* Growth and Development Section, Environmental and Occupational Toxicology Section, Safe Environments Directorate, Health Canada, Environmental Health Centre, Tunney's Pasture, Ottawa, Ontario, Canada K1A 0L2;
Department of Obstetrics and Gynecology, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5;
INRS, Institut Armand Frappier, 245 Blvd. Hymus, Pointe-Claire, Québec, Canada H9R 1G6; and
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina 27710
Received July 19, 2001; accepted October 29, 2001
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ABSTRACT |
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Key Words: organochlorines; dioxin, PCB; spermatogenesis; hepatotoxicity; EROD; PROD; mixture effects.
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INTRODUCTION |
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Current understanding of the toxicity of these compounds is based primarily on toxicity studies performed on laboratory animals exposed to a single toxic agent. These studies demonstrate that many of these compounds have effects on the reproductive tract, through the alteration of endocrine physiology, the liver, and thyroid physiology (Brouwer et al., 1998; Gray et al., 1989
; Liu et al., 1995
; Singh and Pandey, 1989
). The reported effects are seen, in all cases, at relatively high levels of exposure. The human population is ubiquitously exposed to complex mixtures of these contaminants generally at much lower levels of exposure than those routinely examined in animal toxicity studies and the effects of any interactions between such substances on their toxicity is virtually unknown.
To estimate safe levels of exposure for any given compound, for the human population, all available toxicity data for that compound are synthesized into dose rate through a broadly accepted methodology (Barnes and Dourson, 1988) which, theoretically, is the highest rate of exposure that can occur over a moderate length of time that will cause no adverse health impacts. These dose estimates, called minimum risk level, reference dose, tolerable daily intake, etc., are promulgated by various public environmental health organizations and are generally used to gauge specific exposures for their safety. While this approach assumes that there is little interaction between chemical substances in their toxic effects or that the degree of any synergistic increase in toxicity will not exceed the safety factors applied, there have been relatively few studies that have tested these assumptions. Further, there have been a number of studies on the toxic effects of exposure to mixtures on general toxicity (Boyd et al., 1990
; Gyorkos et al., 1985
; Ito et al., 1995
) and several studies examining the effects of complex defined mixtures of ground water contaminants on reproduction (Chapin et al., 1989
; Heindel et al., 1994
, 1995
) or immune function (Germolec et al., 1989
). In the latter study, the effect of a mixture of 25 ground water contaminants was examined on reproduction in mice and rats using a continuous breeding study and only minor effects of the mixture were demonstrated.
In the current study we have examined the toxicity of low doses, relative to doses generally used in animal toxicity experiments, of a complex mixture, containing contaminants that have been demonstrated to be present in human reproductive tissues and fluids, to liver and general physiology and, specifically, to the reproductive and immune systems in the adult male rat.
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MATERIALS AND METHODS |
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Rats were randomly assigned to control (n = 9), 1x (n = 10), 10x (n = 10), 100x (n = 10), or 1000x (n = 10) treatment groups. Rats received corn oil (vehicle) or appropriate treatment in a volume of 1 µl/g body weight by gavage daily for 70 days, equivalent to one full cycle of spermatogenesis. Animals were monitored daily for signs of overt toxicity and body weight was recorded every 23 days.
On the day following the final dose, rats were anesthetized with isofluorane and, after recording body weight, were sacrificed by exsanguination via cardiac puncture followed by decapitation. Blood was collected in Serum Separator Vacutainer tubes (Becton-Dickenson), held for no more than 4 h before serum was collected by centrifugation (3000 x g for 15 min), aliquoted and frozen at 80°C until analysis. Analysis of serum biochemistry was conducted using a Technicon RA-XT (Bayer Diagnostics, Tarrytown, NY) and assay of circulating levels of LH, FSH, and prolactin was conducted using commercial RIA kits (Amersham Pharmacia, Piscataway, NJ). Prior to dissection of the abdominal organs, the body surface was wiped with 70% ethanol, an incision was made into the body cavity and, after the diaphragm was transected, the spleen was removed aseptically and teased into small pieces in complete RPMI-1640 containing 5% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Spleens were kept on ice and shipped to Montréal where splenocyte function was assessed.
