Haloacid Induced Alterations in Fertility and the Sperm Biomarker SP22 in the Rat Are Additive: Validation of an ELISA

Emilie H. Kaydos*, Juan D. Suarez{dagger}, Naomi L. Roberts{dagger}, Kathy Bobseine{dagger}, Robert Zucker{dagger}, John Laskey{dagger} and Gary R. Klinefelter{dagger},1

* North Carolina State University, Department of Environmental and Molecular Toxicology, Raleigh, North Carolina 27606; and {dagger} United States 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 May 10, 2004; accepted July 1, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dibromoacetic acid (DBA) and bromochloroacetic acid (BCA) are prevalent disinfection by-products of drinking water that produce defects in spermatogenesis and fertility in adult rats. Previously we demonstrated that BCA compromises the fertility of cauda epididymal rat sperm and SP22, a sperm membrane protein that is highly correlated with the fertility of these sperm. Herein, we administered DBA and BCA, individually and in combination, to determine whether fertility and levels of SP22 on sperm were diminished in an additive fashion. Moreover, we wished to validate an immunoassay for quantitation of SP22. In a dose finding study, animals were exposed by oral gavage daily for 14 days to: BCA alone at 1.6, 4, and 8 mg/kg; DBA at equimolar levels of 2, 5, and 10 mg/kg; and two binary mixtures of 1.6 mg/kg BCA + 2 mg/kg DBA and 4 mg/kg BCA + 5 mg/kg DBA. The ED50s for the decrease in SP22 quantified by two-dimensional SDS–PAGE were 7.2 and 4.6 mg/kg for DBA and BCA. The ED50s for the decrease in SP22 quantified by ELISA were 8.1 and 5.9 mg/kg for DBA and BCA. The definitive study consisted of 2 and 4 mg/kg DBA, 1.6 and 3.2 mg/kg BCA, and a 2 mg/kg DBA + 1.6 mg/kg BCA mixture. The ED50s for decreases in fertility assessed by intrauterine insemination were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA. Immunolocalization of SP22 in spermatocytes and spermatids, as well as on the cytoplasmic droplet and the equatorial segment of luminal sperm, was decreased by the DBA + BCA mixture. The decrease in SP22 in testicular parenchyma was comparable to that observed for sperm extracts. Based on 2D SDS–PAGE, ELISA, or fertility the haloacid-induced decreases in SP22 or fertility were additive or synergistic. The correlation between SP22 levels by ELISA and fertility was r2 = 0.72 compared to 0.82 for SP22 levels by 2D SDS–PAGE and fertility, validating SP22 quantitation by ELISA.

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


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Drinking water disinfection emerged in the early 1900 s to combat water-borne illnesses like cholera and typhoid fever. Deaths from illnesses associated with drinking water virtually ceased to exist (Mughal, 1992Go), but hundreds of drinking water disinfection by-products (DBPs) have now been identified (Richardson, 2002Go). DBPs such as haloacetic acids are formed when chlorine, ozone, or chloramine from the disinfection process reacts with humic and fulvic acids in source water (Christman et al., 1983Go). EPA's Stage II DBP rule (U.S. EPA, 2003Go) established a maximum contaminant level (MCL) of 60 µg/l for the sum of five haloacids (monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoacetic acid, and dibromoacetic acid) reported as a running yearly average of various sites along the distribution system at a water treatment plant. Currently bromochloroacetic acid (BCA), one of the more prevalent haloacids is not included among the regulated haloacids.

Epidemiology studies are now underway to relate haloacid exposure with compromised semen quality in men. This effort was prompted by the toxicology data demonstrating that the disubstituted haloacids adversely affect male reproduction in rats. Dibromoacetic acid (DBA), dichloroacetic acid (DCA), and BCA have all been shown to delay spermiation in the testis (Klinefelter et al., 2002aGo; Linder et al., 1994Go, 1995Go, 1997Go). Histopathology also reveals atypical residual bodies in the lumen of seminiferous tubules, fusion of round and elongated spermatids, and fusion of mature spermatids. Collectively, these manifestations suggest that spermiogenesis is affected by exposure to haloacetic acids. In our study of BCA (Klinefelter et al., 2002aGo) we evaluated proximal cauda epididymal sperm after 14 days of exposure, because elongating spermatids in stage I of the spermatogenic cycle at the onset of exposure would be expected to present as mature sperm in the proximal cauda epididymidis after 14 days, based on kinetics of spermatogenesis (Sharpe, 1994Go) and transit time in the epididymis (Robb et al., 1978Go). The fertility of these sperm and the sperm membrane proteome were compromised by BCA exposure in this study. That both of the measures were compromised indicated the final stages of sperm membrane modeling are indeed compromised during spermiogenesis. The membrane of proximal cauda epididymal sperm is not compromised by haloacids in a 4-day exposure of the epididymis (unpublished observations).

