Neurotoxicity Produced by Dibromoacetic Acid in Drinking Water of Rats

V. C. Moser*,1, P. M. Phillips*, A. B. Levine{dagger}, K. L. McDaniel*, R. C. Sills{ddagger}, B. S. Jortner§ and M. T. Butt

* Neurotoxicology Division, NHEERL/ORD, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711; {dagger} University of North Carolina, Chapel Hill, North Carolina, 27599; {ddagger} NTP, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 27709; § Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, 24061; and Pathology Associates Division of Charles River Laboratories, Frederick, Maryland, 21701

Received October 10, 2003; accepted January 14, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An evaluation of potential adverse human health effects of disinfection byproducts requires study of both cancer and noncancer endpoints; however, no studies have evaluated the neurotoxic potential of a common haloacetic acid, dibromoacetic acid (DBA). This study characterized the neurotoxicity of DBA during 6-month exposure in the drinking water of rats. Adolescent male and female Fischer 344 rats were administered DBA at 0, 0.2, 0.6, and 1.5 g/l. On a mg/kg/day basis, the consumed dosages decreased greatly over the exposure period, with average intakes of 0, 20, 72, and 161 mg/kg/day. Weight gain was depressed in the high-concentration group, and concentration-related diarrhea and hair loss were observed early in exposure. Testing with a functional observational battery and motor activity took place before dosing and at 1, 2, 4, and 6 months. DBA produced concentration-related neuromuscular toxicity (mid and high concentrations) characterized by limb weakness, mild gait abnormalities, and hypotonia, as well as sensorimotor depression (all concentrations), with decreased responses to a tail-pinch and click. Other signs of toxicity at the highest concentration included decreased activity and chest clasping. Neurotoxicity was evident as early as one month, but did not progress with continued exposure. The major neuropathological finding was degeneration of spinal cord nerve fibers (mid and high concentrations). Cellular vacuolization in spinal cord gray matter (mostly) and in white matter (occasionally) tracts was also observed. No treatment-related changes were seen in brain, eyes, peripheral nerves, or peripheral ganglia. The lowest-observable effect level for neurobehavioral changes was 20 mg/kg/day (produced by 0.2 g/l, lowest concentration tested), whereas this dosage was a no-effect level for neuropathological changes. These studies suggest that neurotoxicity should be considered in the overall hazard evaluation of haloacetic acids.

Key Words: dibromoacetic acid; disinfection by-products; neurotoxicity; behavior; neuropathology; rats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chlorinated disinfection of drinking water, while beneficially killing harmful microbes, has the drawback of producing disinfection by-products (DBPs). Many of these chlorinated and/or brominated DBP compounds have been shown to be carcinogenic, mutagenic, and/or teratogenic in animal studies (Boorman, 1999Go). The two most prevalent classes of DBPs currently found in drinking water are trihalomethanes (THMs) and haloacetic acids (HAAs) (Boorman, 1999Go; Krasner et al., 1989Go). The U.S. Environmental Protection Agency (U.S. EPA) has proposed maximum contaminant levels of 60 µg/l for the total of five HAAs (U.S. EPA, 2003Go), the level that is believed to reduce risk from reproductive and developmental toxicity as well as cancer. However, levels of even single HAAs are sometimes reported to exceed that amount (Boorman, 1999Go; Krasner et al., 1989Go; Pourmoghaddas et al., 1993Go), with wide variations due to location, treatment process, source waters, and season (Egorov et al., 2003Go; Sérodes et al., 2003Go; Singer and Chang, 1989Go).

Dichloroacetic acid (DCA), the most prominent HAA, and dibromoacetic acid (DBA) are structural analogs, yet considerably less is known about DBA. DCA is carcinogenic and a reproductive toxicant in rats (DeAngelo et al., 1996Go; Linder et al., 1997aGo). Direct comparisons between DCA and DBA reveal that DBA produces spermatotoxicity at levels as low as 0.05 and 0.14 mM/kg/day (10 and 30 mg/kg/day), and is in fact similar to, but more potent than, DCA (lowest observable spermatotoxic effect of DCA found at 0.49 mM/kg/day, or 62.5 mg/kg/day; Linder et al., 1994aGo).

DCA is neurotoxic in both laboratory animals and humans (Bhat et al., 1991Go; Cicmanec et al., 1991Go; Katz et al., 1981Go; reviewed in Stacpoole, 1998Go). Administration of high doses of DCA produces peripheral neuropathy in humans, and laboratory studies of DCA in rats and dogs report hindlimb weakness, paralysis, neuropathology, and myelin vacuolation. The neurobehavioral toxicity produced by DCA has been characterized in Fischer 344 rats at dosages similar to those producing reproductive toxicity (Moser et al., 1999Go). In that study, DCA produced neuromuscular toxicity including limb weakness (decreased hindlimb grip strength) and deficits in gait and righting reflex, as well as mild tremors, ocular abnormalities, and a unique chest-clasping response. The neurotoxicity was progressive with continued exposure and was observed at DCA intake levels as low as 16 mg/kg/day in subchronic studies. This dosage is in the range of doses (around 30 mg/kg/day) producing reproductive toxicity (Mather et al., 1990Go; Toth et al., 1992Go).