The liver, thymus gland, left kidney, pituitary gland, adrenal glands, and major organs of the reproductive tract were removed, dissected free of connective tissue, and weighed. Once weighed, 4 pieces of the liver, roughly 1 g each, were frozen in liquid nitrogen and stored at 80°C prior to extraction of microsomes for hepatic enzyme analysis. An additional piece of liver, taken from the median lobe, was fixed in 4% paraformaldehyde for 72 h for histopathological analysis. One testis from each animal was snap frozen in liquid nitrogen for assessment of daily sperm production and the second testis was placed in ice cold M199 and held on ice until dispersion of testicular cells for flow cytometric analysis. As the amount of fluid lost from seminal vesicles due to dissection varied between animals, seminal vesicle weight was determined after all fluid had been extruded from the structure.
Splenocyte Function
The rationale for the selection of the mitogenic assay and the natural killer activity in this study is the following. Because it was not possible to stimulate an immune response by injection of an antigen (possible effects of the immune response on other physiological parameters studied in the experiment) we had to choose parameters allowing the assessment of the immune status of the animals in relation to the putative toxicity of the mixture. The ability of the mitogenic assay and NK activity to predict an immunotoxic outcome is 67 and 69% respectively when these tests are used alone. However, when they are used in combination, the predictability increases up to 79% (Luster et al., 1992). Moreover, in recent studies designed to look at the immunotoxicity of complex mixtures in rat as well as in mouse these immunological markers appeared to be sensitive endpoints to assess the toxic action of chemicals (Fournier et al., 2000
; Lapierre et al., 1999
; Omara et al., 1997
, 1998
, 2000
; Tryphonas et al., 1998a
,b
).
Splenocytes were put in suspension and washed twice in HBSS with centrifugation. Viable leucocytes were isolated using density gradient centrifugation on Ficoll-paque (Amersham Pharmacia, Piscataway, NJ). The splenocytes were collected from the Ficoll-paque interface and washed 3 times in HBSS. After the final wash, the splenocytes were suspended in complete RPMI-1640. The viability of splenocytes was determined by membrane permeability to propidium iodide using a flow cytometer. The cells were collected and labeled with propidium iodide (1.5 µg/ml) before data acquisition by flow cytometry (FACScan, Becton-Dickinson) to distinguish live and dead cells.
Determination of mitogen-induced lymphocyte blastogenesis.
The ability to undergo blastogenesis was measured by [3H]thymidine uptake. Briefly, splenocytes (5 x 105 per well) were cultured for 72 h at 37°C and 5% CO2 with various concentrations of mitogen in 96-well flat-bottom plates. The optimal concentrations used to stimulate splenocytes were 2.5 µg/ml and 5 µg/ml for concavalin A (Con A), and 2.5 µg/ml and 10 µg/ml for phytohemaglutinin (PHA), both being T-lymphocyte mitogens. After a 72-h incubation, 0.5 µCi of [3H]thymidine was added to each well and plates were incubated for a further 18 h. The cells were harvested onto filters with a Tetratex Cell Harvester, and the amount of radioactivity incorporated was quantitated in a scintillation counter, (Beckman LS1801). The results were expressed as disintegration per minute (dpm).
Determination of NK activity.
To determine the possible effects of the mixture on NK cell activity, splenic NK cell-mediated lysis of murine lymphoma cells (Yac-1 cells) was determined by a flow cytometry method (Brousseau et al., 1998; Chang et al., 1993
). Briefly, target cells (YAC) in exponential phase are counted and 107 cells are incubated with 33`-dioctadecycloxacarbocyanine perchlorate (DiO) for 20 min at 37°C with 5% CO2. The cells are then washed and the concentration adjusted at 1 x 106 cells/ml. DiO has an absorption and fluorescence spectra compatible with FITC. Target cells (DiO+) are then incubated with effector cells at different ratios (1/30, 1/60) for 4 h in the presence of propidium iodide (PI). At the end of the incubation period the cells are gently dislodged and data were acquired by FACScan (Becton Dickinson) flow cytometer with a threshold set at FL1 (DIO+ cells) to exclude effector cells and bare cell nuclei that are DiO negative. A total number of 10,000 events are collected. Data were analysed by LYSYS II software (Becton Dickinson) to determine target cell death (cytolethality). When an NK cell damages the membrane of a target cell, PI can no longer be excluded and the target cell is stained by PI to become DiO+, PI+. In contrast, target cells not affected by NK cells will exclude PI to remain DiO+, PI. The results are expressed in percentage of cytotoxicity.
