ppm) had high tissue levels of PCBs (80 ppm) and increased p450 1A1 gene expression, indicating that p450 1A1 can serve as a biomarker of exposure to higher doses of A1254 in adult frogs. Exposure to lower doses (24–100 ppm) did not induce significant p450 1A1 gene expression in this study. In our previous studies, there was no significant change in p450 1A1 in 11- or 18-day-old tadpoles despite very high tissue levels of PCBs (up to 200 ppm). Some of the 18day-old tadpoles showed dramatic increases in p450 1A1 gene expression, but due to a large variability in the response of different animals the result was not significant. Most likely, the p450 system was beginning to mature around 18 days of age. In this report, increased p450 1A1 expression occurred in frogs independent of adverse health effects or changes in survival. Frogs had relatively high tissue levels of PCBs (up to 80 ppm), indicating that the frogs may have a mechanism for coping with chronic exposure to higher doses of A1254.

5. Conclusion In the present report, we demonstrate that longterm dietary exposure to the PCB mixture A1254

produces changes in gene expression in newly metamorphosed frogs. Statistically significant increases in two key nervous system genes, NGF and POMC, occurred after exposure to low doses of A1254 independent of observable adverse health effects. At higher doses, gene expression returns to normal levels, indicating that the frog response to A1254 does not follow a linear dose–response pattern; rather, it more closely follows the inverted U-shaped response to contaminants associated with hormesis (Calabrese and Baldwin, 2002). On the other hand, the p450 1A1 gene expression response showed no change from normal except in the highest dose (200 ppm), indicating that the Ah receptor system in X. laevis frogs may respond only to high tissue levels (80 ppm) of xenobiotics. Interestingly, even at tissue levels of 80 ppm and increased expression of p450 1A1, no adverse health effects were obvious. P450 may thus play a role in protecting frogs from the effects of PCBs. This report demonstrates that increases in expression of specific genes are associated with exposure dose and tissue concentration of PCBs. These results are in agreement with our previous acute exposure studies (Jelaso et al., 2002, 2003), providing further evidence for the use of gene expression changes as biomarkers of exposure.

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