A few studies of DBA mention neurological clinical signs at high dose levels. In oral gavage studies with daily dosing of Sprague-Dawley rats, mild lethargy was observed during the first week of dosing at 270 mg/kg/day (Linder et al., 1994bGo). In another study (Linder et al., 1995Go), subchronic exposure to 250 mg/kg/day DBA produced transient diarrhea and weight loss early on, followed by signs of excitability, awkward gait, atypical movement of limb, and abnormal posturing, and progressing to tremors and immovable hindlimbs. No such effects were observed at the next lower dose, 50 mg/kg/day. In yet another study, a single dose of 1000–2000 mg/kg of DBA produced "excessive drinking," hypomobility (defined as difficulty moving hindlimbs), labored breathing, and mild diarrhea and ataxia (Linder et al., 1994aGo).

Since DBA and DCA are similar in that both produce reproductive toxicity and neurotoxicity at high doses, this study was initiated to examine the potential neurotoxicity of DBA at lower doses and to compare it to DCA, by using a focused neurological screening battery. A thorough neurotoxicological evaluation of DBA could not be found in the literature. We used drinking water as the route of exposure in light of our findings that DCA is more potent when administered in the drinking water compared to gavage (Moser et al., 1999Go). We also reported that Fisher 344 rats are more sensitive to the effects of DCA, and that neurotoxicity is most evident when exposure begins in weanling rats (Moser et al., 1999Go). We therefore designed this DBA study with those experimental parameters. The study was intended to characterize the potential neurotoxicity of DBA during 6-month exposure in the drinking water, using a neurobehavioral screening battery to detect neurological and functional changes, and followed by perfusion fixation at the end of dosing for neuropathological evaluation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Dibromoacetic acid solutions were prepared by Battelle (Columbus, OH) under contract to NIEHS (contract #N01-ES-55395). DBA was purchased from Fluka (St. Louis, MO). Identity of the neat chemical was verified at the beginning and end of the study with NMR, IR, and UV/Vis spectrometry, and the purity was determined to be greater than 99% by functional group titration, Karl Fischer titration, and HPLC/UV. Formulations were prepared by mixing DBA with filtered tap water (Columbus, OH) at the target concentrations of 0.2, 0.6, and 1.5 g/l on a monthly basis. Each batch was determined to be within 10% of target when analyzed by a validated HPLC/UV method (Baughman, 1998Go). Linear regression was used to generate an equation relating the relative response at 220 nm of the DBA peak with that of the internal standard (dichloroacetic acid at 1.2 µg/ml in Milli-Q water). The method achieved a LOD of 0.31 µg/ml and a LOQ of 1.03 µg/ml with an average relative error 1.2% or less, using calibration standards prepared in Milli-Q water over a range of 10.75 µg/ml to 47.98 µg/ml. Stability in tap water had been previously confirmed for 35 days in Nalgene® containers if refrigerated (approximately 5°C) and for up to four days at room temperature in amber glass water bottles (Baughman, 1997Go).

Animals.
Fischer-344 derived rats were obtained from Charles River Breeding Laboratories (Raleigh, NC) at 24 days of age, and housed at the U.S. EPA AAALAC-accredited animal facility. The animal room was maintained at 70 ± 2°F, 50 ± 10% humidity, with a 12-hour light:dark cycle. Since DBA exposure began at 28 days of age, acclimation was limited to several days. Male and female rats (n = 12/concentration/sex) were assigned to treatment groups based on stratification of body weight at 25 days of age. Rats were individually housed in polycarbonate cages with heat-treated pine shavings for bedding and feed (Purina Rat Chow #5001) available ad libitum. All testing was conducted during the light cycle.

Drinking water was provided in 8-oz. (240-ml) amber glass bottles fitted with a Teflon liner inside the cap, and double ball-bearing sipper tubes. Pilot studies showed that these bottles drip less than 0.5 ml/day under simulated conditions. Bottles were weighed and replaced with clean ones twice a week.

Experimental testing.
Initial neurobehavioral testing (functional observational battery, or FOB, and motor activity) took place on days 26, 27, and 28 (32 rats each day, counterbalanced across concentration group, sex, and time of day). In the afternoon of day 28, bottles containing DBA were placed on the cages. Additional neurobehavioral testing occurred during the middle 3 days of weeks 4, 9, 17, and 26 of exposure (corresponding to 1, 2, 4, and 6 months). All rats were weighed weekly during cage-side observations and when tested with the FOB. DBA exposure continued until the rats were perfused.

