* Environmental and Occupational Toxicology Division, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada; Toxicology Research Division, Health Products and Foods Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada
1 To whom correspondence should be addressed at Room 320, Environmental Health Centre, P/L 0803B, Tunney's Pasture, De la Colombine Blvd, Ottawa, Ontario K1A 0L2, Canada. Fax: (613) 957-8800. E-mail: Ih_chu{at}hc-sc.gc.ca.
Received July 29, 2005; accepted September 15, 2005
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ABSTRACT |
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Key Words: systemic toxicity; organochlorine mixtures; Aroclor 1254; PCBs; Great Lakes/St. Lawrence region.
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INTRODUCTION |
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Although studies in which rats were fed with contaminated fish can best mimic human exposure to mixtures of pollutants found in fish, they do have certain limitations. The doses of contaminants given to laboratory rats are limited by the quantity of fish meals consumed by the rats, thus precluding establishing good doseresponse relationships for toxicological effects. Nutrients in fish diets may interact to alter toxicological responses to the pollutants. In view of the constraints of previous POP mixture studies, we conducted a multi disciplinary study to evaluate the toxic effects using a reconstituted POP mixture based on the contaminant profiles in the blood of women with a high fish diet in the Great Lakes/St. Lawrence region (Kearney et al., 1999; Kosatsky, 1999a
, 1999b
). The present study has a number of advantages over previous studies in that it investigates developmental pathology, neurobehavioral pathology, systemic toxicity, brain neurochemistry, and molecular pathology in subsets of rat offspring in the same study. Through investigation of related toxicological end points in subsets of rats, results can be interpreted more meaningfully because some effects may be related to others and occur as sequels to them. Using a reconstituted mixture of POPs to dose animals permits the investigation of the effects at a wide range of doses to establish a clear doseresponse relationship. Further, by investigating the molecular pathological end points of rat brain, the mechanisms of neurotoxicity can be explored. The first in this series of studies reported early developmental effects (Bowers et al., 2004
). The present report describes the clinical, biochemical, liver microsomal enzyme activity and gross and light microscopic findings in the offspring terminated at PND 35, 70, and 350.
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MATERIALS AND METHODS |
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Animal treatment and study design.
Animal treatment and housing conditions were approved by the Animal Care Committee of Health Canada and conformed to the Guidelines of the Canadian Council on Animal Care. Ninety-three nulliparous female Sprague-Dawley rats (200230 g) and 38 male rats (315350 g) were used. Details of animal handling, mating, and rearing have been described previously (Bowers et al., 2004). Briefly, two female Sprague-Dawley rats were mated with one male. Once the mating was confirmed by observation of vaginal plugs, the female rats were removed from the males' cages and housed in individual cages with nesting materials. The pregnant rats were dosed orally with 0.013, 0.13, 1.3, or 13 mg/kg bw/day of the POP mixture from gestational day (GD) 1 to postnatal day (PND) 23. Pups were never dosed directly but were exposed through the dams in utero and during lactation. The pregnant rats were dosed daily with Nabisco Teddy Graham cookies (2 g) laced with measured amounts of the corn oil solution containing the POP mixture. The vehicle control group was given Teddy Graham cookies containing corn oil, and the positive control was dosed with the same kind of cookies laced with 15 mg/kg bw/day Aroclor 1254. At parturition, the pups were counted, and at PND 4, they were culled to 4 male and 4 female pups per litter. Subsets of rat offspring were killed at PND 35, 70, and 350 to collect tissues for analysis. At termination, rats were killed by guillotine, and liver, spleen, brain, kidneys, heart, thymus, testis, uterus, and ovary were dissected and weighed. The organ/body weight and organ/brain weight ratios were calculated, and analyzed statistically. Tissues and blood were collected to measure the biochemical, histopathological, and other systemic end points as described below. Brain molecular biomarkers/end points and brain neurotransmitter levels were measured at all of the three time points. The results will be described elsewhere. Dams were killed at PND 35 for tissue collection and measurement of end points.
Biochemical measurements.
Immediately after guillotine, trunk blood of rats was collected in SST Vacutainer tubes (Becton Dickinson) and allowed to clot at room temperature. Serum was separated by centrifugation at 3000 x g and stored at 80°C until analysis with a Technicon analyzer (Model RA-XT, Technicon-Miles, Mississauga, Ont., Canada). Inorganic phosphate, albumin, creatinine, total protein, alkaline phosphatase, alanine aminotransferase, calcium, cholesterol, glucose, urea nitrogen, uric acid, and lactate dehydrogenase (LDH) were determined in these samples.