Liver microsomal enzyme activity.
To assess the activities of several inducible enzymes, a frozen sample of liver was homogenized in 2.5 volume of ice-cold 0.2M Tris containing 1.15% KCl (pH 7.4) with a teflon-glass homogenizer, to prepare a 33% (w/v) homogenate. Nuclei and mitochondria were removed by centrifugation at 10,000 x g, 4°C for 20 min. This postmitochondrial supernatant, (S9) was recovered and stored at 80°C until assayed. Protein concentration was determined by Bio-Rad Bradford protein assay (Bio-Rad, Hercules, CA) using bovine serum albumin (Fraction V, Sigma Chemical, St. Louis, MO) as the standard. In order to determine the effects of the mixture on the induction of hepatic phase I biotransformation monooxygenases, aliquots of S9 were thawed on ice and assayed for ethoxy- and pentoxy-resorufin-O-dealkylase (EROD and PROD, respectively) activities as previously described (Burke et al., 1985; Lubet et al., 1985
) to estimate the activities of CYP1A1 and CYP2B gene products, respectively. These in turn indicate the activation of the aryl hydrocarbon receptor signalling pathway and phenobarbitol-like xenobiotic response. In addition the activity of 7-benzyloxy-resorufin-O-dearylase (BROD) was analyzed as previously described (Burke et al., 1985
) to indicate the induction of various cytochrome P450 (Namkung et al., 1988
).
Histological examination of liver.
Fixed liver tissue was processed into paraffin and 5 µm-thick sections were cut and stained with hematoxylin and eosin. Liver histopathology was analyzed for 5 animals per treatment group by an observer who was unaware of treatment. Sections were scored using an arbitrary scale from 1 to 16 for degree of severity of histopathological lesions. The scale was broken into 4 categories with 14 being minimal, 58 mild, 912 moderate, and 1316 marked with 1 point increments within each range for focal, diffuse, multifocal, or throughout.
Micronucleus assay of bone marrow.
To assess the degree to which the mixture caused chromosome damage or interfered with mitotic apparatus of developing red blood cells, the incidence of residual chromosome fragments (micronuclei) in polychromatic erythrocytes from femoral bone marrow was determined, as this is a well-established biomarker of chromosome breakage due to DNA damage (Schmid, 1975). At necropsy, bone marrow was washed out of the femoral lumen onto a glass slide in 200 µl of bovine serum, smeared, and allowed to air dry. Slides were fixed in methanol and stained with 0.005% acridine orange in 66 mM phosphate buffer (pH 7.3). Stained slides were examined in a dark room using an Olympus fluorescence microscope with mercury lamp illumination. At least 3000 polychromatic erythrocytes were examined for the presence of micronuclei.
Daily sperm production.
Daily sperm production was estimated according to previously described methods (Blazak et al., 1985). Briefly, the testis was decapsulated and parenchyma weighed and homogenized in 20 ml of STA solution (0.9 % NaCl, 0.01% Triton X-100, and 0.025% sodium azide) using an Ultramicro blender (Waring, New Hartford, CT) on low setting for 2 min. Testis homogenates were then disrupted with a VibraCell sonicating dismembranator (Sonics & Materials, Danbury, CT). Sperm nuclei density in the testis homogenate was estimated by counting at least 10 replicate samples using a haemocytometer. The number of homogenization resistant testis nuclei was divided by 6.1 days to estimate the number of mature spermatozoa released to the epididydimis per day (Blazak et al., 1985
). Results were also expressed as the ratio of DSP to testis weight to estimate the efficiency of sperm production (DSP efficiency).
Sperm chromatin structure assay.
