* Toxicology Research Division,
Food Research Division and
Chemical Health Hazard Assessment Division, Food Directorate, Health Protection Branch, Health Canada, Ottawa, Ontario K1A 0L2, Canada
Received June 14, 2000; accepted September 5, 2000
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ABSTRACT |
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Key Words: trans-nonachlor; cis-nonachlor; chlordane; oxychlordane; toxicity.
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INTRODUCTION |
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The unofficial Canadian tolerated daily intake value for chlordane is 0.05 µg/kg body weight (bw)/day, with an official maximum residue limit of 0.1 ppm for chlordane and oxychlordane (calculated on fat content) for a variety of dairy and meat products (Dr. M. Feeley, personal communication; Canada Food and Drugs Act). The U.S. Food and Drug Administration has established that chlordane and its metabolites should not be present at levels higher than 300 ppb in fruits and vegetables or 100 ppb in animal fat and fish (U.S. Department of Health and Human Services [U.S. DHHS], 1994). The primary focus of the majority of toxicological studies supporting these recommendations is on the potential adverse health effects of the parent chlordane mixture, for which a toxicological profile has been compiled (U.S. DHHS, 1994). However, toxicity data for individual chlordane constituents or metabolites such as trans-nonachlor, cis-nonachlor, or oxychlordane, which are among the most common chlordane-related environmental contaminants and tissue residues, are nonexistent. This study addresses the toxicological data gap for trans-nonachlor and cis-nonachlor. Male and female rats were exposed to trans- and cis-nonachlor by gavage for 28 consecutive days, and multiple toxicological endpoints were examined. The clinical chemistry, hematology, urinalysis, tissue residue analyses and histopathology results have been summarized for these chemicals. Also included in this study were male and female rats exposed to the technical chlordane mixture for comparison to rats treated with cis- and trans-nonachlor.
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MATERIALS AND METHODS |
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Animals.
Male and female Sprague-Dawley rats (45 to 50 days old) were obtained from Charles River Canada, Inc. (Montreal, Quebec). Upon receipt, rats were housed individually in plastic cages (Health Guard System, Research Equipment Company, Inc., Bryan, TX) under conditions meeting the requirements of the Canadian Council for Animal Care. All rats were acclimatized for a minimum of one week before the study. Purina Rodent Chow (Woodstock, Ontario) and water were provided ad libitum throughout the study.
Experimental design.
Male and female rats were randomized and divided into 6 test groups, designated A through F, as indicated in Table 1. Within each group, rats were randomly assigned to receive 1 of 4 test chemical doses: controls (0 mg/kg test chemical; corn oil vehicle only), 0.25, 2.5, or 25 mg test chemical/kg body weight (bw)/day. The highest test doses were chosen based on studies indicating that 50 mg chlordane/kg bw administered by gavage to rats caused deaths after 9 to 12 days, whereas no deaths occurred in rats treated with 25 mg chlordane/kg for 15 days (Ambrose et al., 1953
). Mean starting bws are indicated in Table 1
. For all groups, the number of rats at each dose level was 7. Body weight data were analyzed prior to the beginning of the study to confirm that within each group there were no significant differences in starting body weights between rats assigned to each dose level. For 28 consecutive days, each rat received a single daily gavage dose of test chemical in a volume of 0.5 ml corn oil/100 g bw. Body weights were monitored daily throughout the studies; food and water consumption were monitored biweekly. For urine collection, rats were transferred to Nalgene metabolic cages for 24 h before the first dose and for 24 h after the last dose. On the final day of the study (24 h after the last dose), each rat was anesthetized with isoflurane (Janssen, Toronto, Ontario, Canada), exsanguinated and necropsied. Organ weights were recorded for liver, kidneys, spleen, thymus, adrenals, brain, ovaries, and testes.
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For clinical chemistry, clotted blood was centrifuged at 700 x g for 20 min to prepare serum. A Beckman Synchron CX5 clinical system (Beckman Instruments Canada, Inc., Mississauga, ON) and Beckman reagent kits were used to measure clinical endpoints. The following clinical chemistry parameters were measured in serum from each rat: glucose, blood urea nitrogen (BUN), creatinine, uric acid, total protein, albumin, immunoglobulin A (IgA), IgM, IgG1, IgG2a, IgG2b, IgG2c, total bilirubin, cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), -glutamyltransferase (GGT), ornithine carbamyl transferase (OCT), lactate dehydrogenase (LDH), creatine kinase, amylase, thyroxine (T4), thyroxine uptake, calcium, sodium, potassium, magnesium, chloride, phosphorus, and osmolality.
Renal toxicology.
