Bromochloroacetic Acid Exerts Qualitative Effects on Rat Sperm: Implications for a Novel Biomarker

Gary R. Klinefelter,1, Lillian F. Strader, Juan D. Suarez and Naomi L. Roberts

U. S. Environmental Protection Agency, Office of Research and Development, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD #72, Research Triangle Park, North Carolina 27711

Received November 16, 2001; accepted February 11, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Disubstituted haloacid by-products of drinking water disinfection such as dibromoacetic acid and dichloroacetic acid have been shown to perturb spermatogenesis and fertility in adult male rats. In the present study we sought to establish whether equimolar exposure to bromochloroacetic acid (BCA), a prevalent by-product in finished drinking water, is also capable of disrupting these endpoints, and if so to determine whether the novel biomarker of fertility (SP22) would be correlated with subfertility induced by testicular toxicity. A dose range finding study indicated that body weight was not affected by exposure to 14 daily doses of 72 mg/kg BCA while numerous male reproductive parameters were altered, including decreases in the number and progressive motility of cauda epididymal sperm. In addition, there was an increased incidence of delayed spermiation in the testes of males exposed to 72 mg/kg BCA. In the definitive study, exposures ranged from 8 to 72 mg/kg, the fertility of cauda epididymal sperm was evaluated by in utero insemination, and the two-dimensional profile of cauda sperm membrane proteins was evaluated quantitatively. The morphology of both caput and cauda epididymal sperm was altered by 72 mg/kg BCA. The fertility of cauda epididymal sperm, the percentages of progressively motile sperm and progressive tracks, and two sperm membrane proteins (SP22 and SP9) were decreased significantly by each BCA exposure. While the two sperm proteins and the two measures of progressive motility were each significantly correlated with fertility, only one of these measures (i.e., SP22) had an r value of greater than 0.5. When data for SP22 and fertility were fit to a nonlinear model, r2 was 0.84. Using this exposure paradigm, the no-observed-effect level for BCA is less than 8 mg/kg. Moreover, SP22 may be useful in predicting compromised fertility after exposure to by-products of drinking water disinfection.

Key Words: disinfection by-products; fertility; spermatogenesis; sperm biomarker.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
While the public health benefit of disinfecting drinking water cannot be questioned, the process of disinfection results in the formation of hundreds of chemical by-products that might also pose a health risk. Indeed, several classes of chemical by-products (i.e., trihalomethanes, haloacids, haloaldehydes/nitriles) are formed when the naturally occurring humic and fulvic acids in source water react with chlorine, chloramine, or ozone, and the relative prevalence of individual by-products within each class varies as a function of bromide ion content and pH (Glaze et al., 1989Go, 1993Go; Krasner et al., 1989Go). Regardless of treatment regimen, haloacids constitute one of the most prevalent by-products, and under conditions in which bromide ion level is high (i.e., coastal water) the levels of brominated species such as dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) can reach levels approximating 10–20 µg/l. Current maximum contaminant level goals (MCLGs) are set at 60 µg/l for the sum of S haloacids, excluding BCA. Therefore, it is important to determine whether brominated haloacids are capable of eliciting adverse reproductive outcomes in a dose-related manner, as well as the lowest effect level for observed dose-responsive outcomes.

Previously we demonstrated that adult male rats exposed to DBA daily for 14 or more days manifest both quantitative and qualitative effects on spermatogenesis and epididymal sperm (Linder et al., 1994Go, 1995Go, 1997Go). For example, at 10 mg/kg, there was an increase in delayed spermiation as the total number of retained spermatids increased over 3-fold in stages IX–XII of DBA-exposed testes. Moreover, the weight of the epididymis was decreased at 10 mg/kg due to the decrease in the number of sperm in the caput epididymis (Linder et al., 1994Go). In a subsequent 70-day exposure, these endpoints were less sensitive than in the former study, with comparable effects observed at 50 mg/kg exposure (Linder et al., 1995Go, 1997Go). However, the latter studies provided data to suggest that DBA exposure produced qualitative alterations in sperm function prior to quantitative alterations. For example, when fertility of males was evaluated using in utero insemination (i.e., a fixed number of cauda epididymal sperm injected in utero), only 20% of the males were able to produce a litter after 16 days of exposure to 250 mg/kg DBA. As duration of exposure increased to 31 days, the requisite number of sperm could be obtained from only one of six males, indicating that quantitative effects were now prevailing. Indeed, cauda epididymal sperm numbers were reduced from 240 million to 33 million.