The FOB protocol used in this study consisted of twenty-eight endpoints that evaluated different aspects of nervous system function; a detailed protocol is most recently described in McDaniel et al. (1993)Go. Following brief assessment of the rat in its home cage, the rat was removed from the cage and held in the observer's hand to allow scoring of reactivity and observations of general appearance. The rat was then placed in the open field (top of a laboratory cart with a rim and covered with clean absorbent paper). During three minutes of exploration, the observer counted the number of rears and ranked and/or described any gait abnormalities, arousal, activity level, abnormal motor movements, and excretion level (urination, defecation). Next, sensorimotor responses were ranked following stimulation by a metal clicker, a tail pinch, approach of a pen, and light touch. Aerial righting and the pupillary reflex were tested next. Forelimb and hindlimb grip strength were measured with wire mesh screens attached to a strain gauge. Landing foot splay and core temperature were recorded. Since DCA produces a pronounced and unusual chest-clasping response (see Moser et al., 1999Go), all rats were suspended briefly by the tail to test for this response. This test was only conducted at the end of exposure. Clinical observations and FOB testing were conducted by the same personnel throughout, and they were unaware of the treatment level of the rats.

Shortly after FOB testing, motor activity was measured in an automated device shaped like a figure eight and composed of a series of interconnected alleys with two blind alleys projecting from the central arena (Reiter, 1983Go). Eight phototransmitter-diodes were spaced at specific locations throughout the figure eight, and horizontal activity was defined as the number of photocell interruptions throughout the session. In addition, the central arena had four phototransmitter-diodes located 14 cm above the floor of the chamber to detect vertical activity.

Specific cage-side observations took place each week for the duration of exposure, except for the weeks when the FOB was conducted. Each rat was removed from its cage and evaluated on three specific criteria (based on observations during a range-finding study); any other notable observations were recorded by exception. Each rat was examined for hair loss (if present, the location was noted), and evidence of diarrhea (on the rat or in the cage). Body tone was judged by muscle resistance, and hypotonia or hypertonia was recorded.

Neuropathological evaluation.
The day after the last FOB testing, rats were deeply anesthetized with pentobarbital and perfused via the left ventricle with an initial flushing solution of 0.9% sodium chloride containing 1000 units/l heparin sodium and 1 ml/l of 1% sodium nitrite (approximately 1 min) followed by 4% formaldehyde:1% gluteraldehyde at a rate of approximately 30 ml/min (10–15 min). Tissues from the central and peripheral nervous systems, skeletal muscle, and eyes with optic nerves were harvested and placed into perfusate. The following tissues were prepared from the control and high-concentration rats for histopathologic evaluation: brain (cross sections including the forebrain, center of cerebrum, midbrain, cerebellum, pons, and medulla oblongata), mid-cervical and mid-lumbar spinal cord (cross and oblique sections), skeletal muscle (thigh), eyes with optic nerves, trigeminal ganglion, cervical ganglion with attached dorsal root, cervical ventral root, lumbar ganglion with attached dorsal root, lumbar ventral root, and sciatic, tibial, and sural nerves (cross and longitudinal sections). In addition, brain and spinal cord sections (cervical and lumbar) were prepared and evaluated from the mid-concentration animals, and spinal cord sections (cervical and lumbar) were prepared and evaluated from the low-concentration rats.

The brain and spinal cord sections, skeletal muscle, and eyes with optic nerves were embedded in paraffin, sectioned at approximately 5 µm, and stained with hematoxylin and eosin (H&E). Additional brain sections were stained with luxol-fast blue/periodic acid Schiff's stain (LFB/PAS) and anti-glial fibrillary acidic protein (GFAP). Sections of sciatic, tibial and sural nerves, trigeminal ganglion, cervical and lumbar ganglion with attached dorsal roots, and cervical and lumbar ventral roots were embedded in glycomethacrylate (GMA), sectioned at approximately 2–3 µm, and stained with H&E.

At microscopic evaluation, all tissue changes were graded by using a semi-quantitative scale as follows: normal (‘0’); minimal (‘1’) indicated a microscopic change that was very focal in the tissue section or affected multifocal isolated portions of the tissue; mild (‘2’) indicated a change affecting <10% of the tissue section; moderate (‘3’) indicated a change affecting 10–40% of the tissue; severe (‘4’) indicated a change affecting >40% of the tissue. More specifically, with regards to nerve fiber degeneration in the spinal cord, minimal (‘1’) indicated that single, scattered axons were affected; mild (‘2’) indicated multiple small clusters of axons or up to 10% of the total axons were affected; moderate (‘3’) indicated that 10–40% of the axons were affected, and severe (‘4’) indicated that greater than 40% of the axons were affected. Concerning the diagnosis of cellular vacuolation, minimal (‘1’) indicated one or a few scattered vacuolated cells in the section, and mild (‘2’) indicated more than a few or at least one cluster of vacuolated cells.