The methodology for urinalysis has been described previously (Suzuki et al., 1995). Briefly, urine volume, osmolality, and total protein were measured in whole urine. A 1-ml aliquot of each urine sample was applied to Sephadex G-25 PD-10 columns (Pharmacia LKB, Baie D'Urfe, Canada) and eluted through with 8 ml of saline to remove low molecular weight enzyme inhibitors. The first 4-ml fraction collected from the column was analyzed for creatinine using the method of Heinegard and Tiderstrom (1973). The enzymes, N-acetyl-ß-D-glucosaminidase (NAG) and
-glutamyltranspeptidase (GGT) were measured in the next 4-ml fraction by the methods of Leaback and Walker (1961) and Dierickx (1980), respectively. Transport of the organic anion, p-aminohippuric acid (PAH), and the cation, tetraethylammonium (TEA) in kidney slices was measured as described previously (Suzuki et al., 1995
). Kidneys were weighed and prepared for transport assays immediately after the rat was exsanguinated. Transport was expressed as a ratio between the amount of ion transported into the slice and the amount remaining in the medium (S/M). A decrease in this ratio represents reduced transport function.
Pathology.
For light microscopy, tissues were fixed in 10% neutral buffered formalin (pH 7.0). Paraffin sections (4 µm) were stained with hematoxylin and eosin. The following tissues were examined: adrenals, bone marrow, esophagus, heart, kidneys, liver, lungs, pancreas, skeletal muscle, spleen, thymus, thyroid, ovaries, uterus, testes, epididymis, prostate, coagulatory gland, and seminal vesicles. To identify hepatocellular vacuole contents, liver sections were stained with periodic acid Schiff (PAS) for water soluble polysaccharides or oil red O for lipids (Luna, 1968).
Residue analyses.
Whole blood was collected from male rats at necropsy for residue analyses; female rats were too small to provide sufficient blood for residue analyses in addition to clinical chemistry and hematology. Liver (1 g) and adipose tissue from the abdominal fat pad (1 g) were collected from male and female rats. All tissues, including whole blood, were frozen in tightly sealed vials at 20°C until extraction. All samples were analyzed for the following residues: cis-nonachlor, trans-nonachlor, cis-chlordane, trans-chlordane, oxychlordane, and heptachlor. Adipose, liver, or blood samples were extracted with acetone:hexane (2:1) and the extract filtered through glass wool. The solvent was concentrated, dried over sodium sulfate, and evaporated to dryness. The residue was then chromatographed on 2% water deactivated Florisil (Fisher Scientific, Fair Lawn, NJ) to remove lipid. Analytes were eluted with 2% dichloromethane in hexane and quantified by gas chromatography on a DB-5 capillary column using a Varian Star 3400 chromatograph fitted with an electron capture detector (Varian, Walnut Creek, CA). Recoveries of oxychlordane, cis-nonachlor, trans-nonachlor, cis-chlordane and trans-chlordane spiked into corn oil were 95% or greater, while that of heptachlor was 82%. Corn oil, spiked at from 2 to 4 ppm, was run with each set of 10 samples to verify satisfactory recoveries.
Statistics.
Statistical analyses were done using SigmaStat (Jandel Scientific, San Rafael, CA). For each test chemical, data from control and treated rats were compared using one-way analysis of variance (ANOVA) for multiple comparisons, followed by Dunnett's test for pairwise comparisons if necessary. For nonparametric data, multiple comparisons were made using the Kruskal-Wallis ANOVA on ranks, followed by Dunnett's (for equal sample sizes) or Dunn's (for unequal sample sizes) pairwise tests if necessary. Data comparisons were considered significant if p 0.05.
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RESULTS |
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Body Weight and Food and Water Consumption
In female rats receiving cis-nonachlor (Group A), daily food consumption was significantly lower at the 25 mg/kg dose level (Table 1), but this did not translate into changes in total weight gain or final bw (Table 2
). In female rats receiving trans-nonachlor (Group B), daily food consumption was significantly depressed in the 25 mg/kg dose group (Table 1
). In conjunction with the rapid weight loss and death of 3 rats at this dose level, total weight gain and final bw in these rats were significantly lower than in corresponding controls (Table 2
). Technical chlordane had no effect on weight gain or final bw in female rats (Table 2
, Group C), even though food consumption was significantly lower than controls at the 25 mg/kg dose level (Table 1
). In males treated with cis-nonachlor (Group D), both weight gain and food consumption were significantly higher for all doses compared to controls (Tables 1 and 2
), but this did not result in significantly higher final bws (Table 2
). In trans-nonachlor-treated males (Group E), food consumption was significantly higher in the 0.25 and 2.5 mg/kg dose groups (Table 1
), but weight gain and final bw were unaffected (Table 2
). Food consumption was significantly depressed in male rats receiving 25 mg/kg technical chlordane (Table 1
, Group F), but the apparent drop in bw at this dose level was not significant (Table 2
).