A 23-g piece of fresh liver was removed, homogenized in 2.5 volumes of ice-cold 0.05 M tris/1.15% KCl buffer, pH 7.4, and centrifuged at 10,000 x g. The supernatant was separated and assayed for ethoxyresorufin-O-de-ethylase (EROD), pentoxyresorufin-O-de-alkylase (PROD), and benzoyloxyresorufin-O-de-alkylase (BROD) activities according to the method of Lubet et al. (1985).
Measurement of sperm function.
Resistant spermatid nuclei were examined by a previously described method (Wade et al., 2002) in male offspring on PND 70 and PND 350 to determine effects of the mixture on sperm functions. Briefly, either one whole decapsulated testis or one cauda epididymis was weighed and homogenized in 20 ml of a solution made of 0.9 % NaCl, 0.01% Triton X-100, and 0.025% sodium azide using a Waring Ultramicro blender (VWR-Canlab, Mississauga, Ontario) on low setting for 2 min. Homogenates were then disrupted with a VibraCell sonicating dismembranator (Sonics & Materials, Danbury, CT). The density of remaining condensed spermatids (testis) and sperm nuclei (cauda) in the homogenates was estimated by counting at least 6 fields of a hemocytometer.
Histopathology.
Detailed histopathological examination was made on decapitated rat offspring, and organs and tissue were removed and fixed in 10% neutral buffered formalin and processed for Paraplast paraffin embedding. Sections of 4 µ thickness were cut and stained with hematoxylin and eosin. The tissues were assessed by the pathologist with prior knowledge of vehicle control and treated groups, and the pathological results were not subjected to peer review. The severity of changes in nonlymphoid tissues was graded subjectively by the pathologist, who categorized lesions and assigned values ranging from 0 to 5 (0 for normal, 1 for minimal, 2 for mild, 3 for moderate, 4 for marked, and 5 for severe). Enhanced immunopathological examination was conducted on spleen and thymus as described by the ICICIS Group (1998). Spleen was divided into the following zones for detailed assessment: periarteriolar lymphoid sheath (PALS), follicle, marginal zone, and red pulp. Thymus was divided into cortex, medulla, and cortex/medulla ratios for assessing treatment effects. The pathological changes in spleen and thymus were graded subjectively according the following criteria: grade 1, normal; grade 2, normal to 25% difference from normal; grade 3, 2550% difference from normal; and grade 4, >than 50% difference from normal. The differences could be either an increase or decrease and correspond to cellularity and/or areas. Thyroid glands could not be obtained for histological examination because rats were killed by guillotine in order to collect brain for biogenic amines and molecular biomarker analysis. Termination of rats with a guillotine was used because anesthesia followed by exsanguination is known to disrupt biogenic amine analysis (Nakai et al., 2005
). Lymph nodes are not reported because insufficient numbers were examined. Based on gross pathological observation and biochemistry data, rats at PND 350 appeared to be normal and were not examined microscopically. Histopathological evaluation was not performed on the highest dose mixture group (13 mg/kg bw) because of the limited number of rats that survived at PND 35 and beyond.
Statistical analysis.
Organ and body weight data were analyzed by analysis of variance (ANOVA) and when significant differences (p 0.05) were observed, Dunnett's test was used to compare each of the dose groups and the vehicle control. Tukey's HSD test was used for other pairwise comparisons. Biochemical data were analyzed by ANOVA followed by Duncan's multiple range test.
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RESULTS |
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Postnatal Day 70.
Organ weight changes were observed only in Aroclor-treated groups and consisted of the following:
Postnatal Day 350.
The Aroclor groups showed increased relative liver weights (control male: 2.4 ± 0.30%; treated male: 2.7 ± 0.35%; control female: 2.4 ± 0.23%; control female: 2.8 ± 0.40%), and absolute (control: 3.5 ± 0.42 g; treated: 3.8 ± 0.43 g) and relative testis weights (control: 0.84 ± 0.09%; Aroclor: 1.0 ± 0.14%). The weights of other organs were not affected.
Biochemical Measurements
Serum cholesterol showed a treatment-related increase in highest dose mixture males and Aroclor groups at PND 35 (Table 2). Urea nitrogen was increased in the surviving male rats of the 13 mg/kg mixture group at PND 35, and in females at PND 70. The female rats of the highest dose and Aroclor groups had decreased LDH values at PND 70 but not at other time points. No effects were observed on inorganic phosphate, albumin, creatinine, total protein, alkaline phosphatase, alanine aminotransferase, calcium, glucose, and uric acid.