To understand molecular events underlying the qualitative changes resulting from exposure to DBA, a comparative ex vivo/in vitro study was undertaken in which protein synthesis in isolated seminiferous tubules was examined (Holmes et al., 2001Go). For in vitro exposures, concentrations mimicking those found in testicular interstitial fluid following in vivo exposure were tested. The synthesis of four individual proteins was compromised, and identification of these proteins is underway. Based on Western blot data, one of these proteins is SP22, a novel sperm protein that is expressed in postmeiotic germ cells (Klinefelter and Welch, 1999Go; Welch et al., 1998Go) and correlates with the fertility of cauda epididymal sperm (Klinefelter et al., 1997Go).

In the present study we sought to characterize the reproductive toxicity attributed to exposure to BCA, another brominated analogue of dichloroacetic acid that is actually more prevalent in disinfected drinking water than DBA (Krasner et al., 1989Go). For this, we selected doses of BCA that were equimolar representations of previously tested DBA exposures. In addition, we wanted to determine whether reductions in fertility following in utero insemination were associated with changes in the sperm protein profile, particularly SP22.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose Finding Study
Animals and dosing.
Ninety-day-old Sprague-Dawley rats (Charles River Breeding Laboratories Inc., Raleigh, NC) were allowed to acclimate for 2 weeks to room conditions of 12-h light/dark, 22 ± 1°C, 50 ± 10% relative humidity, and housed two per cage in an AAALAC-approved animal facility. Animals were ranked by weight and randomly distributed into 4 groups of 12 rats each. BCA of 90% purity was obtained from Carbolabs Inc. (Bethany, CT). For dosing, BCA was dissolved in distilled water and pH was adjusted to approximately 6.5 using descending concentrations of NaOH (5N, 1N, and 0.5N NaOH). Rats were administered 14 daily doses of either 0 (water control), 24, 72, or 216 mg/kg by po gavage in a dose volume of 5 ml/kg; volumes were adjusted biweekly for body weight. The doses of 24, 72, and 216 mg/kg BCA were selected as these represent the molar equivalents of the 30, 90, and 270 mg/kg DBA doses tested previously in our laboratory (Linder et al., 1994Go).

Organ and hormone assessments.
While under a surgical plane of halothane anesthesia and prior to vascular perfusion fixation, animals were hemicastrated and the left testis and epididymis were excised, trimmed, and weighed. Rats not selected for perfusion fixation were killed by CO2 asphyxiation followed by cervical dislocation and both testes, both epididymides, and the seminal vesicles were handled similarly. For ex vivo assessment of testosterone production (Klinefelter et al., 1994Go), the tunica albuginea was removed from the left testis and 50 mg pieces of parenchyma were incubated in 1.0 ml of Medium 199 (M199) buffered with 0.71 g/l sodium bicarbonate and 2.1 g/l N-2-hydroxylethyl piperazine-N`-2-ethanesulfonic acid (HEPES) and containing 0.1% protease-free BSA and 25 mg/l soybean trypsin inhibitor, pH 7.4. After a 30-min incubation period, media were removed and replaced with fresh medium. T production was assessed by incubating parenchyma (4, 50 mg pieces per testis) in duplicate either with or without hCG stimulation (100 mIU/ml) for 2 h at 34°C. After 2 h, medium was removed and frozen at -70°C until T assay. T was assayed by RIA protocol as described by Kelce et al. (1997). Briefly, a 100 µl sample aliquot was assayed using additions of [3H] testosterone (DuPontNEN; 10,000 dpm in 100 µl), T-antibody (ICN; 1:10,000 in 100 µl) and 400 µl DPBS-Gelatin. Samples were then vortexed and incubated overnight at 4°C. The next day 1 ml of activated charcoal was added to each sample, and after 20 min each sample was vortexed and centrifuged (10 min at 2000 x g). OPTI-fluor scintillation cocktail (Packard Bioscience, The Netherlands) was added and radioactivity was quantified by scintillation counting for 2 min (Beckman LS 5000 TD, Irvine, CA).

In addition, blood was removed from each animal while under a surgical plane of anesthesia by cardiac puncture and serum obtained using serum separator tubes was frozen at -70°C prior to assay for testosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH), and prolactin (PRL). For this, serum T was measured using a Coat-A-Count RIA kit obtained from Diagnostic Products Corporation (Los Angeles, CA). Minimum detectable limit was 0.20 ng/ml and inter- and intra-assay coefficients of variation were 10.8 and 5%, respectively. LH, FSH, and PRL RIA assays were performed using the following materials supplied by the National Hormone and Pituitary Agency for LH, FSH, and PRL, respectively: iodination preparation I-9, I-9, I-6; reference preparation RP-3, RP-3, RP-3; and antisera S-11, S-11, S-9. Radiolabeling was done with 125I (DuPont/New England Nuclear) using a modification of the chloramine-T method of Greenwood et al. (1963). Labeled PRL was separated from unreacted iodide by gel filtration chromatography as described by Goldman et al. (1986). Samples were pipetted with appropriate dilutions to a final assay volume of 500 µl with 100 mM phosphate buffer containing 1% bovine serum albumin (BSA). Standard reference preparations were serially diluted for the standard curves. Primary antisera (200 µl) in 100 mM potassium phosphate, 76.8 mM EDTA, 1% BSA, and 3% normal rabbit serum were pipetted into each assay tube, vortexed, and incubated at 5°C for 24 h. Iodinated hormone (100 µl) was then added, and each sample was vortexed and incubated for 24 h. Second antibody (Goat Anti-Rabbit Gamma Globulin, Calbiochem) at a dilution of 1 unit/100 µl was added and samples were vortexed and incubated 24 h. After centrifugation (1260 x g, 30 min), the supernate was aspirated, and the sample tube with pellet was counted on a gamma counter. For LH, FSH, and PRL, the intra- and inter-assay coefficients of variation were approximately 2.4 and 9.1%, respectively.