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Organ weights
Liver weight was consistently elevated in Groups A through F at the 25 mg/kg dose level, regardless of whether the data were expressed as total liver wet weight (g) or as % final bw (Table 3). In male rats treated with trans-nonachlor (Group E), both liver wet weight and liver % bw were also elevated at the 2.5 mg/kg dose level. In male rats treated with technical chlordane (Group F), liver % bw was elevated in the 2.5 mg/kg dose group. Overall, trans-nonachlor had the greatest effect on liver weight. In both females and males, liver wet weights were 2.1 x higher than comparable controls, whereas livers from rats treated with cis-nonachlor and technical chlordane ranged from 1.5x to 1.8x larger than controls. Kidney weights were elevated in male rats but not in female rats (Table 3
). cis-Nonachlor treatment (Group D) resulted in significantly elevated kidney weights at the 2.5 mg/kg dose level, and both elevated kidney weight and kidney % bw at the 25 mg/kg dose level. Kidney weight (Group E) or kidney % bw (Group F) were significantly elevated in males treated with trans-nonachlor or technical chlordane, respectively, at the 25 mg/kg dose level. Spleen, thymus, adrenal, brain, ovary, and testis weights were unaffected in all groups (data not shown).
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Statistically significant serum clinical chemistry changes due to cis-nonachlor, trans-nonachlor, and technical chlordane are summarized in Tables 4, 5, and 6, respectively. Most clinical chemistry changes were observed in the 25 mg/kg dose group for all of the test chemicals. One of the most consistent changes was elevated serum cholesterol at the 25 mg/kg dose level in male and female rats. Alanine aminotransferase (ALT) was elevated in trans-nonachlor-treated rats only (Table 5
), whereas
-glutamyltransferase (GGT) and ornithine carbamyl transferase (OCT) were unaffected in all rats (data not shown). Serum triglycerides were significantly depressed in male rats treated with technical chlordane (Table 6
). Total serum protein was significantly elevated by all of the test chemicals, in conjunction with depressed albumin/globulin (A/G) ratios (Tables 46
). Serum calcium and/or magnesium levels were elevated in males treated with cis-nonachlor (Table 4
) and technical chlordane (Table 6
), and in males and females treated with trans-nonachlor (Table 5
). Thyroxine uptake was significantly depressed in trans-nonachlor-treated rats (Table 5
), and both thyroxine uptake and thyroxine levels were depressed in rats treated with technical chlordane (Table 6
).
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In the thyroids of control rats and of rats treated with 0.25 mg/kg cis-nonachlor, trans-nonachlor, or technical chlordane, spherical follicles with squamous to cuboidal epithelium were evident. In 2 male control rats (Group D), in a few male rats at the 2.5 mg/kg dose level for trans-nonachlor and chlordane, and in male and female rats at the 25 mg/kg dose level for cis-nonachlor, trans-nonachlor, and chlordane, numerous aspherical follicles with cuboidal to columnar epithelium were evident in the thyroid. In some high-dose rats, the aspherical follicles dominated. Follicular changes in the thyroids of rats treated with 25 mg/kg trans-nonachlor can be seen in Figure 2. Thyroid lesion incidence is summarized in Table 7
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DISCUSSION |
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Mean daily food and water consumption were significantly lower in female rats treated with 25 mg/kg trans-nonachlor, which was consistent with a significant decrease in both mean weight gain and mean final bw. In all other groups, significant changes in food consumption, water consumption, or weight gain did not result in significant changes in the final bws of treated rats compared to control rats. It is likely that the cumulative effects of changes in food and water consumption over a longer treatment period would have eventually resulted in decreased bws, but these effects did not appear within the time frame of the present study.