The degree of condensation of sperm nuclei is related to fertilizing potential (Evenson and Melamed, 1983) and is a parameter indicative of toxicant-induced disruption of spermatogenesis (Evenson, 1986
; Evenson et al., 1980
). Sperm chromatin condensation was measured by differential acridine orange fluorescence of acidified sperm, in which condensed and decondensed DNA fluoresce in the green or red ranges respectively. The susceptibility of sperm nuclei to denaturation by acid is estimated by the ratio of red fluorescence to total fluorescence (
t). The values of
t for normal sperm fall within a tight range and toxicant-induced damage increases the number of cells falling outside this range such that the percent of cells with
t more than 2 x SD outside this range (% cells outside of main population) or the variability of
t in this variant population are inversely correlated with function (Evenson, 1986
; Evenson et al., 1980
).
To assess t, epididymal sperm suspensions were prepared by homogenizing one cauda epididymis from each animal in ice cold TNE buffer (10 mM Tris, 0.9% NaCl, and 0.5 mM EDTA, pH 7.4) using an Ultra-Turrax homogenizer (Tekmar, Cincinnati, OH) and stored at 4°C for no more than 5 days prior to analysis of sperm chromatin structure by flow cytometry. The Sperm Chromatin Structure Assay (SCSA) utilizes the differential fluorescence of the nucleic acid-binding dye acridine orange (AO) that fluoresces red or green when bound to either denatured or condensed chromatin, respectively. To assess the effect of treatment on sperm chromatin condensation, sperm chromatin structure was assessed in acid-treated sperm nuclei using a flow cytometric method described previously (Foster et al., 1996
).
Flow cytometric analysis of testis cells.
One fresh testis from each animal was decapsulated into 3 ml of fresh M199 and minced with iris scissors. After all testes were minced, the resulting cell suspensions were filtered through 105 µm diameter plastic mesh. Filtered suspensions were pelleted at 3000 x g for 10 min, resuspended in 70% ethanol, and stored at 20°C for subsequent determination of spermatic cell populations based on DNA staining (Suter et al., 1997). Briefly, fixed cells were washed twice in phosphate buffered saline (PBS) and resuspended in PBS. The cell suspension was then passed sequentially through a 25-gauge needle and then a nylon mesh (75 µm mesh opening, Costar Netwell, Costar, Cambridge, MA). A 1 ml aliquot of the filtered cell suspension was stained by adding Tween 20 (0.3% final concentration), RNAase (bovine pancreatic, 10 U/ml, Sigma), and propidium iodide (50 µg/ml, Molecular Probes, Portland, OR) and incubated at 4°C in the dark overnight. Samples were analyzed flow cytometrically with excitation at 488 nm and emmission fluorescence at 630 nm. A minimum of 10,000 cells were examined at a flow rate of 200 cells/s and the results analyzed using List-View (Phoenix Flow Systems, San Diego, CA). Reproducibility of sample manipulation and instrument performance was routinely evaluated for both flow cytometric methods utilized in this study. An aliquot from a single control sample was analyzed for each measurement at least 4 times during each session and coefficients of intra- or intersession variability did not exceed 15% for any measure.
Statistical analyses.
Animals were killed between 0900 and 1300 h on either of 2 consecutive days with the number of animals per treatment group balanced between days. Data that were expressed as percent were normalized by arcsine transformation prior to analysis by ANOVA. Unless otherwise indicated, all data were analyzed by two-way ANOVA with mixture dosage and day of necropsy being the 2 factors tested. Analysis of effects of mixture on variability of t was performed using O'Brien's test of homogeneity of variance (O'Brien, 1979
) Where significant effects were identified for treatment, multiple comparisons were tested using Student Neuman Keuls post hoc test. All data sets were tested for homogeneity of variance and normal distribution and, where homoscedasticity or normality were not indicated, data were retested after log transformation. Where either homoscedasticity or normality tests were not satisfied after this transformation, data were analyzed, for mixture dose treatment effects, but not effect of day of sacrifice, using Kruskal-Wallis ANOVA on ranks test followed by Dunn's test for multiple comparisons. The accepted level of significance was set at p
0.05 while tests with a p value between 0.05 and 0.1 were viewed as identifying a trend. All statistical analyses were performed using SigmaStat software version 2.0 (SPSS, Chicago).