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Effects of the mixture on hepatic EROD, PROD, and BROD activities in the offspring are shown in Figure 1. There was a significant increase in EROD activity in the 1.3 and 13 mg/kg bw mixture and Aroclor 1254 groups of both genders at PND 35. At PND 70, only the 13 mg/kg mixture and Aroclor groups of both sexes showed increased activity. The effect on EROD was more persistent in male rats treated with the highest dose mixture and Aroclor, as elevation in the activity of this enzyme remained evident at PND 350. An increased PROD activity was seen in the Aroclor-treated offspring of both genders at PND 35, whereas elevation of this enzyme activity was observed in female rats exposed to the highest dose mixture at PND 35 and PND 70. The BROD activity was increased in the highest dose mixture rats of both genders at PND 35, PND 70, and PND 350. Although female, not male, rats showed a statistically significant increase in BROD at PND 350, the magnitude of the increases appeared to be the same for both genders. Aroclor 1254 caused elevations in BROD activity of both males and females at PND 35 (Fig. 1).
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Histopathology
Treatment-related histological changes were observed in the liver, spleen, and thymus. There was also a trend of treatment-related changes in the ileum.
Liver.
In vehicle rats there was a background level of very mild inflammation characterized by random sinusoidal granulomas. There was a dose-dependent increase in severity of this inflammation in POP mixturetreated animals (Table 3; Fig. 2). The degree of inflammation was increased in the PND 35 and PND 70 Aroclor groups. Mild hepatocellular hypertrophy was common in all mixture groups and was slightly more pronounced in the Aroclor animals at PND 35 and PND 70. At PND 35, 6 of 12 rats in the 1.3 mg/kg group showed slight bile duct hyperplasia. Mild hepatocellular vacuolation with variable lobular distribution occurred in a few rats in all the treated groups. Among Aroclor-treated animals terminated at PND 35, there was a low incidence of rats with cholangiofibrosis, cirrhosis, fibroacinar formation, and oval cell proliferation (Table 3). In the PND 70 Aroclor group, fibroacinar formation occurred in 2 of 4 males and 2 of 8 females. One female had 2 foci of altered hepatocytes (1 clear cell, the other eosinophilic). One male had bridging fibrosis with cholangiofibrosis.
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Testis.
Examination of testis homogenates showed no effects on sperm functions.
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DISCUSSION |
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The effects observed were attributed to the combined effects of the total POP mixture rather than to any individual components therein, because at the low levels present in the mixture, individual components are not known to cause any effects.
Over the years, different approaches have been adopted to investigate the effects of mixtures, each of which has its strengths and weaknesses. Chu et al. (1984) and Arnold et al. (1998)
used diets supplemented with POP-contaminated salmon. Administering fish oil as the source of concentrated POPs represents another approach to investigating the effects of mixtures (Stern et al., 2002
; Roegge, 2005
). Because of the limited quantity of fish and fish oil that can be incorporated into diet, more definitive toxicological effects of high-dose exposure could not be demonstrated. Nevertheless, mild treatment-related effects such as hepatic enzyme inductions were demonstrated in these studies (Chu et al., 1984
; Iverson et al., 1998
; Stern et al., 2002
). While the dose levels of the POP mixtures in these studies cannot be readily compared because different units of doses were employed (e.g., TEQs, and % fish diets versus mg test substance/kg bw), an examination of the chemical components of the dosed materials in these studies revealed similar profiles of pollutant components existing among these test materials, with PCBs and DDE/DDT being the major chemical components.
Rat offspring had increased liver microsomal EROD, PROD, BROD activities at doses of 1.3 mg/kg and above. An increase in EROD and MROD activities was reported in F0 and F1 rats given a 20% lyophilized Lake Ontario fish diet (Iverson et al., 1998), and in rats receiving subchronic exposure to the high concentration of fish oil, which was equivalent to 34 times the exposure following human fish consumption (Stern et al., 2002
). Effects on hepatic microsomal enzymes are not considered a serious adverse effect. Rather, they are considered an adaptive response to metabolizing and detoxifying xenobiotics. Concurrent with the enzyme inductions, mild vacuolation and hypertrophy of hepatocytes, likely representing increased smooth endoplasmic reticulum due to induced cytochrome enzymes, were observed in these animals. Rats terminated at PND 35 had the strongest microsomal effects as compared to those terminated at later dates. Because exposure stopped at PND 23, this cessation of exposure allowed rats to recover, as evidenced by a lack of enzyme inductions in the 1.3 mg/kg mixture group at PND 70 and beyond. Consistent with a lack of enzyme induction in these rats at PND 70, histological changes such as hypertrophy and vacuolation were less severe than those observed earlier. Adaptive liver changes of this nature have been reported after subchronic exposure to PCB congeners (Chu et al., 1995
). In the present study, the Aroclor control group exhibited adaptive changes as animals aged. In addition, Aroclor elicited histological changes such as bile duct hyperplasia, cholangiofibrosis, cirrhosis, and fibroacinar formation.