Sperm measures.
Sperm motility and morphology were evaluated on six animals per group. Sperm motion analysis was done on cauda epididymal sperm collected in supplemented Hank's Balanced Salt Solution using a diffusion technique (Klinefelter et al., 1991Go). After dispersion of sperm, an aliquot was further diluted in the same medium, carefully inverted several times to mix, loaded into a 100 µm deep cannula (VitroCom Inc., Mt. Lakes, NJ), and placed on the heated (36°C) stage of the Hamilton Thorne IVOS. Images consisting of 30 frames each from 6 fields, collected at 60 frames/s, were saved to an optical disk for later analysis. Velocity analysis was carried out using the Tox IVOS version 10.8q and no tracks with less than 20 points were accepted. Progressive sperm were defined as those whose average path velocity (VAP) was greater than or equal to 100 and straightness (STR) was greater than or equal to 50%. Percent motile was scored manually by looking at the playback and counting motile and total sperm for each of the six fields.

Morphology was evaluated on sperm from both the cauda and caput epididymis. Cauda sperm were taken from the original dilution for motility and diluted 1:3 with 10% neutral buffered formalin. Caput sperm were diluted in 500 µl of motility medium and allowed to disperse, and 1 ml of 10% formalin was added. Five hundred sperm from each cauda and caput sample were scored for morphological abnormalities as described previously (Linder et al., 1992Go). Briefly, in wet preparations using phase-contrast optics, sperm were categorized as one of the following: (1) normal head and tail, (2) isolated heads (whether the head was misshapen or not), (3) head-only defects (i.e., misshapen head with normal tail), (4) tail defects (i.e., normal head with abnormal tail or misshapen head with abnormal tail), and (5) fused sperm.

The right testis (with the tunica albuginea removed) and epididymis from six animals per group were frozen for later determination of testicular spermatid head counts (TSHC) and epididymal sperm counts. For TSHC the testis was thawed, suspended in 8 ml of 0.9% saline containing 0.05% Triton X-100 and 0.01% Merthiolate (STM), homogenized for 10 s in a polytron, and sonicated for 30 s in an ice bath. The sample was vortexed and diluted 1:9 with STM. Six counts were made per sample with a hemacytometer (both sides of three chambers) and the counts were averaged. Sperm counts were done on the cauda and caput epididymis using the Tox IVOS HTM-Ident (Hamilton Thorne Research, Beverly, MA), which is an automated system that uses a DNA-specific dye and fluorescence illumination to identify sperm. Epididymal sperm count samples were prepared and counted as described previously (Strader et al., 1996Go). Briefly, thawed cauda tissue was minced with a scissors in 1.5 ml STM, diluted to 40 ml with STM, and homogenized for 2 min in a Waring blender, and a 200 µl aliquot was placed in an IDENT Stain reaction vial and allowed to sit for at least 5 min. Caput samples were processed similarly but diluted to 20 ml with STM. Counting was done with a 20 µm deep Cell-Vu chamber. Twenty fields from three chambers were counted for each sample and the counts were averaged.

Histopathology.
The right testis and epididymis from six animals per group were fixed in situ by vascular perfusion via the descending aorta with Dulbecco's phosphate buffered saline (DPBS) followed by 5% glutaraldehyde in a 0.05 M collidine buffer with 0.1 M sucrose. After 10–15 min of perfusion, tissues were excised and placed in this fixative for 24 h. Tissues were then washed in three changes of 0.05 M collidine buffer, dehydrated through alcohols, and embedded in GMA (JB-4 Plus, Polysciences, Inc.). Blocks were sent to Pathology Associates International, Frederick, MD, for sectioning at 2 µm and staining with H&E or PAS counterstained with Gill's hematoxylin. Testis sections were examined by light microscopy for delayed spermiation, formation of atypical residual bodies, and germ cell depletion.