A unique feature of the work in our laboratory is the evaluation of male fertility by intrauterine insemination (IUI). In rodents, spermatogenesis is highly efficient, with 90% of the sperm produced being qualitatively normal (Amann, 1986Go). A chemical exposure that compromises sperm quality may not render a rat infertile by natural mating unless virtually all sperm available for ejaculation are affected. By contrast, spermatogenesis is inefficient in humans, with up to 50% of the sperm that are produced being qualitatively abnormal (Johnson, 1986Go). Thus, a chemical insult could render a man infertile more readily than a male rodent. Indeed, there may be credence in reports over the past decade that semen quality in men is declining due to environmental exposures (Carlsen et al., 1992Go; Veeramachaneni, 2000Go). It seems prudent for toxicologists to increase the sensitivity of fertility assessments in the male rat to better approximate a potential human effect scenario.

A long-term goal of our laboratory has been to identify a sperm biomarker that is predictive of fertility and could be used in epidemiology studies to address the potential decline in semen quality. As mentioned above, our earlier study of BCA revealed that both fertility and the sperm membrane proteome were compromised. Of two proteins significantly affected, one (SP22) was highly correlated with fertility. When fertility and SP22 were fit to a nonlinear equation, the r2 value was 0.84 (Klinefelter et al., 2002aGo). The SP22 protein has now been shown to be present on sperm from all species examined including human, and antibodies to SP22 significantly inhibit fertilization both in vivo and in vitro (Klinefelter et al., 2002bGo) The SP22 mRNA (Welch et al., 1998Go) and protein (Klinefelter et al., 2002bGo) are expressed in post-meiotic germ cells and elongated spermatids. Thus, this protein is a potential molecular target for chemical insults that target spermiogenesis. The goals of this study were to: (1) determine whether haloacid-induced alterations in fertility and SP22 were additive; (2) validate the use of an ELISA for quantitation of SP22; and (3) determine whether haloacid-induced diminutions in SP22 were present in the testis as well as on epididymal sperm.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Adult male and female Sprague-Dawley rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN) at 90 and 60 days of age, respectively. The U.S. EPA NHEERL Institutional Animal Care and Use Committee approved all procedures. Males were housed one per cage while the female counterparts were housed two per cage. All were allowed to acclimate for 14 days to room conditions of 12 h light/dark, 22 ± 1°C, 50 ± 10% relative humidity in an AAALAC-approved animal facility. Males were ranked by weight and randomly placed into dosing groups to ensure equivalent weight distribution across all groups. Due to the size of the study, two animals from each dosing group were placed in experimental blocks; the dose-finding and definitive studies consisted of eight and four blocks, respectively.

Chemicals and dosing. Custom synthesized dibromoacetic acid (99 + % purity) was obtained from Aldrich Chemical Company (Milwaukee, WI). BCA of 90% purity was obtained from Carbolabs Inc. (Bethany, CT). Both DBA and BCA were dissolved in distilled water to make 1% stock solutions; pH was adjusted to 6.5 using descending concentrations of NaOH (5, 1, 0.1, 0.01, and 0.001 N NaOH). Stock solutions were then used for all dose formulations. After combining DBA and BCA, the pH of the mixtures was verified and adjusted by NaOH as necessary. It was previously determined that these chemicals were stable for up to 2 weeks at room temperature. Stock and dosing solutions were both stored in amber glass bottles to reduce their exposure to light, and kept at 4°C.

Dose-Finding Study
Experimental design. 72 male rats were acclimated, weighed, and stratified into treatment groups. As indicated in Figure 1, the males were placed into one of the following dose groups: 0 (distilled water); 10, 5, and 2 mg/kg DBA; 8, 4, and 1.6 mg/kg BCA; a mixture of 5 mg/kg DBA + 4 mg/kg BCA; or a mixture of 2 mg/kg DBA + 1.6 mg/kg BCA. These doses were selected based on data from a pilot study demonstrating that the sperm biomarker SP22 was diminished significantly within this range of exposures. Animals within each dose group were randomly placed into one of eight experimental blocks. Animals were assigned a number and identified with a corresponding ear tag. Each male was gavaged daily for 14 consecutive days. Doses were adjusted biweekly according to body weight.



