Toxicological Effects of Gestational and Lactational Exposure to a Mixture of Persistent Organochlorines in Rats: Systemic Effects

Ih Chu*,1, Wayne J. Bowers*, Don Caldwell{dagger}, Jamie Nakai*, Olga Pulido{dagger}, Al Yagminas*, Michael G. Wade*, David Moir*, Santokh Gill{dagger} and Rudi Mueller{dagger}

* Environmental and Occupational Toxicology Division, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada; {dagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
A large multi-disciplinary study was conducted to investigate the systemic, neurodevelopmental, neurochemical, endocrine, and molecular pathological effects of a mixture of reconstituted persistent organochlorine pollutants (POP) based on the blood profiles of Canadians residing in the Great Lakes/St. Lawrence region. This report outlines the overall study design and describes the systemic effects in rat offspring perinatally exposed to the POP mixture. Maternal rats were administered orally 0, 0.013, 0.13, 1.3, or 13 mg/kg bw/day of the mixture from gestational day (GD) 1 to postnatal day (PND) 23. Positive and negative controls were given Aroclor 1254 (15 mg/kg bw/day) and corn oil (vehicle), respectively. The rat pups were reared, culled to 8 per litter, and killed on postnatal days 35, 70, and 350, at which time tissues were collected for analysis. Exposure to high doses of the mixture elicited clinical, biochemical, and pathological changes and high mortality rates in rat offspring. Aroclor 1254 produced similar effects but a lower mortality than was seen in POP mixture groups. Biochemical changes consisted of increased liver microsomal activities and elevated serum cholesterol. Hepatomegaly was observed in the highest dose group of the mixture and in the positive control. Liver, thymus, and spleen were the target organs of action. Microscopic changes in the liver consisted of vacuolation and hypertrophy, and those in the thymus were characterized by reduced cortical and medullary volume. The spleen showed a treatment-related reduction in lymphocyte density and lymphoid areas. This study demonstrates that exposure to the POP mixture up to 13 mg/kg/day perinatally produced growth suppression, elevated serum cholesterol, increased liver microsomal enzyme activities, and immunopathological changes in the thymus and spleen, and lethality. Most of the effects were seen at dose levels much higher than expected human exposure.

Key Words: systemic toxicity; organochlorine mixtures; Aroclor 1254; PCBs; Great Lakes/St. Lawrence region.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Concern over the potential adverse health effects of ingesting Great Lakes fish contaminated with persistent organochlorine pollutants (POPs) has led many researchers to investigate the toxic effects of these compounds. Epidemiological studies have shown that ingestion of fish contaminated with PCBs and other persistent organochlorines during pregnancy is associated with a lower growth rate and intellectual capacity in infants (Jacobson et al., 1990Go; Jacobson and Jacobson, 1997Go; Rogan and Gladden, 1991Go; Stewart et al., 2003Go). Laboratory studies testing POP mixtures in animals have also reported neurological deficits and systemic effects (Arnold et al., 1998Go; Chu et al., 1984Go; Daly et al., 1998Go; Hany et al., 1999aGo, 1999bGo; Iverson et al., 1998Go; Seegal et al., 1998Go). Daly et al. (1998)Go have demonstrated that offspring of female rats that consumed a diet containing 30% Lake Ontario salmon, known to contain POP contaminants, showed an increased reactivity to frustrative non-reward. Seegal et al. (1998)Go have shown that rats fed a diet containing 20% Lake Ontario fish perinatally and as adults had significant reductions in brain biogenic amine functions. Although there were demonstrated neurochemical effects in the rats fed the diet containing contaminated Lake Ontario salmon, neurobehavioral effects for memory as measured using Morris water maze and radial arm maze were inconclusive (Pappas et al., 1998Go). Arnold et al. (1998)Go have shown that F1 and F2 generations of rats fed diets containing 20% Lake Ontario salmon had increased relative kidney and liver weights but showed no changes in reproduction. Similarly, in a 13-week feeding study Chu et al. (1984)Go reported that rats of both sexes fed diets containing 2.9% and 5.8% Lake Ontario salmon exhibited an increase in ethoxyresorufin-O-de-ethylase activity relative to animals fed the control diet, and this effect was reversed after 13 weeks on a clean diet. Similar effects on liver microsomal enzyme activities have been observed by Iverson et al. (1998)Go, who reported a significant increase in liver methoxyresorufin-O-demethylase, phenoxyresorufin-O-de-alkylase, and benzoyloxyresorufin-O-de-alkylase activities in rats fed contaminated Lake Ontario fish.