Dose Response Study
Animals and dosing.
This study was conducted in two comparable experimental blocks. Ninety-day-old Sprague-Dawley rats, obtained from Charles River Breeding Laboratories Inc., Raleigh, NC (block 1) or Harlan Sprague-Dawley, Indianapolis, IN (block 2), were allowed to acclimate for 3–4 weeks to room conditions of 12 h light/dark, 22 ± 1°C, 50 ± 10% relative humidity, and housed two per cage. In each experimental block, animals were ranked by weight and randomly distributed into 4 groups of 10 rats each. An 8 mg/kg dose was chosen as the lowest dose since all doses in the dose finding study were effective. Rats were given 14 daily doses of either 0 (water control), 8, 24, or 72 mg/kg administered as in the dose finding study.

Organ and hormone assessments.
Animals were euthanized with CO2 (block 1) or decapitation (block 2). Blood was removed from each animal per group by cardiac puncture, and serum obtained using serum separator tubes was frozen at -70°C prior to assay for T, LH, FSH, and PRL, as described above. The left testis and epididymis on each of the 10 animals per group were trimmed and weighed. Seminiferous and interstitial fluid from the left testis was collected as described previously (Rehnberg, 1993Go) for testosterone determination. Briefly, the testes were removed and 100–200 µl of interstitial fluid was collected from a nick in the caudal pole of the testis by centrifugation (60 x g for 90 min, 4°C). Approximately 100 µl of seminiferous tubule fluid was recovered from the same testis by removal of the tunica albuginea, multiple rinsing of the parenchyma with DPBS, syringe-extrusion of the parenchyma, and centrifugation (2500 x g, 30 min, 4°C).

Sperm measures.
The left epididymides were used for sperm morphology, sperm motility, sperm protein extraction for two-dimensional gels, and for fertility assessment using artificial insemination (AI). For this, the cauda epididymis of each male was placed in a 35 mm culture dish containing 2 ml of medium 199 (M199; Sigma, M-3769 with Earle's salts and phenol red-free) buffered with 26 mM sodium bicarbonate and containing 3 mg/l (0.1X) DL-methionine, 0.2% protease-free BSA, 10 mM sodium pyruvate, 1 mM nonessential amino acids, 12 mg gentamicin sulfate, an insulin/transferrin/selenium mixture, and 200 nM testosterone and 200 nM dihydrotestosterone, pH 7.2. On the day of the in utero inseminations (see below), 0.25 mg/ml bovine lipoprotein (Sigma, L-3626) was added to the medium. Sperm that diffused after piercing the epididymal tubule with a #11 scalpel blade were allowed to disperse for 5 min at 34°C, 5% CO2. A 50 µl aliquot was diluted with 450 µl of fixative (10% formalin in DPBS with 10% sucrose, pH 7.4) and counted using a hemacytometer; sperm concentrations ranged from 20–30 x 106/ml. After removal of sample for insemination, aliquots were removed for sperm motion and morphology analyses as described in the dose range finding study. For this study, sperm were tracked 1 s at 60 frames per s, with a minimum track length of 30 frames (1/2 s).

Sperm remaining were subjected to protein analysis (Klinefelter et al., 1997Go). For this, sperm (10–40 x 106) were transferred to a microcentrifuge tube and washed twice by centrifugation (3000 x g, 10 min) in Hanks' Balanced Salts Solution buffered with 4.2 g/l HEPES and 0.35 lg/l NaHCO3 and containing 0.9 g/l D-glucose, 0.1 g/l sodium pyruvate, and 0.025 g/l soybean trypsin inhibitor, pH 7.4 with freshly-added 0.2 mM phenylmethylsulphonyl fluoride (PMSF; Sigma, #P-7626). After the final wash, sperm were extracted for 1 h at room temperature with 1 ml of 80 mM n-octyl-B-glucopyranoside in 10 mM Tris, pH 7.2 containing freshly-added PMSF. Following a final centrifugation (10,000 x g, 5 min), the supernatant was removed and frozen (-70°C).

Prior to two-dimensional gel electrophoresis, samples were thawed and each extract was concentrated with 1 mM Tris buffer, pH 7.2, by two centrifugations (3000 x g, 45 min, 4°C) in Ultrafree-4 centrifugation filter units (Millipore). Protein concentration was determined using a Pierce protein assay kit. Sample volumes containing 30 µg protein were lyophilized and protein was solubilized for 30 min at room temperature in 45 µl of sample buffer consisting of 5.7 g urea, 4 ml 10% NP-40, 0.5 ml ampholytes (Serva; 3–10 only), and 0.1 g dithiothreitol per 10 ml. Isoelectric focusing (750 V, 3.5 h) was carried out in mini isoelectric focusing gels consisting of 6.24 g urea, 1.5 g acrylamide (30% acrylamide, 1.2% bisacrylamide), 2.25 ml 10% NP-40, and 0.65 ml ampholytes (3–10 only) per 10 ml. Molecular weight separation was carried out in mini 14% acrylamide gels (200 V, 45 min). Gels were soaked in 50% methanol and silver stained using a Daiichi silver staining kit (Integrated Separation Systems).