Additional processing was conducted in select DBA-treated rats in order to further investigate the finding of cellular vacuolation in the spinal cord. Paraffin-embedded blocks of spinal cord tissue (the same blocks from which sections had been examined via light microscopy) from a representative high-concentration male, high-concentration female, and a control female were cut with a razor to section the tissue into approximately 3x3x3 mm blocks. These blocks were deparaffinized, and the tissues were buffer-rinsed, post-fixed in osmium tetroxide, dehydrated, infiltrated with epoxy resin, and embedded in epoxy resin. Thick sections (1 micron plastic sections) were prepared, stained with toluidine blue, and reviewed. Grids for evaluation via transmission electron microscopy (TEM) were prepared from the areas of interest and examined.

Data analysis.
All initial analyses included a grouping factor of concentration and gender, with time of testing as a repeated (within-subject) factor. When significant overall effects were obtained, step-down analyses were conducted. If there were no interactions with gender in the model, then the data for males and females were combined for subsequent analyses. Where the overall concentration-by-time interaction was significant, analyses at each time point were conducted to determine which time points were significant, followed by post hoc analyses to determine which exposure groups differed from control. Analyses of data averaged across all test times were conducted if the overall analyses showed a significant concentration effect but no interaction with time.

Continuous data (e.g., body weight, fluid intake, grip strengths, etc.) were analyzed by a general linear model (GLM; SAS, 1990). Rank-order data (e.g., gait score, sensorimotor response scores, pathology scores, etc.) were analyzed by using a categorical modeling procedure (CATMOD; SAS, 1990) that fits linear models to functions of response frequencies, which was then analyzed by weighted regression. Incidence of pathological findings were analyzed by using Fisher's exact test (FREQ; SAS, 1990). In all cases, resulting probability values <0.05 were considered significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Analysis of the DBA concentrations in the drinking water throughout the 6-month study indicated that actual concentrations were slightly higher, being 103–104% of the target concentrations. Concentrations varied by less than 3% from month to month. The mean actual and range of each concentration (g/l) was: 0.205 (0.197–0.212), 0.627 (0.614–0.636), and 1.558 (1.518–1.605). Average actual concentration values were used to calculate DBA intake on a mg/kg basis.

Fluid intake was calculated for each rat twice weekly and averaged, and the overall average water intake for each concentration group is presented in Table 1. Control intake increased from 16.6 and 14.4 ml/day (males and females, respectively) within the first 6 weeks of study and stabilized thereafter at about 21 and 17 ml/day (males and females). Analysis of the weekly data showed a significant three-way interaction of concentration, gender, and time (F(75,2200) = 2.60, p < 0.0001). Subsequent analyses indicated that, in male rats, the high concentration level suppressed intake initially (first week intake = 13.6 ml/day) and for just a few days in the second month of dosing. On the other hand, female rats drinking the high concentration showed lower intake (about 14 ml/day) only in the last 2 months of study. Male rats drinking the middle concentration drank significantly more (about 25 ml/day) throughout the exposure period, but in females, a similar increase at that concentration was only observed in the first 3 weeks of exposure (averaging 21 m/day). One female rat in the middle-concentration group consistently drank considerably more throughout the study (34.1 ml/day compared to the average of 17.8 ml/day in the rest of that group, and 17.1 ml/day in controls). This one rat was closely monitored and no evidence of spillage (e.g., wet bedding) was ever observed. For intake calculations only, the data from that rat were excluded.


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TABLE 1 Nominal Concentrations of DBA in Tap Water

 
Body weight was measured weekly; weekly values are presented in Figure 1, and the averages across the study are shown in Table 1. Since the rats were 28 days of age at the beginning of the study, there was considerable weight gain over the first 3 months. As with intake, there was a significant three-way interaction of concentration, gender, and time (F(78,2288) = 3.41, p < 0.0001). In both sexes, lower body weight was significant in the high-concentration group. In males, the high-concentration group was not different from controls over the last 5 weeks of the study, whereas these differences were significant throughout the study in females. Overall, weight gain in the high-concentration group was 295.6 and 146.7 g compared to control weight gains of 320.8 and 163.8 g in males and females, respectively.



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FIG. 1. Body weight (top: male, bottom: female), plotted as a function of age. Inset graphs indicate the DBA intake calculated from weekly fluid intake and body weight. *significantly different from control.