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RESULTS |
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Reproductive and Endocrine Effects
There was remarkably little indication of toxicity of the mixture to the wide variety of reproduction-related endpoints examined in this study. Other than effects seen on epididymis weight and epididymal sperm content, no treatment-related effects were seen on the weights of other organs of the male reproductive tract (Table 2), daily sperm production (either per testis or per g of testis; Table 6
), relative numbers of spermatogenic cell populations (Figs. 3A3D
), various parameters related to nuclear condensation in sperm (Figs. 4A4D
), or serum levels of LH, FSH, or prolactin (Table 7
). However, exposure to the mixture caused a dose related reduction in both absolute (not shown) and relative total and caput, but not cauda, epididymis weight (Table 2
).
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Exposure to the complex mixture had little impact on epididymal sperm chromatin condensation as there were no effects on mean t (Fig. 4A
) or on the variability of
t values within each animal (Fig. 4B
). The number of cells for which
t values differed greatly from the mean value for each animal (percent cells outside the main population; Fig. 4C
) was significantly reduced in animals in the 10x treatment group, relative to all other groups. In addition these animals showed a slight increase in the SD of this group of cells although this effect was only significant relative to animals from higher dose groups.
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DISCUSSION |
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The quantities used in formulating the doses of most components of the mixture are promulgated by public environmental health agencies in Canada (Health Canada) or the U.S. (Agency for Toxic Substances and Disease Registry, U.S. Environmental Protection Agency) as guidelines to acquaint physicians or public health professionals with the maximum exposure levels that would not be expected to cause increased risk to health. The sole exception was the dose selected for TCDD that is based on the highest dose of TCDD shown to be without effect on the endpoint shown to be most sensitive to TCDD toxicity (NOAEL of 1 ng/kg/day; Feeley and Grant, 1993; Murray et al., 1979
). Other than TCDD, the safe levels used to formulate the dosing mixtures were calculated using a modification of basic risk assessment methodology used to determine reference dose for lifetime exposure by the U.S. EPA (Barnes and Dourson, 1988
). This process involves the identification of the highest NOAEL for the most sensitive endpoint in the most sensitive species, where toxicity data are available for multiple species, and dividing this value by an appropriate uncertainty factor. For example, the TDI estimate for 1,2,4-trichlorobenzene, of 0.0023 mg/kg/day, is based on a NOAEL of 22.9 mg/kg/day inhalation exposure for 13 weeks on a variety of general toxicity endpoints (CEPA, 1993
). This value was then divided by 10 for uncertainty in extrapolating from rodent to human, 10 to account for human variability, 10 for extrapolation from a relatively short-term study to longer-term exposures, and 10 because this study provides the only data on the mammalian toxicity of this compound. The use of these values as a predictor of risk assumes that toxicity will not be modified by coexposure to other substances or that any increase in potency from such interaction will not exceed the safety factor used. For all compounds included in the mixture, with the exception of cadmium, the safe levels are derived from animal studies and have safety factors equivalent to or in excess of 100. The chronic MRL for cadmium is derived from human data on renal toxicity (NOAEL = 2.1 µg/kg/day) corrected by a safety factor of 10 (ATSDR, 1999
). If one assumes that the available toxicity data for a given compound is a complete reflection of the compound's hazard, the calculated MRL should be less than 1/100 the dose that causes toxicity in the animal models (generally rodents) used to generate the data. In the present study, hepatic EROD activity was significantly elevated in 10x exposed animals. This suggests that the low doses used to derive the mixture formulation were not entirely without effect and implies that the assumption that coexposures are irrelevant is not valid. Alternatively, the endpoints used in determining the NOAEL on which these MRLs are based are less sensitive than the endpoints that were altered by the 10x mixture. It should also be noted that the MRLs used for some of the components have since been replaced with reduced estimates of safe exposure. For example, when the dosing mixture was first formulated, the safe level of dioxin used was based on the interim TDI for PCBs that was 1 µg Arochlor 1254 per kg body weight per day (Grant, 1983
). Since that time, ATSDR has promulgated a MRL for chronic exposure for PCBs (as Arochlor 1254) of 20 ng/kg/day (ATSDR, 1997
). In addition, the dose of dioxin of 1 ng/kg/day used in the present study, based not on a MRL type estimate but on a NOEL from a developmental toxicity study, is 3 orders of magnitude above the more recently promulgated MRL of 1.0 pg TEQ/kg/day (ATSDR, 1998
).