A Kepler two-dimensional gel analysis system (Large Scale Biology Corp., Rockville, MD) was used for background correction, spot matching, and spot area quantitation. Images were acquired by transmittance at 80 µm spatial resolution and 4096 gray levels on an Ektron 1412 scanner and converted to 256 gray levels. The Kepler system uses a combination of digital filtering and two-dimensional least-square Gaussian fitting for background subtraction, spot detection, and modeling. Quantitation was done by fitting two-dimensional Gaussian distributions to the density distribution of the spot area following background subtraction.

In utero insemination.
The procedure for in utero insemination of cauda epididymal sperm has been described previously (Klinefelter et al., 1997Go). Briefly, a cohort of females was synchronized with 80 µg sc of LHRH agonist (Sigma, # L4513) 115 h prior to insemination. Just after room lights turn-off on the day of proestrus, these females were paired with sexually experienced, vasectomized males for 30 min. Typically a copulatory plug could be found at the bottom of a wire-bottom cage if repeated intromissions occurred. Receptive females, and males representing each of the study treatment groups, were taken to the surgical suite.

Within 15 min, each uterine horn was injected with a volume containing 5 x 106 sperm, a value that results in approximately 75% fertility in control males. A single female was inseminated per male. All inseminations were performed while the recipient female was in a surgical plane of halothane anesthesia. Uterine horns were exposed through a low, mid-ventral incision. A fine, curved forceps was used to elevate and create some tension on the uterine horn, while sperm (0.1 to 0.2 ml) were injected through an 18 g iv catheter attached to a 0.5 ml syringe. Injection sites were cauterized immediately upon withdrawal of the needle. Nine days later, inseminated females were anesthetized and killed via cervical dislocation. The implanted embryos and corpora lutea of pregnancy were counted. Fertility of each male was expressed as a percentage equivalent to the number of implants/number of corpora lutea x 100.

Statistics.
The various data were analyzed using two-way analysis of variance (SAS; PROC GLM, 1985) for treatment effects. Dunnett's multiple comparison test was used to determine significance (p < 0.05). A correlation analysis was performed to determine whether significant (p < 0.05) correlations existed between sperm proteins and fertility. The background-corrected area of SP22 and fertility data were fitted to a nonlinear function that defines a sigmoid curve that approaches a horizontal asymptote as follows:

(1)
in which FSP22 is the fertility at a given SP22 concentration, F0 is the fertility when SP22 equals 0. A and B are constants where A is the initial increase in fertility and B is the rate of exponential decay of the increase in fertility. Curve fitting was conducted using the Marquardt curve fitting algorithm available in SAS (PROC NLIN, 1985). This fitting procedure calculated the parameters A and B, adjusting to minimize the simultaneous sum of squares for the model with respect to the data points.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose Range Finding Study
Body weights of adult male rats exposed to 24 and 72 mg/kg BCA for 2 weeks were comparable to those in the control group. However, exposure to 216 mg/kg BCA did result in a significant reduction in body weight (Table 1Go). The weights of the testis, epididymis, or seminal vesicles were not influenced by BCA exposure. While homogenization-resistant spermatid numbers were not altered by BCA, a significant decline in epididymal sperm reserves was observed at 72 and 216 mg/kg (Table 1Go). This decline was more apparent for cauda epididymal sperm (p < 0.05) than caput epididymal sperm, although sperm numbers in each region declined in a dose-related manner.


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TABLE 1 Effects of BCA on Organ Weights and Sperm Numbers
 
Dose-related diminutions in serum LH, FSH, and prolactin were observed in males exposed to BCA, with significance achieved in the 72 and 216 mg/kg BCA treatment groups for LH (Table 2Go). There were no significant differences in serum T or the ability of testis parenchyma to produce T in vitro in the absence or presence of hCG.


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TABLE 2 Effects of BCA on Serum Hormones and Ex vivo Testis Testosterone Production
 
The percentage of motile and progressively motile cauda epididymal sperm was diminished in a dose-related fashion by BCA, with significance achieved at 72 and 216 mg/kg (Table 3Go). Similarly, velocity parameters (i.e., average path, straight line, and curvilinear velocities) and linearity were diminished significantly at these BCA doses. The percentage of morphologically normal caput epididymal sperm decreased from 94.8% in controls to 31.2% in animals exposed to 216 mg/kg BCA (Table 4Go). Similarly, the percentage of morphologically normal cauda sperm decreased from 98.3% in controls to 33% in animals exposed to 216 mg/kg BCA. Caput epididymal sperm abnormalities were characterized by an increased number of intact sperm with misshapen heads or tail defects. Cauda epididymal sperm abnormalities were characterized by an increased number of isolated heads.