 
DBA intake changed considerably over the study as a function of changing fluid intake and increasing body weight, shown in the insets in Figure 1. The average intakes across the study are listed in Table 1. Note that these average intakes do not accurately reflect the range of dosages received by the animals due to the marked decrease of DBA intake on a mg/kg basis over the first few months. Indeed, within the first 8–10 weeks of the study, DBA intake decreased by more than 50%. For example, intake in high-concentration male rats in the first week was over 350 mg/kg/d, whereas by the tenth week intake was about 130 mg/kg/d. It is also evident in Figure 1 that there was overlap in the consumed dosages for all three treatment groups; for example, the middle concentration produced intakes at the beginning of the study that were higher than the intakes of the high concentration later in the study.

Weekly observations of the rats revealed a concentration-related increased incidence of diarrhea (unformed, liquid stools) and hair loss that was confined to the top of the head between the ears. These changes were most prominent in the first month of exposure, peaking at two to three weeks after exposure began, and then decreasing for the remaining months. Diarrhea occurred in all exposure groups in males (25%, 58%, and 92% of rats drinking 0.2, 0.6, and 1.5 g/l, respectively), and in the two higher concentration groups in females (25% and 83% at 0.6 and 1.5 g/l). In contrast, the incidence of diarrhea in control male rats was 0–17%, mostly occurring in the fifth and sixth months, and 0% in control female rats. Hypotonia was observed after about two weeks of exposure and continued throughout in the high-concentration groups (peak occurrence 75–100%). Forehead hair loss was evident in both genders in almost all rats (83–92%) in the two higher concentration groups but was observed only in the first month of exposure. We could identify no possible reasons for this hair loss, and the only metal edges in the cages (against which the rats could rub) were the tips of the sipper tubes. Hypotonia and forehead hair loss were observed in only one female control rat at one time throughout the study.

Overall results of the FOB data are presented in Table 2. For almost all endpoints, there were no significant interactions with gender; therefore, male and female data were combined for subsequent analyses. There were, however, mostly concentration-by-time interactions, such that step-down analyses were conducted at each time point to determine effective concentrations.


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TABLE 2 DBA Effects on Endpoints of the FOB

 
Several measures assessing neuromuscular function were altered by DBA. Grip strength, shown in Figure 2, was decreased throughout exposure. Forelimb grip strength appeared more sensitive in that the mid and high concentrations were significant at all time points, whereas hindlimb grip strength was decreased throughout by the high concentration only (the mid concentration was only significant at the last test). As a percent of control values at each test time (see insets in Fig. 2), the DBA-induced decrease in grip strength values was similar across all time points. Changes in gait were evident in the high-concentration group throughout exposure, and at the two higher concentrations at the last test time. Again, these changes did not show marked worsening over the six months. Maximum severity of gait abnormality in the high-concentration rats was recorded as only "somewhat" to "moderately" abnormal. There were no treatment-related changes in landing foot splay or righting reflex.



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FIG. 2. Grip strength (top: forelimb, bottom: hindlimb). Male and female data combined (no sex-by-treatment interaction). Kilograms to release (X ± SEM) are plotted as a function of time of exposure. Inset graphs indicate the mean percent of control values at each time point. * significantly different from control.

 
Specific sensorimotor responsiveness was decreased by DBA. Table 3 lists the distribution of tail-pinch response scores at each test time during exposure. The responses to the tail pinch and click were decreased in all exposure groups. These changes were evident as early as the first test time but did not progress while exposure continued. In addition, the effects were not large in magnitude (i.e., most rats showed a dampened response as opposed to a lack of response). The touch response was transiently increased, but only at one time point and in the lowest dose group. Approach response was not altered.


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TABLE 3 Distribution of Tail-Pinch Scores in Each Concentration Group Across Test Times

 
DBA generally decreased activity levels and reactivity to nonspecific stimuli. Total activity counts, presented in Figure 3, were decreased in the high-concentration group only. In contrast to the other endpoints, activity levels continued to decline as a percent of control values over the six months (from 88% to 53% of control values). Both horizontal and vertical counts showed similar patterns of effect. Home-cage activity and removal reactivity were also similarly decreased. Only three endpoints showed significant concentration-by-gender interactions: open-field rearing, handling reactivity, and arousal. Rearing was altered only in female rats, being decreased in the latter months of exposure; however, the mid-concentration group only showed increased rearing at the first time point. Handling reactivity was transiently decreased in males at the beginning of exposure, whereas females showed decreased reactivity at the end of exposure. Arousal was transiently decreased in both genders in a non-concentration-dependent manner.



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FIG. 3. Motor activity (X ± SEM) are plotted as a function of time of exposure. Male and female data combined (no sex-by-treatment interaction). *significantly different from control.

 
The chest clasp response, which was only tested at the end of the study, was present in 42% of high-concentration females but no males. Body temperature was also significantly decreased in the high-concentration rats (no gender interaction), but the differences from control values were small (0.1–0.4°C).