Effects on serum biochemical profiles were confined to changes in cholesterol, LDH, and urea nitrogen levels. An elevation of serum cholesterol is usually associated with increased hepatic microsomal induction, a finding that has been reported in our previous studies with PCBs and 2,3,7,8-TCDD (Chu et al., 2001). Elevated serum LDH is usually associated with liver injury where this enzyme is released into blood, and no specific tissue injuries have been related clinically to decreased level of this enzyme. Decreased LDH has been previously reported in rats given POP-contaminated fish (Villeneuve et al., 1981
). There was also a report that organochlorine inhibited LDH activity in vitro by co-precipitation of this enzyme (Meany and Pocker, 1979
). Urea is an end product of protein metabolism, and it is removed from the blood by the kidneys. Increased serum urea nitrogen in the Aroclor group and the high-dose mixture groups may indicate compromised kidney function, although histopathological examination reveals no kidney damage.
The liver is one of the target organs affected by POP mixture treatment. However, the histological effects are minimal to mild in nature, characterized by increased lymphocytic inflammation, hepatocellular hypertrophy and vacuolation, and bile duct hyperplasia. At these dose levels, no functional changes such as serum cholesterol or other changes were noted. Treatment with Aroclor produced cholangiofibrosis and intestinal metaplasia, which are interesting changes and are examples of what may result when livers are exposed to a mito-inhibitory, non-necrotizing hepatotoxin (Tatematsu et al., 1985). More severe liver changes occurred at the highest dose where mottled liver and increased liver weights were seen (the relative weights being 130% and 100% over the control values for male and female, respectively, Table 1) at PND 35.
Thyroid, known to be a target organ of PCB treatment, could not be examined in the present study. However, Bowers et al. (2004) demonstrated that serum levels of T3, T4, and T3 uptake in offspring were not affected at mixture doses up to 1.3 mg, suggesting that no severe morphological changes occurred in the thyroid up to 1.3 mg/kg. As previously reported, Aroclor and 13 mg/kg of the mixture reduced T4 levels, and also altered thyroid morphology in offspring (Bowers et al., 2004
).
Suppression or stimulation of cell-mediated immunity may be reflected as hypocellularity or hypercellularity of T-dependent regions in lymph nodes and spleen (ICICIS, 1998). In the present study, reductions in lymphoid density and lymphoid areas in spleen and thymus were observed and considered a treatment-related effect. Although the immunopathological changes were seen at doses as low as 0.013 mg/kg, a dose-dependent effect could not be demonstrated. Germolec et al. (2004)
reported that accuracy of using extended histopathology to identify immunologic chemicals is the highest when it is combined with functional tests. It is apparent that until immune function tests are carried out, the effect level of immunotoxicity could not be ascertained based on immunopathological data alone. As a consequence of our study design, peripheral hematological parameters, including leukocyte and lymphocyte count as indices of functional changes, could not be obtained. Tryphonas et al. (1998)
reported a decrease in white blood cells and lymphocytes in F2 female rats treated with the 20% Lake Ontario fish diet, suggesting that immunological parameters may be affected.
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SUMMARY |
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A more precise assessment of effects observed in rats and relating them to human blood levels requires residue analysis of blood of dams, which is currently underway. Human placental intake of the chemicals has been estimated by assuming 50% of the dose retained in the mother and 50% transferred to the fetus (Health Canada, 1998). Based on this estimation, the lowest dose (0.013 mg/kg) used in the present study would be approximately 1.7 times the intake in human infants (Health Canada, 1998
; Bowers et al., 2004
). Considering the systemic effects at 1.3 mg/kg, this would suggest that human intake (0.013 mg/kg) of the POP mixture is much lower than the levels demonstrated to produce systemic toxicity in rats.
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ACKNOWLEDGMENTS |
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