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TABLE 3 Effects of BCA on Cauda Epididymal Sperm Motion Parameters
 

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TABLE 4 Effects of BCA on Epididymal Sperm Morphology and Testis Histology
 
In addition to these qualitative changes in epididymal sperm, a histological evaluation of the testes from BCA-treated animals revealed a dose-related increase in the number of step 19 spermatids retained in stages X and XI of the spermatogenic cycle (Table 4Go). This increase was statistically significant in testes from the 72 and 216 mg/kg treatment groups. Moreover, there was a dose-related increase in the number of atypical residual bodies found in stages X and XI but these were not quantified. In addition to the increased size of these residual bodies, their localization shifted from basal migration to luminal release as the dose of BCA increased.

Dose Response Study
Body weights of adult male rats exposed to 8, 24, and 72 mg/kg BCA for 2 weeks were comparable to those in the control group (Table 5Go). While testis weight and seminal vesicle weight were not influenced by these BCA exposures, the weight of the epididymis was reduced significantly at 72 mg/kg. In contrast to the dose finding study, serum LH was not decreased significantly at 72 mg/kg. Indeed, no hormone measures were altered in this study (Table 6Go).


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TABLE 5 Effects of BCA on Body Weight and Organ Weight
 

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TABLE 6 Effects of BCA on Serum Hormones (LH, FSH, Prolactin) and Intratesticular Testosterone (T)
 
Consistent with the dose finding study, sperm motion parameters were significantly compromised by BCA exposure. While the percentage of motile sperm was decreased only at 72 mg/kg, both the percentage of tracks that were progressive and progressive motility were decreased at 8, 24, and 72 mg/kg BCA (Fig. 1Go). Likewise, the morphology of caput and cauda epididymal sperm was compromised, but changes were observed only at the highest exposure, i.e., 72 mg/kg (Table 7Go). Alterations in sperm morphology were similar to those in the dose finding study, i.e., increased incidences of sperm with tail defects in the caput epididymis, and isolated heads in the cauda epididymis.



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FIG. 1. Graph depicting the BCA-induced changes in motion parameters of cauda epididymal sperm. Values represent the group mean and standard errors; n = 20 animals per group. *Indicates statistical significance (p < 0.05).

 

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TABLE 7 Effects of BCA on Epididymal Sperm Morphology
 
The fertility of cauda epididymal sperm, assessed by in utero insemination, was dramatically altered by each BCA exposure (Fig. 2Go). Indeed, cauda epididymal sperm from control males has a fertility rate of 75% compared to 33, 44, and 37% for sperm from males in the 8, 24, and 72 mg/kg BCA groups, respectively. A quantitative evaluation of the two-dimensional profile of 120 proteins extracted from the plasma membrane of cauda epididymal sperm revealed that two proteins decreased significantly following BCA exposure (Fig. 3Go). These two proteins (SP22 and SP9) were diminished significantly in extracts of sperm in each BCA treatment group, but the shape of the dose response for the mean SP22 values across groups paralleled fertility, whereas the means for SP9 did not. Indeed, the Pearson correlation coefficients (r) were 0.53 (p < 0.001) and 0.23 for fertility versus SP22 and SP9, respectively. The r values were 0.27 (p < 0.04), 0.30 (p < 0.02), and 0.15 for fertility versus tracks progressive, progressive motility, and motility, respectively (Table 8Go).



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FIG. 2. Graph depicting the BCA-induced changes in fertility of cauda epididymal sperm. Values represent the group mean and standard errors; n = 20 animals per group. *Indicates statistical significance (p < 0.05).

 


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FIG. 3. (Top) Silver stained profiles of two-dimension denaturing gels of protein in detergent extracts of cauda epididymal sperm from control (left) and BCA-exposed (right) animals. The 2 proteins (SP22 and SP9) that were diminished by exposure are indicated. (Bottom) Graph depicting the BCA-induced changes in the cauda epididymal sperm proteins designated SP22 and SP9. Values represent the group mean and standard errors; n = 20 animals per group. *Indicates statistical significance (p < 0.05).

 

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TABLE 8 Relationships between Sperm Measures and Fertility
 
Based on the similar treatment-related responses in fertility and SP22, and the apparent correlation between fertility and SP22, the data were fitted to a nonlinear, threshold response model (Fig. 4Go). The resulting correlation coefficient r2 between fertility and SP22 was 0.84. While predictive analysis was not performed, it is apparent that when the background-corrected, integrated optical density of SP22 in a two-dimensional denaturing gel is 2500 units or less, fertility is generally less than 50%. At around 3000 units a "threshold" is reached where fertility is observed to range from 5 to 100%. When SP22 is greater than 4000 units fertility is consistently greater than 50%.