Nerve fiber degeneration was diagnosed when axonal segments were noted to have variable combinations of the following: segmental dilation of myelin sheaths, accumulations of axonal debris, foamy macrophages within dilated myelin sheaths, and nuclear debris. The fiber degeneration was primarily noted in white matter, and was seen at both cervical and lumbar levels (the only cord regions studied). The incidence and average severity of nerve fiber degeneration are listed in Table 4. The increased incidence of this finding was statistically significant (Fisher's exact test, p < 0.05) in the cervical and lumbar spinal cord of high-concentration males and females, and also in lumbar spinal cord of the mid-concentration females.


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TABLE 4 Incidence and Severity of Spinal Cord Degenerating Fibers and Cellular Vacuolation

 
A unique lesion in this study, termed cellular vacuolation, was characterized by the presence of large (10–20 microns), well-demarcated, multilocular vacuoles that appeared (at light microscopy) to be contained within a cell, cellular process, or dilated myelin sheath. These vacuoles were noted only in spinal cords from DBA-exposed rats; incidence and severity are listed in Table 4. These vacuoles, illustrated in Figure 4, were noted within gray (dorsal and ventral horns) and white matter areas (lateral tracts), but they were generally more common within the gray matter. The vacuoles were generally associated with a homogeneous, eosinophilic matrix (presumably cytoplasm or axoplasm) that appeared to be compressed or displaced by the vacuoles. Cellular vacuolation was observed at the mid and high concentrations in both sexes, but females were slightly more severely affected than males, particularly at the mid concentration. The lumbar spinal cord was slightly more severely affected than the cervical spinal cord in both sexes.



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FIG. 4. Cellular vacuolation, noted only in treated (mid- and high-dose) rats. Top panel: Lateral white matter tracts of the spinal cord in a high-dose female. The long arrow indicates one of two, prominent, clear vacuoles apparently confined within a cellular structure. The short arrow indicates the eosinophilic matrix (presumably cytoplasm or axoplasm) displaced by the vacuoles. Bottom panel: Gray matter area of the spinal cord in a mid-dose female. The long arrow indicates a multilocular structure within a cell or mylin sheath. Note the close proximity to several neurons (N) in the section. The short arrow indicates the eosinophilic matrix displaced by the vacuoles.

 
Electron photomicrographs (increasing magnification) from a high-concentration female are shown in Figure 5. The vacuoles were generally round and of variable size. They were well demarcated from adjacent structures, but not enclosed within a membrane (i.e., not within cellular organelles), and were contained within a cell or cell process that was surrounded by an intact cell membrane. Myelin was not present surrounding the vacuolated cell or cell processes. The matrix surrounding the vacuoles was granular to filamentous and contained numerous mitochondria but no visible rough endoplasmic reticulum, indicating that the vacuoles were probably not within neuronal cell bodies. The exact nature of the vacuoles could not be specifically determined.



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FIG. 5. Electron micrograph of cellular vacuoles (same area with increasing magnification) in a high-dose female. (A) Magnification: x2,300. Arrows point toward a cluster of well-defined, clear, variably sized multilocular vacuoles that appear to be in a single cell. Note the normal appearing neurons (N) and axonal structures nearby. There are also the myelin artifacts, probably due to histological re-processing of the tissue (see Materials and Methods). (B) Magnification: x10,800. There are numerous myelinated axons filling the area in the lower left with multiple vacuoles in the upper right. The axons contain numerous mitochondrial profiles. Vacuoles are clearly within a cell that is bordered by a membrane (arrow), although each individual vacuole is not membrane bound. There is no evidence of myelin around the cell containing the vacuoles. (C) Magnification: x72,600. There are multiple mitochondria (m) embedded in a filamentous matrix adjacent to the vacuoles. The vacuoles are not immediately surrounded by a membrane; these diffuse borders are marked by arrows.

 
Retinal degeneration with associated optic nerve hypoplasia was noted in a control male and female. An epidermal cyst was observed adjacent to the lumbar spinal cord in a control and a high-concentration male. Minimal nerve fiber degeneration noted in the sciatic nerve of a control female was interpreted to be a spontaneous change. Thus, no treatment-related changes were seen in brain, eyes, peripheral nerves or peripheral ganglia. Furthermore, there were no differences in GFAP immunoreactivity across treatment groups.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All rats survived the 6-month exposure to DBA, and only the high-concentration group showed depressed weight gain in both sexes (approximately 9% decrease, averaged across time). While the high-concentration group drank less than controls at various times in the study, the mid-concentration male rats drank more. A similar finding was reported in another drinking water study in Fischer 344 rats, in which rats exposed to 1 g/l DBA showed increased water intake (George et al., 2000Go), as well as in an oral gavage study at high doses (Linder et al., 1994bGo). On the other hand, developmental studies reported by Christian et al. (2001Go, 2002Go) in Sprague-Dawley rats did not report increased fluid intake. DBA also produced transient diarrhea, as was reported in other studies with much higher doses (Linder et al., 1994bGo, 1995Go). The hair loss, and particularly the specificity of the location, was not mentioned in other studies, and the cause is unknown.