Technical chlordane causes increased liver weights in rats (Khasawinah and Grutsch, 1989; Ogata and Izushi, 1991
). as was confirmed in this study. This change is considered to be an adaptive response associated with increased liver microsomal enzyme activity and not an adverse effect (U.S. DHHS, 1994). The present study confirmed that oral exposure to trans- and cis-nonachlor had similar effects on rat livers, although trans-nonachlor was more potent. Both chemicals caused increased liver weights, which were highest in rats exposed to trans-nonachlor. The observation of liver cell hypertrophy in treated rats was consistent with increased liver weight, as was the observation of hepatic drug metabolizing enzyme induction in treated rats in the present study (I. Curran, unpublished data). Technical chlordane, oxychlordane, trans-chlordane and cis-chlordane have previously been shown to induce rat hepatic microsomal enzymes (Campbell et al., 1983
). The increased incidence of lipid-filled vacuoles in livers of treated rats in the present study is consistent with increases in liver total lipids, triglycerides, and phospholipids measured in rats gavaged with 100 mg chlordane/kg bw for 4 days (Ogata and Izushi, 1991
). Increased serum cholesterol, which is associated with hepatic changes, was more pronounced in rats treated with trans-nonachlor than in those treated with cis-nonachlor or technical chlordane. Increased serum alanine aminotransferase (ALT), which can be indicative of hepatotoxicity, was observed in male rats treated with trans-nonachlor. This increase was statistically significant but the degree of elevation was modest and there was no corresponding hepatocellular necrosis in liver sections.
Renal changes were sex-specific and were statistically significant in male, but not female rats. Elevated kidney weights in male rats were a general index of renal changes. Standard urinalysis endpoints such as protein, NAG, and osmolality were not sufficiently sensitive to indicate any corresponding functional changes. Changes in blood urea nitrogen were inconsistent and did not correspond to changes in kidney weights. However, changes in organic ion transport in renal cortical slices have been used extensively to study nephrotoxic compounds (Berndt, 1987) and significant depression of this parameter in male rats confirmed that changes in kidney weights were accompanied by altered kidney function. Uptake of the organic cation TEA was inhibited by 32, 24, and 17% in trans-nonachlor, cis-nonachlor, and chlordane-treated male rats, respectively. Since uptake of the organic anion PAH was unaffected, the results suggest that damage to renal tubular cells was specific to the cationic transport system and was not due to nonspecific, membrane-related perturbations.
Morphological changes were observed in kidneys from male rats but not females. This was consistent with the sex-related nature of renal functional changes. However, a comparison of changes in kidney morphology and other indices of renal toxicity in rats treated with cis-nonachlor, trans-nonachlor, or chlordane revealed several inconsistencies. First, there were no visible renal lesions in male rats treated with cis-nonachlor, in spite of increased kidney weights and decreased TEA transport in these animals. This implies that there was not necessarily a direct relationship between the renal toxicity endpoints used in this study and the observed structural changes. Second, chlordane-treated rats had the highest incidence of visible kidney lesions, indicating that components of chlordane other than cis-nonachlor and trans-nonachlor were partly responsible for renal changes. Overall, the consequences of renal changes were not immediately apparent from the healthy outward appearance of male rats and from the lack of consistent changes in urine volume and water consumption.
Treatment with each of the test chemicals resulted in elevated serum total protein at the highest test dose, with trans-nonachlor causing the most pronounced changes. The lack of significant or consistent changes in serum and urine osmolality and urine output indicates that dehydration was not responsible for increased serum total proteins. Serum immunoglobulins can be ruled out as contributors to increased serum total protein levels, as they were not significantly altered by any of the test chemicals. In trans-nonachlor-treated rats, increased serum albumin appears to account in part for elevated total protein, but this is not the case for rats treated with cis-nonachlor and technical chlordane, indicating that more in-depth analyses are necessary to determine which proteins are elevated in treated rats. Serum magnesium, and to a greater extent serum calcium, were elevated in some treatment groups. Both of these ions can be elevated by increases in plasma carrying proteins (Riley and Cornelius, 1989), so it is possible that elevated serum total protein levels play a role in the observed increases in serum calcium and magnesium.
The induction of hepatic microsomal enzymes by chlorinated hydrocarbons, including chlordane, has been shown to disrupt thyroid hormone metabolism (Capen, 1994). It is possible that microsomal enzyme induction played a part in the reduction of serum thyroxine (T4) and thyroxine uptake in chlordane-treated rats and in reduced T4 uptake in trans-nonachlor-treated rats. However, altered protein levels may also have had an effect on serum T4 and T4 uptake as albumin is an important T4 binding protein in rats (Capen, 1992
; Döhler et al., 1979
). The interaction of these factors and their relationship to the increased incidence of irregularly shaped follicles in the thyroids of rats given cis-nonachlor, trans-nonachlor, or chlordane remains to be determined. It is possible that the thyroid changes observed in the present study were transient. Barrass et al (1993) used bromodeoxyuridine labeling to measure increased cellular proliferation in the rat thyroid, which peaked after 5 days of receiving 50 ppm chlordane in the diet, but which was not evident after day 99. Increased thyroid cell proliferation was not accompanied by histopathological changes in sections stained with hematoxylin and eosin. In addition, there were no histopathological changes in the thyroids of rats ingesting chlordane in the diet at levels up to 25 ppm for 130 weeks (Khasawinah and Grutsch, 1989
).