The compounds used to formulate the mixture were chosen to reflect some of the persistent anthropogenic compounds, which have been shown to be present in the tissues of Canadians, to determine if exposure to relatively low levels of mixtures of these may have health consequences. While the lowest doses were below those shown to cause any toxicity, the administered doses are much higher than the estimated average daily exposure to some of these substances for Canadian populations (Birmingham et al., 1989; CEPA, 1993
; Health Canada, 1998
).
The mixture was clearly hepatotoxic at the highest dose administered as indicated by the increased liver weight and modified liver histology. In addition, hepatic activity of EROD and BROD, but not PROD, were increased in a dose-related fashion. The induction of hepatic EROD activity is characteristic of exposure to arylhydrocarbon receptor agonists such as dioxins or coplanar PCBs (Van den Berg et al., 1998), both of which are present in the dosing mixture. The lack of induction of PROD activity suggests that it was not as sensitive as BROD for detecting CYP2B induction. BROD was selected as an endpoint to provide additional indication of cytochrome P450 induction, although the interpretation of the increased catabolism of this substrate due to mixture treatment is difficult as this substrate is also catabolized by CYP1A1, CYP3A, and CYP2B (Namkung et al., 1988
). As the activity of EROD, exclusively a substrate of CYP1A1 activity, was markedly increased by the mixture, the concomitant increase in BROD activity may simply reflect an increase in CYP1A1 activity, likely in response to the TCDD and, to a lesser extent, the Aroclor in the mixture. The cumulative dose of TCDD to which 10x MRL animals have been exposed (700 ng/kg) is comparable to doses of dioxin that have been shown to induce EROD activity in rats in chronic exposure studies (200 ng/kg, Walker et al., 1999
) but more than 10-fold higher than the lowest dose shown to induce toxicity to the reproductive tract in rats exposed in utero to a single dose of TCDD on day 15 of gestation (64 ng/kg; Mably et al., 1992a
,b
).
Other biochemical parameters sensitive to dioxin or PCB exposure include serum glucose, total protein, and albumin (Chu et al., 1994, 2001
). Depletion of serum glucose is thought to occur via direct inhibition of phosphoenol pyruvate carboxykinase, a key enzyme of gluconeogenesis, by these compounds that ultimately leads to a wasting syndrome (Viluksela et al., 1995
). The reduction in serum glucose seen in the 1000x animals, although not quite statistically significant, is consistent with many previous studies of relatively long-term dioxin or PCB exposure (e.g., Chu et al., 1994
; Gorski et al., 1990
) and will likely impact on intermediary metabolism in these animals. The increase in total protein and albumin may be the result of hypertrophic changes in the liver in response to the mixture and the proliferation of endoplasmic reticulum implied by the obvious cellular hypertrophy (Fig. 2
) and increased cytochrome P450 activity. Alternatively, increased serum protein and albumin levels could indicate mild dehydration that is consistent with negative water balance seen in rats treated with a single dose of TCDD, similar to the total dose administered to the high dose animals over the 70-day treatment period (50 µg/kg vs. 70 µg/kg in the present study; Potter et al., 1986
). Other changes seen in the serum are not clearly defined. Increases in calcium and phosphorus and the reduction in uric acid and urea nitrogen may indicate some impaired kidney clearance. This is consistent with the observed increase in kidney weight in the 1000x animals although, in the absence of kidney histopathology or assessment of urinary parameters no definitive conclusions can be drawn. Finally, the reduced serum LDH in the highest doses is interesting and may indicate the inactivation of this enzyme by highly induced mixed function oxidase reactions (Fucci et al., 1983
) or a direct inhibition by one or more of the mixture components.