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FIG. 1. Diagram depicting the design of both the dose finding and definitive study. In both studies adult rats (n = 8 per group) were administered DBA and/or BCA via gavage daily for 14 days. The doses of DBA, the molar equivalents of BCA, and the endpoints evaluated at the end of each study are shown.

 
Necropsy evaluations. Blood was obtained from each animal via cardiac puncture while under a surgical plane of anesthesia, and rats were euthanized via cervical dislocation. Serum was obtained using serum separator tubes and then frozen at –70°C prior to testosterone assay. The left epididymides were used to recover proximal cauda sperm for membrane protein extraction and a quantitative evaluation of the SP22 biomarker. For this, the cauda epididymidis of each male was placed in a 35-mm culture dish containing 2 ml of sperm isolation buffer [95 ml/l 10x Hanks Balanced Salts Solution (HBSS), 0.35 g/l NaHCO3, 4.2 g/l HEPES, 0.9 g/l glucose, and 10 ml/l 100x Na pyruvate, pH 7.4]. After piercing the proximal cauda epididymal tubule with a #11 scalpel blade, sperm that diffused out were allowed to disperse for 5 min at 34°C, 5% CO2.

2D SDS–PAGE and ELISA for SP22. Proximal cauda epididymal sperm were used for a quantitative sperm membrane protein evaluation. (Klinefelter et al., 2002aGo). For this, sperm (10–40 x 106) were transferred to a microcentrifuge tube and washed twice by centrifugation (3000 x g, 10 min) in sperm isolation buffer with freshly added 0.2 mM phenylmethylsulphonyl fluoride (PMSF; Sigma). After the final wash, sperm were extracted for 1 h at room temperature with 1 ml of 80 mM n-octyl-B-glucopyranoside (OBG) 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 2-D gel electrophoresis, samples were thawed, and each extract was concentrated with 1 mM Tris buffer, pH 7.2, by two centrifugations (3,000 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 11% acrylamide gels (200 V, 45 min). Gels were soaked in 50% methanol and silver stained using a PlusOne Silver Staining Kit (Amersham Biosciences, Piscataway, NJ).

A Kepler 2-D 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 (i.e., the background-corrected, integrated optical density, I.O.D.).

After 2D electrophoresis, the remaining sperm extract was used for SP22 quantitation via ELISA. For this, 96-well tissue culture plates (Costar 3595 96-well cell culture; Corning Inc., Corning, NY) were used. A standard curve was generated using serial dilutions of antigen (i.e., full-length rat recombinant SP22 [rSP22; Klinefelter et al., 2002bGo]), 0, 0.01, 0.05 0.1, 0.5, 1, 5, and 10 ng in 50-µl/well; all dilutions were in NaHCO3, pH 9.5. Initially, sperm extracts diluted in Dubecco's Phosphate Buffered Saline (DPBS; Gibco, Grand Island, NY) were plated at 0, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 µg in 50 µl/well. Evaluation of the response in control extracts indicated 10 µg provided a maximal response with the least variance so 10-µg protein loading was used for all samples subsequently. Duplicate wells were used for both the SP22 standards and each sperm extract. The plates were stored overnight at 4°C to maximize antigen absorption. The following day, unbound antigen was removed by inverting the plate and shaking gently. A blocking step consisted of addition of milk protein (caseinate or dry milk powder) in DPBS (150 µg/well) followed by incubation for 1 h at 37°C. Sheep anti-rSP22 diluted 1:1000 in DPBS + 1% BSA was added (50 µl/well) and allowed to bind during incubation for 1 h at 37°C. After three washes with DPBS + 1% BSA (200 µl/well), peroxidase conjugated rabbit anti-sheep antibody (Pierce Immunopure 31480, Rockford, IL) diluted 1:500 in DPBS + 1% BSA was added (50 µl/well) and allowed to incubate for 1 h at 37°C. After four washes with DPBS w/1% BSA, the peroxidase substrate ABTS (Pierce, #37615) was added (100 µl/well). The reaction was allowed to develop over a 15–20 min period. Absorbance was read using FLUOstar Galaxy software (BMG Labtechnologies Inc., Durham, NC) at 405 nm and values were expressed as absorbance or optical density (O.D.) Sample values were always found to fall within the range of the standard curve. While it was possible to express SP22 in ng per sample, we wanted to compare the absorbance values obtained between the two detection methods (2D SDS–PAGE and ELISA).