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FIG. 4. Data (n = 60) for fertility and the integrated, background-corrected area (I.O.D.) of SP22 were fitted to the following equation:

(2)
in which FSP22 is the fertility at protein concentration SP22, F0 is the fertility a 0 protein concentration. A and B are constants (A is the initial increase in fertility and B is the rate of exponential decay of the increase in fertility). The dotted lines represent the 95% confidence limits around the fitted line. The overall correlation r2 = 0.843.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our previous work has demonstrated that DBA, a prevalent haloacetic acid, produces profound alterations in spermatogenesis and fertility (Linder et al., 1995Go, 1997Go); the lowest observed adverse effect level based on these studies is 10 mg/kg. At this dose there was a modest increase in delayed spermiation and reduced ability for successful, repeated mating. The occurrence of BCA in drinking water is often four times greater than that of DBA (Krasner et al., 1989Go). Thus, it is important to establish whether BCA, like DBA, is capable of perturbing spermatogenesis and fertility, and if so, if it is equally effective at equimolar exposures.

Both DCA and DBA have been shown to produce similar quantitative and qualitative alterations in spermatogenesis in the rat. These alterations include delayed spermiation resulting in reduced numbers of epididymal sperm, formation of atypical residual bodies, fusion of spermatids and sperm, and misshapen spermatids and sperm (Klinefelter et al., 2001Go). Collectively, these alterations suggest that Sertoli cell-germ cell communications are disrupted by haloacid exposure resulting in defects in the process of spermiation (i.e., phagocytosis of residual spermatid cytoplasm during spermatid elongation, separation of elongating spermatids, and release of mature spermatids).

In the present study of BCA, a dose finding evaluation indicated that the lowest tested dose at which body weight was unaffected was 72 mg/kg. Consistent with our previous findings for DBA and DCA, both quantitative and qualitative sperm alterations were observed in adult males exposed to 72 mg/kg BCA for 14 days. Quantitatively, there were significantly decreased numbers of sperm in the cauda epididymis and a significant increase in the number of testicular spermatids retained beyond stage VIII of the spermatogenic cycle. Qualitatively, there was a significant increase in the number of cauda epididymal sperm with tail defects and significant decreases in the sperm motion parameters of these sperm. To what extent, if any, alterations in sperm morphology can account for alterations in sperm motion or fertility is unknown. Finally, there was a dose-related increase in the number of tubules with atypical residual bodies. These residual bodies were evident in testes of animals exposed to 24 mg/kg BCA. Based on this, as well as evidence of delayed spermiation at 24 mg/kg, the dose of 8 mg/kg was selected as the lowest dose for the definitive study.

In the definitive study the weight of the epididymis was decreased significantly at 72 mg/kg BCA, presumably due to a decrease in epididymal sperm numbers. Epididymal sperm numbers were not enumerated in this study as sperm were needed for fertility and sperm protein analyses. There was a significant increase in the number of cauda epididymal sperm representing all classes of sperm morphology abnormalities from males exposed to 72 mg/kg BCA compared to an increase in only tail defects at 72 mg/kg in the dose finding study. This is most likely attributed to the fact that number of animals evaluated in the definitive study was 20 compared to 6 in the dose finding study. Similarly, whereas sperm motion parameters were not affected at doses less than 72 mg/kg in the dose finding study, the percentage of progressive tracks and the percentage of progressively motile sperm were reduced by each dose of BCA in the definitive study.

The fertility of sperm from the cauda epididymis was reduced significantly for males exposed to each dose of BCA. There was no dose response associated with the significant decline in fertility. Indeed, the average fertility of cauda sperm from animals in 8 mg/kg treatment group was somewhat less than the means for animals in the 24 and 72 mg/kg treatment groups. Sperm motion parameters (i.e., percentage of progressively motile sperm and percentage of tracks progressive) were correlated with fertility and statistically significant, yet the r values for these measures were low (i.e., 0.27 and 0.30), indicating only weak associations. Of the 120 detergent-extracted cauda epididymal sperm proteins resolved and identified on two-dimensional denaturing gels, only two were signficantly correlated with fertility. One protein (SP9) had a molecular weight of approximately 28 kDa and a pI of 5.5; the other protein (SP22) had a molecular weight of 28 kDa and a pI of 5.25. While these proteins were diminished significantly in extracts of cauda epididymal sperm from animals in each BCA treatment group, the shape of the dose response for SP22 was more similar to that observed for fertility. Thus, it is not surprising that the r value for SP9 was 0.23 compared to 0.53 for SP22. Indeed, when the background-corrected integrated optical density of SP22 and fertility for each animal was fitted to a nonlinear threshold model, the resulting correlation (r2) between SP22 and fertility was 0.84. While SP9 was not well correlated with fertility, it is noteworthy that SP9 is now known to be a charged variant of SP22 (Welch et al., 1998Go). Indeed, this is our second study in which SP22, but not its more basic variant, was correlated with fertility.