In contrast to the toxic effects on liver, the mixture had little effect on the reproductive physiology of adult male animals. This conclusion is based on a broad assessment of reproductive physiology including an examination of several endpoints related to spermatogenesis (relative proportions of testicular cell populations, daily sperm production, epididymal sperm reserves, and sperm nuclear condensation), indicators of endocrine control of reproduction (serum levels of LH, FSH, and prolactin) and terminal weights of reproductive organs. The only organ that appeared to be influenced by the mixture was the epididymis, where the relative weight of the whole or caput were significantly reduced at the two highest doses. In addition, the mixture had an apparently biphasic effect on sperm transit through the epididymis as cauda sperm reserves were significantly increased at the 1x dose but returned to control levels at higher doses. The significance of this nondose-related finding is unclear. Although no statistically significant effects were indicated on the majority of other reproductive endpoints, it should be noted that the power of the statistical tests for most of these endpoints was low. This could imply that endpoints for which the mixture-induced changes were close to (e.g., testis weight) or just at statistical significance (epididymis weights and sperm content) may have shown a stronger response if more animals had been used in the study. Some of the components of the mixture (dioxin, PCB, methoxychlor) have previously been shown to compromise reproductive competence when administered to male rats, although these effects occurred at much higher doses than were present in the dosing mixtures administered in the current study and/or involved the initiation of dosing at a younger age (Chapin et al., 1997; Cooke et al., 1996
; Gray et al., 1989
, 1995
; Welshons et al., 1999
). It should be noted that fetal, neonatal, and immature animals are more sensitive to some mechanisms of action of reproductive toxicants, so the possibility that the mixture may have effects in developing animals is currently being determined in our laboratory.
In addition to reproductive endpoints, the current study also evaluated the mutagenic and immunotoxic effects of the mixture. The effects of the mixture on immune function were also minimal having clear toxic effects only at the highest dose tested. As described above, the ability of the mitogenic assay and NK activity to predict an immunotoxic outcome is 67 and 69% respectively when these tests are used alone. However, when they are used in combination, the predictability increases up to 79% (Luster et al., 1992). Despite this, it is very hard to translate immunotoxic outcomes into a direct measurable decrease of resistance to infections; they represent definitely adverse effects representing a serious hazard.
While considerable research has been devoted to the immunotoxicity of single exposures to numerous chemicals, less emphasis has been given to the toxicity of mixtures. Most human exposures to chemicals involve mixtures at relatively low levels, and concurrent exposure to multiple chemicals can increase or decrease the toxicity of single chemicals (Teuschler and Hertzberg, 1995). Human volunteers with TCDD body burdens of > 60 ppt, and those with reference TCDD of < 20 ppt, or workers exposed to dibenzofurans showed no evidence of immunosuppression from immunological tests performed (Webb et al., 1989
; Zober et al., 1992
). In a previous study, Omara et al. (1997) demonstrated that in vitro exposure of rat lymphocytes to low levels of Aroclor PCB mixtures, PCDD/PCDF mixtures, or MeHg/PCB/PCDD/PCDF mixtures, produced no additive immunotoxicity as assessed by leukocyte cytolethality, and T- and B-cell mitogen-stimulated lymphocyte proliferation, macrophage phagocytic activity, natural killer (NK) and cellular immune response by the one-way mixed leukocyte reaction (MLR). On the other hand other types of mixtures such as particulate air pollution, mixtures of contaminant as present in fish flesh or marine mammals blubber were demonstrated immunotoxic in rodents, especially in mice. The dose range at which certain immunological effects are observed in experimental animals are exposed to chemicals should be taken into consideration before extrapolating data to human exposure situations. A number of observations also suggest that rat and human immune systems appear to be less susceptible than mice to halogenated aromatic hydrocarbons (HAHs), particularly TCDD, which is considered the most toxic of the HAHs (Lang et al., 1994
; Mocarelli et al., 1986
; Smialowicz et al., 1994
; Zober et al., 1992
).
The results of this study demonstrate that while estimates of safe levels of exposure, applied to a mixture of persistent contaminants, provide apparently good protection for the function of the male reproductive tract, these studies raise concern over their applicability for hepatic and thyroid effects. However, MRL promulgation is a reactive process and the actual values tend to creep downward as new toxicological evidence comes forward as demonstrated by the reduced estimates of safe levels of exposure for PCBs and dioxins. The current study suggests that the MRLs, TDIs, or RfD set by the U.S. ATSDR, U.S. EPA, and CEPA provide adequate protection for adult male animals, for those systems examined, from the adverse effects of these persistent contaminants.
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ACKNOWLEDGMENTS |
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NOTES |
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