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 dose–response 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., 1999Go; Kosatsky, 1999aGo, 1999bGo). 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 dose–response 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., 2004Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Materials.
Details of the composition of the POP mixture, sources of rats, and chemical analysis of dosing solutions have been described previously (Bowers et al., 2004Go). Briefly, the mixture consists of 26 chemicals containing 10 non-PCB organochlorines (63.25% by weight) including p,p'-DDE, p,p'-DDT, hexachlorobenzene, and 16 selected PCB congeners (36.75%). The relative amount of each of the 26 chemicals in the mixture is based on the relative amount found in the blood of women with a high fish diet in the Great Lakes/St. Lawrence region. Other chemicals and solvents were of reagent grade purchased commercially.

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 (200–230 g) and 38 male rats (315–350 g) were used. Details of animal handling, mating, and rearing have been described previously (Bowers et al., 2004Go). 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 2–3-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)Go.

Measurement of sperm function.
Resistant spermatid nuclei were examined by a previously described method (Wade et al., 2002Go) 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)Go. 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, 25–50% 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., 2005Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Animal Observation
Growth and mortality data have been presented in detail in a previous publication (Bowers et al., 2004Go), and are briefly summarized here. The highest dose mixture groups had 3 males and 2 females out of approximately 15–16 pups per group that survived treatment to PND 35. The body weights of the surviving pups were 67% and 70% of vehicle control males and females, respectively (Table 1). Growth suppression was seen in the 1.3 mg/kg mixture group of males terminated at PND 35. Aroclor groups exhibited growth suppression at both PND 35 (Table 1) and PND 70 (control male: 398 ± 37 g; treated male: 387 ± 36 g; control female: 243 ± 23 g; treated female: 230 ± 19 g), but no increased mortalities were recorded relative to vehicle controls. Growth rates could not be measured meaningfully after PND 70, because at this point rats were placed on food restriction in order to perform behavioral measurements. The growth rates of other treatment groups were not affected, and clinical observations revealed no abnormalities.


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TABLE 1 Organ Weights (wet weight in grams, and percent body weight) of Rats Exposed Perinatally to Persistent Organochlorine Pollutant (POP) Mixture and Terminated at Postnatal Day 35a

 
Organ Weight
Postnatal Day 35.
The relative liver weights in the highest dose (13 mg/kg) mixture, being more than doubled of vehicle control values, were not analyzed statistically (Table 1). The 1.3 mg/kg mixture group showed decreased absolute spleen weight in both genders and increased relative brain weight in males. The Aroclor treated groups showed increased absolute and relative liver (liver/bw) weights, as well as decreased absolute weights of kidney, spleen, heart, brain, thymus, testes, and uterus (Table 1).

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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TABLE 2 Serum Biochemical Effects of POPs in Rat Offspring

 
Serum biochemical profiles in dams were also determined to provide some comparison of toxic effects in dams and offspring. Of the serum biochemical parameters measured in dams, only cholesterol levels were increased in the 13 mg/kg bw mixture and Aroclor groups (control: 72 ± 6 mg/dl; 13 mg/kg mixture: 146 ± 20 mg/dl; Aroclor: 108 ± 21 mg/dl). Other serum biochemical values of the dams were normal.

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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FIG. 1. Effects of the POP mixture on liver microsomal enzyme activities of rat offspring. An asterisk indicates significant difference at p ≤ 0.05.