The filtered tap water provided in this study was the same as that prepared for National Toxicology Program (NTP) studies of DBA. Analyses indicated that, at all sampling times, the control water contained less than 0.31 µg/ml, the limit of detection for DBA. The water was not, however, analyzed for the presence of other DBPs, and thus there is the possibility that other DBPs or a mixture thereof could have influenced these findings. While the concentration dependencies of the behavioral and pathological findings suggest that they were indeed a result of DBA exposure, the possibility of other interactions cannot be ruled out.

The pattern of changing fluid intake and rapid growth produced a high intake of DBA, particularly over the first month, which then decreased rapidly and stabilized over the last three months (Figure 1). The behavioral data also showed that most effects emerged early, when the DBA intake was high, and remained stable thereafter. This pattern was in marked contrast to the progressive nature of DCA effects (Moser et al., 1999Go). The DBA pattern could be indicative of prolonged effects resulting from the high initial intake. Alternatively, the magnitude of behavioral effects may have reached an asymptote after the first month. Both DBA and DCA inhibit their own metabolism, leading to nonlinear kinetics and higher levels following repeated doses (Gonzalez-Leon et al., 1997Go; Schultz et al., 1999Go). On the other hand, DBA was not shown to accumulate with drinking water exposure (Christian et al., 2001Go). Since exposure began in adolescence, it is possible that the younger rats were more sensitive than adults, and the results may have differed if we had begun exposure in adulthood.

There were both similarities and differences in the behavioral effects of DBA and DCA (Moser et al., 1999Go), although we had originally hypothesized that they would appear similar. Both compounds altered neuromuscular function (e.g., decreased grip strength and gait changes). Chest clasping was observed with DBA, but only in females, and this was not as prominent as with DCA. DBA produced no tremors or changes in the righting reflex, which was seen with DCA. In further contrast to DCA, DBA altered sensorimotor function and decreased motor activity. Earlier studies (Linder et al., 1994bGo, 1995Go), using high oral gavage doses, also reported alterations in hindlimb function. Even the highest concentration used in the present study (1.5 g/l, leading to intake averaging 161 mg/kg/day) was not as high as these earlier studies (>=250 mg/kg/day), which could explain the less severe effects reported here. Overall, the neurobehavioral alterations observed with DBA were of mild to moderate severity. For example, rats showed significantly less response to the sensory stimuli, yet all rats still responded. Furthermore, grip strength was decreased approximately 25–35% throughout the study, which was less than the 40–50% decrease observed with DCA. While differences of this magnitude may not be obvious to the casual observer, they would nonetheless impact the normal neurobehavioral function of the rat

DBA effects on behavioral function could not be fully attributed to body weight changes or other general toxicity. For example, forelimb grip strength was decreased by both the mid and high concentrations, but only the high concentration suppressed weight gain. Furthermore, numerous studies conducted in our laboratory have shown dissociation between body weight and grip strength (see for example, Moser et al., 1998Go). Treated rats generally looked in good health, other than the evidence of diarrhea (mostly in the cage) and the transient hair loss. Finally, the specific neuromuscular and sensorimotor deficits observed in these rats cannot be explained by nonspecific toxicity, especially considering that the lower-concentration groups did not even show altered body weight gain.

Nerve fiber degeneration is a relatively common change in the spinal nerve roots and the dorsal spinal cord tracts of aging rats of multiples strains, including the Fischer 344s (Mitsumori and Boorman, 1990Go). However, those changes are generally most pronounced in animals greater than one year of age, whereas the rats at the end of this study were only 7 months old. Further complicating the interpretation of nerve fiber degeneration in the spinal cord are the artifacts that are inherent in paraffin-processed tissue. In this study, spinal cord nerve fiber degeneration, although observed in control animals, was present at incidences that indicated a treatment effect. The actual pathogenesis of the axonal degeneration was not apparent. We cannot rule out that nerve fiber degeneration may have occurred earlier in the study, but was no longer evident at the terminal sacrifices. Given the minimal to mild severity of nerve fiber degeneration in the DBA-treated rats, it was not possible to directly attribute any of the neurobehavioral abnormalities to the spinal cord changes. However, the absence of an obvious association does not invalidate the significance of either set of findings.