One of the most consistent changes in rats treated with cis-nonachlor, trans-nonachlor, and technical chlordane was increased serum amylase, primarily at the 25 mg/kg dose level. This effect was most pronounced in rats treated with trans-nonachlor. Increased circulating amylase is associated with pancreatic damage, which results in leakage of pancreatic enzymes into the peripancreatic area and their subsequent absorption into the general circulation (Short, 1961). The absence of histopathological changes in the pancreas of treated rats rules out pancreatitis, which is the condition most often associated with elevated serum amylase. Other possibilities for future consideration include direct effects on serum amylase at the level of enzyme production, release, and degradation that may affect the half-life of circulating amylase.
The primary metabolite of cis-nonachlor, trans-nonachlor, and technical chlordane was oxychlordane, as indicated by tissue residues. In cis- and trans-nonachlor-treated rats the parent compounds also accumulated in adipose tissue and liver at higher levels than oxychlordane. This was also observed by Hirasawa and Takizawa (1989), who showed that in mice, nonachlor metabolism is slower than chlordane metabolism and large amounts of unchanged nonachlors are retained in the tissues. Since trans-nonachlor has been shown to be rapidly metabolized in rat liver microsomes in vitro (Tashiro and Matsumura, 1978), it is possible that the proportion of cis- or trans-nonachlor to oxychlordane residues would have decreased in rat tissues over a longer treatment period. In addition to cis- and trans-nonachlor, oxychlordane is an important metabolite of other components of the technical chlordane mixture, including cis- and trans-chlordane (Barnett and Dorough, 1974
). This supports the observation that oxychlordane was the most abundant tissue residue in liver and adipose tissue in rats treated with technical chlordane.
The pattern of residue accumulation in rats treated with cis- and trans-nonachlor in this study was similar in several respects to the pattern of accumulation observed in rats treated with cis- and trans-chlordane (Barnett and Dorough, 1974). First, residue levels were highest in adipose tissue. Second, treatment with trans-nonachlor resulted in higher tissue oxychlordane levels than treatment with cis-nonachlor. Finally, residue levels were higher per gram of adipose tissue in females than in males, whereas residue levels per gram of liver were similar for both sexes. In the present study the mean final bws of female rats reached a maximum of 259.8 g, compared to 465.6 g in male rats. Assuming that male rats had more total body fat at necropsy than females and that chlordane-related compounds preferentially localize to adipose tissues, it is possible that increased fat residues in females were the result of test chemical localization into a smaller pool of fat tissue. This cannot be confirmed because total body fat percentages in male versus female rats were not determined in this study. High adipose tissue residue levels appeared to correlate with overt toxicity, as the highest levels were measured in trans-nonachlor-treated female rats. Since organochlorine compounds can be transferred to the fetus via the placenta and to the infant via breast milk (Skaare et al., 1988
), a possible sex-related difference in tissue organochlorine accumulation merits further attention. Although dietary surveys indicate that women in their childbearing years in aboriginal communities are not always the maximum consumers of organochlorine contaminants in wildlife food (Kuhnlein et al., 1995
), the potential for greater residue accumulation in body fat of females based on sex-related differences in size and percentage of body fat are factors to be considered when estimating the impact of dietary organochlorine exposure.
Based on the present study, the target organs and effects of cis-nonachlor, trans-nonachlor, and technical chlordane in rats were generally similar. However, trans-nonachlor accumulation in adipose tissue was greater than cis-nonachlor when rats were administered each chemical under identical conditions of dose and exposure. This is consistent with the observation that in the Arctic food chain, trans-nonachlor has been measured in fat or muscle at levels 5 to 8 times higher than cis-nonachlor levels in fish, seal, and polar bears (Muir et al., 1988). In addition, human milk and breast adipose tissue has been shown to be contaminated with trans-nonachlor at levels ranging from 2 to 12 times higher than cis-nonachlor levels (Dearth and Hites, 1991b
; Polder et al., 1998
). Furthermore, in the present study trans-nonachlor was overtly toxic to female rats and had more pronounced effects than cis-nonachlor on some clinical chemistry and histopathological endpoints. These results indicate a need for further characterization of the long-term effects of trans-nonachlor exposure as well as a need for further examination of sex-related differences in responses to trans-nonachlor.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Toxicology Research Division, Postal Locator 2204D2, Food Directorate, HPB, Health Canada, 2E Banting Building, Ottawa, Ontario K1A 0L2, Canada. Fax: (613) 941-6959. E-mail: genevieve_bondy{at}hc-sc.gc.ca.
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