 
Gross Pathological Observation
Gross pathological changes were rare except for mottled liver and hydronephrosis. Mottled livers were observed in the 13 mg/kg mixture and Aroclor groups at PND 35 and PND 70. The incidence of mottled liver for PND 35 pups is as follows: male-control, 0/22; 13 mg/kg mixture, 2/4; Aroclor, 4/20; female-control, 0/23; 13 mg/kg mixture, 1/2; Aroclor, 4/21. For PND 70 rats the incidence of mottled liver is as follows: male-control, 0/22; 13 mg/kg mixture, 0/1; Aroclor, 3/20; female-control, 0/22; 13 mg/kg mixture, 3/4; Aroclor, 7/21. Hydronephrosis was observed sporadically in both control and treated animals at different time periods, including 1 in the vehicle control group, 5 in the 0.013 mg/kg group, 4 in the 0.13 mg/kg group, 3 in the 1.3 mg/kg group, and 1 in the Aroclor group. The renal effect was judged not to be treatment-related as it occurred in both the control and the treated groups (Chandra and Frith, 1993Go). No other gross pathological changes were observed. No gross changes were observed in rats killed at PND 350.

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 mixture–treated 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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TABLE 3 Incidence and Severity of Liver Lesions in Rat Offspring Exposed to the Mixture

 


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FIG. 2. Liver from Sprague-Dawley rats. A. Vehicle. Arrowhead, central vein; arrow, portal area. 10x objective. H&E. B. Liver from an animal receiving the 1.3 mg/kg per day mixture, showing diffuse mild hepatocellular hypertrophy. Arrow, granuloma; arrowheads, hyperplastic bile ducts. 10x objective, H&E original stain.

 
Spleen.
Lesions were not noted in the PND 35 mixture-fed animals, but mild to moderate involution of T-dependent (periarteriolar lymphoid tissue) and B-dependent (follicles) regions, characterized by reduced lymphoid cell densities, was common among the Aroclor animals (data not shown). In the PND 70 groups, all mixture-fed and Aroclor-fed animals showed similar mild to marked involution characterized by reduction in cellularity in B- and T-dependent areas (Fig. 3; Table 4). A dose–response relationship was not evident for these involution lesions.



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FIG. 3. Spleen from Sprague-Dawley rats. A. Vehicle. Arrow, normal follicle; arrowheads, normal periarteriolar lymphoid sheaths. 10x objective, H&E stain. B. Spleen from an animal receiving the 1.3 mg/kg/day mixture, showing small hypocellular follicle (arrow) and small hypocellular sheath (arrowhead). 10x objective, H&E original stain.

 

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TABLE 4 Spleen Lesions of Rat Offspring Perinatally Exposed to POP Mixture and Terminated at PND 70

 
Thymus.
All PND 35 groups were within normal limits, except for 1 Aroclor-fed rat which had a slight reduction in cortical lymphocyte density (data not shown). In all PND 70 mixture and the Aroclor groups, mild to moderate involution, characterized by decreased lymphoid density in cortex and medulla and decreased cortical area, was common (Fig. 4; Table 5). There was no clear dose–response relationship for these changes.



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FIG. 4. Thymus from Sprague-Dawley rats. A. Vehicle. B. Thymus from an animal receiving the 1.3 mg/kg/day mixture, with a mild decrease in cortical lymphoid density and increased cortical macrophages (arrows). 10x objective; H&E original stain.

 

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TABLE 5 Thymic Lesions of 70-Day-Old Rats Perinatally Exposed to POP Mixture

 
Ileum.
At PND 35 all mixture-fed groups were within the normal limit. In the Aroclor group terminated at the same time, few appropriate sections of Peyer's patches were available for evaluation; however, mild B- and T-dependent area involution was noted in 2 males (data not shown). At PND 70 all mixture-fed and the Aroclor groups showed mild involution characterized by mild reductions in cellularity in B and T-dependent tissues. A dose–response relationship was not evident. Granulomatous inflammation within Peyer's patches was common in all mixture-fed groups and the Aroclor group, and was seen in 1 vehicle animal (data not shown). The cause of this inflammatory lesion is not known; periodic acid–Schiff and acid fast stains were negative for any infectious agent.