Ex vivo testosterone production. For ex vivo assessment of testosterone (T) production, the tunica albuginea was removed from the left testis, and quarters of parenchyma were incubated in 1.0 ml of Medium 199 (M199; Sigma, M-3769 with Earle's salts) 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. T production was assessed by incubating parenchyma in duplicate, either with or without hCG stimulation (100 mIU/ml) for 2 h at 34°C. After 2 h, the medium was removed and frozen at –70°C until T assay. T in medium recovered from parenchyma incubation and serum were 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.

Definitive Study
Experimental design. Forty-eight 90-day-old male Sprague-Dawley rats were acclimated, weighed, and randomly placed into treatment groups. As indicated in Figure 1, the male rats were placed into one of six dosing groups: 0 (water control), 2 and 4 mg/kg DBA, 1.6 and 3.2 BCA, or a mixture of 2 mg/kg DBA + 1.6 mg/kg BCA. These doses were selected based on the demonstrated effectiveness of these doses in the dose-finding study. Animals within each dose group were randomly assigned to one of four experimental blocks and identified as described above. Each male was gavaged daily for 14 consecutive days. Doses were adjusted biweekly according to body weight. Ninety 60-day-old female Sprague-Dawley rats were acclimated to ensure an appropriate number of females were in proestrus (i.e., receptive) the day of in utero insemination.

Necropsy evaluations. Animals were euthanized while under a surgical plane of anesthesia via cervical dislocation. The testis, epididymides, and the seminal vesicles were excised, trimmed, and weighed. The proximal cauda epididymal sperm were used for quantitative evaluations of SP22 and fertility assessment via in utero insemination. The proximal cauda epididymal sperm were recovered as described above for the pilot study, but a 50-µl aliquot of the liberated sperm 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 to 30 x 106/ml. After removal of sample equivalent to at least 10 million sperm for insemination, remaining sperm were used for SP22 quantitation (i.e., 2D-SDS–PAGE and ELISA) as described above.

In utero insemination (IUI). Assessment of the fertility of proximal cauda epididymal sperm by IUI has been described previously (Klinefelter et al., 2002aGo). Briefly, a cohort of females was synchronized with 80 µg sc of LHRH agonist (Sigma) 115 h prior to insemination (day 1). Just after room lights turn off on the day of proestrus (day 5), 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 of sperm diffusion from the proximal cauda epdidymis, each uterine horn was injected with a volume containing to 5 x 106 sperm, a value which 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, midventral 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.

Immunocytochemistry. The right testis from males at sacrifice was removed and immersion fixed in 30 ml Bouin's solution: saturated picric acid (Sigma), 37% formaldehyde solution (Fisher), and glacial acetic acid. After fixation for 24 h, the solution was changed to 70% ethyl alcohol saturated with lithium carbonate: 70% ethanol, 1.9 g/3.5 l Li2CO3. This solution was changed until all picric acid was removed from tissues. Tissues were stored in glass scintillation vials with 70% ethanol at 4°C until processing. Testes were removed from 70% ethanol, dehydrated in a graded series of ethanol and xylene, and embedded in paraffin (Paraplast Plus; Oxford Labwares, St. Louis, MO). Parrafin blocks were allowed to cool overnight and then stored at 4°C until sections (5 µm) were obtained. After sectioning, slides were held at room temperature overnight and stored at 4°C until immunocytochemical analysis.

Testis sections were deparaffinized, rinsed in distilled water and placed in the microwave at low power for 60 s in 0.1 M sodium citrate buffer (Polysciences), pH 6, for antigen retrieval. Sections were then washed two times with DPBS for 3 min, and blocked for 1 h at 34°C with sterile DPBS containing 1% protease-free BSA (Sigma), 10% rabbit serum (Vector Laboratories, Inc., Burlingame, CA), and 0.3% Triton X-100 (Surfact-Amps X-100, Pierce, Rockland, Il). To quench endogenous peroxidase activity, sections were incubated for 30 min at room temperature in 30% hydrogen peroxide in methanol and washed with DPBS twice. Sections were then incubated overnight at 4°C in affinity-purified sheep anti-rSP22 Ig (1:200), while DPBS alone was placed on sections serving as controls. The next day slides were washed with DPBS twice and incubated for 1 h with FITC conjugated rabbit anti-sheep Ig (FL-6000), diluted 1:25 in DPBS. After two washes with DPBS, sections were coverslipped with anti-fade mounting medium (H-1000; Vector Laboratories, Burlington, CA).