In our original study demonstrating that SP22 was highly correlated with fertility (Klinefelter et al., 1997Go), toxicants that compromise the structure and/or function of the epididymis (i.e., ethane dimethanesulphonate, chloroethylmethanesulphonate, epichlorohydrin, hydroxy-flutamide) were tested. Subsequent to identification of SP22 we determined that testis-specific SP22 mRNA is expressed initially within pachytene spermatocytes and later in round and elongating spermatids (Klinefelter and Welch, 1999Go), and that SP22 protein expression begins in round spermatids and continues thereafter (Klinefelter et al., 2002Go). Thus, SP22 appears to represent a "moonlighting" protein (Jeffery, 1999Go) as it is found within the cytoplasm of round spermatids, and later on the plasma membrane overlying the equatorial segment of the sperm head. Moreover, using polyclonal and monoclonal antibodies directed against full-length recombinant and native SP22, respectively, we established that SP22 plays a pivotal role in the fertilization process (Klinefelter et al., 2002Go). Finally, using isolated seminiferous tubule cultures, we demonstrated quantitative diminutions in four specific cytosolic proteins synthesized by the seminiferous epithelium following both in vivo and in vitro exposures to DBA (Holmes et al., 2001Go). Of the four proteins affected, three are currently being identified; the fourth is SP22. Whether any of the other three proteins are involved in the haloacid-induced manifestations mentioned above remains to be determined. Northern blot analysis indicated that SP22 mRNA was not altered in testes of BCA exposed animals (data not shown). Therefore, it seems reasonable to suspect that exposure to BCA altered the translation and subsequent expression of SP22 in the later stages of spermatogenesis, i.e., during spermiogenesis.

What, if any, role disruption of the endocrine axis plays in mediating the haloacid-induced lesions in spermatogenesis and fertility remains unknown. It is well known that normal spermatogenesis is dependent on androgen stimulation (Sharpe, 1994Go), and recent research on the effects on haloacids like DBA and BCA in the female indicate that these chemicals disrupt steroidogenesis in vitro at concentrations comparable to those found in blood following effective in vivo exposures (Goldman and Murr, 2002Go). In the present study, LH levels were reduced significantly in the dose finding study; however, this effect did not repeat in the definitive study. Moreover, the levels of testosterone in serum, testicular interstitial fluid, seminiferous tubule fluid, and media following ex vivo incubation of testicular parenchyma were unchanged. This apparent lack of testosterone deficiency is consistent with the finding that the synthesis of seminiferous tubule proteins was compromised by DBA during androgen-supplemented culture (Holmes et al., 2001Go). Very recent work indicating the nuclear form of SP22 may be a positive regulator of the androgen receptor (Takahashi et al., 2001Go) suggests that these haloacids may compromise androgen-dependent maintenance of spermatogenesis indirectly, i.e., after SP22 is compromised. Still, it is possible that relatively subtle alterations in the pulsatility of testosterone release coupled with androgen-independent mechanisms may act in concert to disrupt spermatogenesis.

Numerous epidemiological studies have demonstrated an association between increased levels of disinfection by-products of drinking water disinfection, specifically trihalomethanes, and adverse reproductive outcomes such as spontaneous abortion (Waller et al., 1998Go), low term birth weights (Gallagher et al., 1998Go), and stillbirths (Dodds et al., 1999Go). Based on the available toxicology data, particularly the reproductive toxicology data (Klinefelter et al., 2001Go), there is a need to evaluate future epidemiology findings based on haloacid exposures (Reif and Bachand, 2001Go). There is also a need to conduct epidemiology studies to examine potential associations between exposure to haloacids and adverse male reproductive effects. Moreover, as fertility and sperm membrane protein composition, specifically SP22, appear to be highly sensitive to haloacid exposure, and preliminary data from recent clinical studies indicates that SP22 levels are diminished in most men presenting with infertility (unpublished data), incorporation of this sperm biomarker into such studies is advisable.


    ACKNOWLEDGMENTS
 
The authors extend their gratitude to Dr. Robert Chapin of DuPont Pharmaceuticals for expert evaluation of histopathology in the dose range finding study. In addition, the authors extend their appreciation to Cristy Lambright and Dr. Tammy Stoker for their skillful assistance with the hormone assays.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 541-4017. E-mail: klinefelter.gary{at}epa.gov. Back

The information in this document has been funded wholly (or 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 reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


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 MATERIALS AND METHODS
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 DISCUSSION
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