Testis.
Examination of testis homogenates showed no effects on sperm functions.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Administration of the POP mixture to pregnant rats up to 13 mg/kg/day elicited a broad spectrum of effects in their offspring, including growth suppression, mortality, hepatomegaly, serum biochemical changes, liver microsomal enzyme induction, and histopathological changes in the liver, thymus, and spleen. Some of the effects observed in the offspring at the 13 mg/kg bw may be due to the toxicity in dams. However, at the 1.3 mg/kg bw dose, no toxic effects were seen in dams. Thus, except for the highest dose groups, the effects observed in the offspring up to 1.3 mg/kg bw were due to perinatal exposure to the POP mixture, and included growth suppression, liver microsomal enzyme induction, and histopathological changes in the liver, thymus, and spleen.

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)Go and Arnold et al. (1998)Go 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., 2002Go; Roegge, 2005Go). 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., 1984Go; Iverson et al., 1998Go; Stern et al., 2002Go). 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., 1998Go), 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., 2002Go). 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., 1995Go). 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., 2001Go). 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., 1981Go). There was also a report that organochlorine inhibited LDH activity in vitro by co-precipitation of this enzyme (Meany and Pocker, 1979Go). 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., 1985Go). 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)Go 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., 2004Go).

Suppression or stimulation of cell-mediated immunity may be reflected as hypocellularity or hypercellularity of T-dependent regions in lymph nodes and spleen (ICICIS, 1998Go). 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)Go 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)Go 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.


    SUMMARY
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
In establishing a no observable adverse effect level (NOAEL), all relevant toxic end points need to be considered. Liver hypertrophy, vacuolation, and inflammation at low doses (i.e., at 0.013 mg and 0.13 mg/kg), rated minimal on the severity scale, could not be considered adverse effects. Effects at the 1.3 mg/kg level are of some concern because not only the liver morphology changed from minimal to mild, but also there was an observable transient decreased growth rate in male pups at PND 35 (Table 1).

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, 1998Go). 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, 1998Go; Bowers et al., 2004Go). 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.


    ACKNOWLEDGMENTS
 
The authors thank Bruce Martin, Susan Kelly, Kelly Brennan, Tanya Hoeksma, and Yasmin Dirieh for technical assistance. This study was supported in part by the Toxic Substances Research Initiative from Health Canada and Environment Canada (grant no. 209).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 SUMMARY
 REFERENCES
 
Arnold, D. L., Bryce, F., Miller, D., Stapley, R., Malcom, S., and Hayward, S. (1998). The toxicological effects following the ingestion of Chinook salmon from the Great Lakes by Sprague-Dawley rats during a two-generation feeding-reproduction study. Reg. Toxicol. Pharmacol. 27, S18–S27.[CrossRef][ISI][Medline]

Bowers, W. J., Nakai, J. S., Chu, I., Wade, G. M., Moir, D., Yagminas, A., Gill, S., Olga, P., and Mueller, R. (2004). Early developmental neurotoxicity of a PCB/organochlorine mixture in rodents after gestational and lactational exposure. Toxicol. Sci. 77, 51–62.[Abstract/Free Full Text]

Chandra, M., and Frith, C. H. (1993). Non-neoplastic renal lesions in Sprague-Dawley and Fischer-344 rats, Exp. Toxicol. Pathol. 45, 4339–4447.

Chu, I., Villeneuve, D. C., Valli, V. E., Ritter, L., Norstrom, R. J., Ryan, J. J., and Becking, G. C. (1984). Toxicological response and its reversibility in rats fed Lake Ontario or Pacific Coho salmon for 13 weeks. J. Environ. Health Sci. B19, 713–731.[ISI]

Chu, I., Villeneuve, D. C., Yagminas, A., Lecavalier, P., Håkansson, H., Ahlborg, U. G., Valli, V. E., Kennedy, S. W., Bergman, A., Seegal, R. F., et al. (1995). Toxicity of PCB 77 (3,3',4,4'-tetrachlorobiphenyl) and PCB 118 (2,3,4,4',5-pentachlorobiphenyl) in the rat following subchronic dietary exposure. Fundam. Appl. Toxicol. 26, 282–292.[CrossRef][ISI][Medline]