Earlier studies of DCA reported neuropathology at high dose levels. Bhat et al. (1991)Go reported vacuolization in the brain, mostly associated with myelinated axons, as well as a positive GFAP response. Others also reported vacuolization in myelinated tracts in the brains of both dogs and rats (Cicmanec et al., 1991Go; Katz et al., 1981Go). In several different reports, Spencer and coworkers have reported degenerative changes in scattered neurons, and vacuolar demyelination in spinal cord, as well as gray matter vacuolation (Spencer et al., 1981Go; Spencer and Bischoff, 1982Go; Spencer and Schaumburg, 1985Go). In the present study, DBA produced neural degeneration, as well as neuronal vacuolization, but these findings were restricted to the spinal cord. Unlike previous reports of DCA, no brain lesions or gliosis were observed. Thus, there may be similarity between the neuropathology produced by DCA and DBA, but further analysis of DCA-treated spinal cord tissue is necessary to confirm this.

The most unusual morphologic abnormality noted in this study was the presence of intracellular vacuoles in the spinal cord sections from mid- and high-concentration animals. At the light microscopic examination, these vacuoles were believed to be a component of the overall nerve fiber degeneration associated with DBA treatment. TEM examination was used as an adjunct to the light microscopic evaluation of this morphologic change, even though only a few samples were evaluated. Furthermore, it was understood that the use of previously paraffin-embedded spinal cord sections for examination via TEM could result in extensive artifact. However, the increased probability of identifying vacuolated cells from specimens with already-identified lesions was deemed an acceptable compromise, and the specimens were of diagnostic value.

The exact constituency and cellular nature of the vacuoles could not be definitively determined. Neuronal cell bodies were largely ruled out due to the lack of Nissl substance in the vacuole-containing structures. Similarly, glial cells were ruled out because of the lack of any cellular characteristics that indicated glia. Dendrites and axons were not ruled out as a possible location, and indeed, the cytoplasm surrounding the vacuoles was consistent with axon cytoplasm. The lack of any discernable myelin surrounding the cells containing the vacuoles indicated the structures were not myelinated axons, or that the surrounding myelin was removed during the six months of the study. There were no detectable glial cell responses (astrocytosis or microgliosis) at light and electron microscopic evaluations. Thus, these cellular vacuoles are a unique finding that merits further evaluation.

DCA produces a myriad of metabolic changes, many of which have been proposed to produce its toxicity. Some of these metabolic effects have led to its therapeutic use for diabetes, hyperlipidemia, and other metabolic disorders (reviewed in Stacpoole, 1998Go). The hypothesis that stimulation of thiamine-dependent enzymes would lead to thiamine deficiency, and its concomitant neurological complications, was supported in studies using thiamine supplementation to reverse the effects of DCA (Stacpoole et al, 1990Go). A more recent line of research has revealed that DCA, as well as DBA, inactivates glutathione transferase zeta (GSTZ) and thereby inhibits tyrosine catabolism and, in addition causes hepatic glycogen accumulation and altered serum insulin and insulin-signaling proteins (Anderson et al., 1999Go; Kato-Weinstein et al., 1998Go, 2001Go; Lantum et al., 2003Go; Lingohr et al., 2001Go). Several of these studies (although in mice) were conducted using drinking water concentrations similar to those used in the present study. These actions are gaining acceptance as potential mechanisms of DCA-induced hepatic carcinogenicity; however, the extent to which these metabolic alterations influence neuropathology and behavioral changes, such as those reported here, is unclear. Clearly, further research on DBA neurotoxicity should address such mechanisms.

In the present study, the lowest concentration tested (0.2 g/l), which produced an average intake of about 20 mg/kg/day, produced sensorimotor changes. Thus, a no-effect level (NOEL) could not be established for the neurobehavioral effects. This concentration was, however, a NOEL for the neuropathological changes. This effective level is in the same dose range as that which produces spermatotoxicity, the most sensitive reproductive endpoint reported to date (Christian et al., 2002Go; Linder et al., 1997bGo). These levels are considerably higher than human exposure to haloacetic acids via drinking water as currently regulated, yet regulatory assessments for exposure to haloacetic acids are based currently on potential reproductive toxicity and carcinogenicity. The findings from this study serve to raise awareness that neurotoxicity is another endpoint of interest in the toxicological evaluation of DBA, and that both neurotoxicity and reproductive toxicity should be considered collectively when assessing noncancer risk attributable to the haloacetic acids.


    ACKNOWLEDGMENTS
 
Ms. A. Levine was funded by the EPA/UNC Toxicology Research Program, Training Agreement CT902908, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill. The authors thank Drs. G. Klinefelter and K. Rheul for reviewing an earlier version of this manuscript.


    NOTES
 
The information in this document has been funded in part by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at Neurotoxicology Division (MD B105-04), U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4849. E-mail: moser.ginger{at}epa.gov

Portions of this research were presented at the annual Society of Toxicology meeting, March 16-21, 2002


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