Chu, I., Lecavalier, P., Håkansson, H., Yagminas, A., Valli, V. E., Poon, P., and Feeley, M. (2001). Mixture effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and polychlorinated biphenyl congeners in rats. Chemosphere 43, 807–814.[CrossRef][ISI][Medline]

Daly, H. B., Stewart, P. W., Lunkenheimer, L., and Sargent, D. (1998). Maternal consumption of Lake Ontario salmon in rats produces behavioral changes in the offspring. Toxicol. Ind. Health 14, 25–39.[ISI][Medline]

Germolec, D. R., Kashon, M., Nyska, A., Kuper, C. F., Portier, C., Kommineni, C., Johnson, K. A., and Luster, M. I. (2004). The accuracy of extended histopathology to detect immunotoxic chemicals. Toxicol. Sci. 82, 504–514.[Abstract/Free Full Text]

Hany, J., Lilienthal, H., Sarasin, A., Roth-Härer, A., Fastabend, A., Dunemann, L., Lichtensteiger, W., and Winneke, G. (1999a). Developmental exposure of rats to a reconstituted PCB mixture or Aroclor 1254: Effects on organ weights, aromatase activity, sex hormone levels and sweet preference behaviour. Toxicol. Appl. Pharmacol. 158, 231–243.[CrossRef][ISI][Medline]

Hany, J., Lilienthal, H., Roth-Härer, A., and Winneke, G. (1999b). Behavioral effects following single and combined maternal exposure to PCB 77 (3,4,3',4'-tetrachlorobiphenyl) and PCB 47 (2,4,2',4'-tetrachlorobiphenyl) in rats. Neurotoxicol. Teratol. 21, 147–156.[CrossRef][ISI][Medline]

Health Canada. (1998). Persistent environmental contaminants and the Great Lakes Basin population: An exposure assessment, Public Works and Government Services Canada, Cat. No. H46-2/98–218E.

ICICIS Group (International Collaborative Immunotoxicity Study Group). (1998). Report of validation study of assessment of direct immunotoxicity in the rat. Toxicology 125, 183–20.[CrossRef][ISI][Medline]

Iverson, F., Mehta, R., Hierlihy, L., Gurofsky, S., Lok, E., Mueller, R., Bourbonnais, D. H., and Spear, P. A. (1998). Microsomal enzyme activity, glutathione S-transferase-placental form expression, cell proliferation, and vitamin A stores of rats consuming Great Lakes salmon. Reg. Toxicol. Pharmacol. 27, S76–S89.[CrossRef][ISI][Medline]

Jacobson, J. L., Jacobson, S. W., and Humphrey, H. E. (1990). Effects of exposure to PCBs and related compounds on growth and activity in children. Neurotoxicol. Teratol. 12, 319–326.[CrossRef][ISI][Medline]

Jacobson, J. L., and Jacobson, S. W. (1997). Evidence for PCBs as neurodevelopmental toxicants in humans. Neurotoxicology 18, 415–424.[ISI][Medline]

Kearney, J. P., Cole, D. C., Ferron, L. A., and Weber, J. P. (1999). Blood PCB, p,p'-DDE, and mirex levels in Great Lakes fish and waterfowl consumers in two Ontario communities, Environ. Res. 80, S138–S149.[CrossRef][ISI][Medline]

Kosatsky, T., Przybysz, R., Shatenstein, B., Weber, J. P., and Armstrong, B. (1999a). Fish consumption and contaminant exposure among Montreal-area sport fishers: A pilot study, Environ. Res. 80, S150–S158.[CrossRef][ISI][Medline]

Kosatsky, T., Przybysz, R., Shatenstein, B., Weber, J. P., and Armstrong, B. (1999b), Contaminant exposure in Montrealers of Asian origin fishing the St. Lawrence River: Exploratory assessment, Environ. Res. 80, S159–S165.[CrossRef][ISI][Medline]

Lubet, R. A., Nims, R. W., Mayer, R. T., Cameron, J. W., and Schechtman, L. M. (1985). Measurement of cytochrome P-450 dependent de-alkylation of alkoxyphenoxazones in hepatic S9 and hepatocyte homogenates: Effects of dicumarol. Mutat. Res. 142, 127–131.[CrossRef][ISI][Medline]

Meany, J. E., and Pocker, Y. (1979). The in vitro inactivation of lactate dehydrogenase by organochlorine insecticides. Pestic. Biochem. Physiol. 11, 232–242.[CrossRef][ISI]

Nakai, J. S., Elwin, J., Chu, I., and Marro, L. (2005). Effects of anaesthetics/terminal procedures on neurotransmitters from non-dosed and Aroclor1254-dosed rats. J. Appl. Toxicol. 25, 224–233.[CrossRef][ISI][Medline]

Pappas, B. A., Murtha, S. J., Park, G. A., Hewitt, K., Seegal, R. F., and Jordan, S. A. (1998). Neurobehavioral effects of chronic ingestion of Great Lakes Chinook salmon. Reg. Toxicol. Pharmacol. 27, S55–S67.[CrossRef][ISI][Medline]

Roegge, C. S., Widholm, J. J., Engeseth, N. J., Wang, X., Brosch, K. O., Seegal, R. F., and Schantz, S. (2005). Delayed spatial alternation impairments in adult rats following dietary n-6 deficiency during development. Neurotoxicol. Teratol. 27, 485–495.[CrossRef][ISI][Medline]

Rogan, W. J., and Gladden, B. C. (1991) PCBs and DDE and child development at 18 and 24 months. Ann. Epidemiol. 1, 407–413.[Medline]

Seegal, R. F., Pappas, B. A., and Park, G. A. (1998). Neurochemical effects of consumption of Great Lakes salmon by rats. Regul. Toxicol. Pharmacol. S68–S75.

Stern, N., Öberg, M., Casabonna, H., Trossvik, C., Manzoor, E., Johansson, N., Lind, M., Öberg, J., Feinstein, R., Johansson, A., et al. (2002). Subchronic toxicity of Baltic Herring oil and its fractions in the rat II: Clinical observations and toxicological parameters. Pharmacol. Toxicol. 91, 232–244.[CrossRef][ISI][Medline]

Stewart, P. W., Reihman, J., Lonky, E. I., Darvill, T. J., and Pagano, J. (2003). Cognitive development in preschool children prenatally exposed to PCBs and MeHg. Neurotoxicol. Teratol. 25, 11–22.[CrossRef][ISI][Medline]

Tatematsu, M., Kaku, T., Medline, A., and Farber, E. (1985). Intestinal metaplasia as a common option of oval cells in relation to cholangiofibrosis in liver of rats exposed to 2-acetylaminofluorene. Lab. Invest. 52, 354–362.[ISI][Medline]

Tryphonas, H., McGuire, P., Fernie, S., Miller, D., Stapley, R., Bryce, F., Arnold, D. L., and Fournier, M. (1998). Effects of Great Lakes fish consumption on the immune system of Sprague-Dawley rats investigated during a two-generation reproductive study. Regul. Toxicol. Pharmacol. 27, S28–S39.[CrossRef][ISI][Medline]

Villeneuve, D. C., Valli, V. E., Norstrom, R. J., Freeman, H., Sanglang, G. B., Ritter, L., and Becking, G. C. (1981). Toxicological response of rats fed Lake Ontario or Pacific Coho salmon for 28 days. J. Environ. Health B16, 649–689.

Wade, M., Foster, W., Younglai, E. V., McMahon, A., Leingartner, K., Yagminas, A., Blakey, D., Fournier, M., Desaulniers, D., and Hughes, C. L. (2002). Effects of subchronic exposure to a complex mixture of persistent contaminants in male rats: Systemic, immune and reproductive effects. Toxicol. Sci. 67, 131–143.[Abstract/Free Full Text]





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