Images were captured with a Nikon Eclipse (E800, Nikon Instruments, Melville, NY) equipped with a digital SPOT camera (Southern Micro Instruments, Atlanta, GA) and corresponding SPOT RT version 3.2 software (Diagnostics Instruments, Sterling Heights, MI). FITC staining was quantified using Photoshop software to determine diameters and areas of a traced image. By using the drawing function, stained areas were highlighted to determine the appropriate integrated optical density. A total of twelve slides representing two per animal, three animals per treatment group (control and the 2 mg/kg DBA + 1.6 mg/kg BCA mixture) were analyzed.

Proteomic evaluation of testicular parenchyma. Testicular parenchyma was frozen in liquid nitrogen and held at –70°C. Upon thawing, parenchyma (approx. 0.4 g) was homogenized using an Ultra-Turrax T25 homogenizer (Janke & Kunkel, IKA Labortechnik Staufer; Germany) in 5 ml 10 mM Tris buffer containing 1 mM EDTA, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% OBG, pH 7.2, to which 0.2 mM PMSF was freshly added. Each homogenate was desalted and concentrated with 1 mM Tris Buffer, pH 7.2, and assayed for protein before quantitative 2D SDS–PAGE analysis as described above. To quantify SP22 by immunblotting, single dimension gels were equilibrated for 15 min in transfer buffer (3.0 g Tris base, 14.4 g glycine, 200 ml methanol brought to 1 l with distilled water) along with fiber pads, filter paper, and the nitrocellulose membrane. The transfer cassette was maintained at 4°C during transfer (1 h at 100 V). After transfer, the nitrocellulose membrane was incubated for 30 min in 1% protease-free BSA in DPBS containing 10% blocking serum at 34°C. The membrane was then incubated overnight at 4°C in sheep anti-rat recombinant SP22 (rSP22) diluted 1:1000 in DPBS. After two washes the membrane was incubated for 1 h with peroxidase-conjugated anti-sheep Ig diluted 1:50,000 in DPBS. After two additional washes, the membrane was incubated for 5 min in Super Signal working solution (Pierce, Rockford, IL). The membrane was overlaid with cellophane, transferred to a film cassette and exposed (1–5 min) to CL-Xposure film (Pierce). In addition to quantitation of SP22 on immunoblots, SP22 was also quantified in testicular parenchyma by ELISA as described above.

Statistics. The various data were analyzed using two-way analysis of variance (SAS; PROC GLM, 1985) for treatment effects. Where overall significance (p < 0.05) was indicated, the least-square means were compared for significant (p < 0.05) treatment related differences. Additivity between two treatments in a mixture was analyzed using a 2 x 2 factorial design via SAS (PROC GLM). Additivity was only indicated when this analysis indicated no significant interaction (p < 0.05) between two treatments. When a significant interaction was indicated, synergy was assumed.

To determine correlations between fertility, SP22 quantified by ELISA, and SP22 quantified by 2D-SDS–PAGE, animals were selected in which data existed for each of these endpoints. Outliers were determined as follows: First, any animals with no corpora lutea were eliminated, since the LHRH synchronization procedure gave a false positive for proestrus. Next, all animals with a fertility value of zero and an SP22 I.O.D. (2D SDS–PAGE) >300 were eliminated. These values were outliers based on their high I.O.D. and low fertility values. Finally, any animals with both fertility and O.D. (ELISA) greater than the highest control were eliminated. These values were definite outliers for the experiment and would give an incorrect representation of the data.

To determine linear correlations for graphs, a regression line with equation and correlation coefficient was inserted using PROC GLM. The SP22 values from ELISA, and 2D SDS–PAGE were fitted to a nonlinear function that forms a sigmoid curve with a horizontal plateau:

where y equals either O.D. values of SP22 via ELISA or I.O.D. values obtained from 2D SDS–PAGE. Both A and B are constants; A represents the initial increase in y when SP22 equals 0, while B represents the rate of exponential decay of the increase in y. The Marquardt curve fitting algorithm from SAS was used to fit the curve, calculate the parameters A and B, and adjusted to minimize the sum of squares for the model with respect to data points.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dose Finding Study
There were no changes in levels of testosterone either in the serum, or produced by testicular parenchyma ex vivo without or with maximal hCG stimulation (not shown). Similarly, there were no changes in the weights of the testes, epididymides, or seminal vesicles of males in any of the exposure groups (not shown).

The sperm membrane protein SP22 was quantified in detergent extracts of proximal cauda epididymal sperm by both 2D SDS–PAGE and ELISA. Quantitation of the SP22 spot in 2-D gels (Fig. 2A) revealed significant diminutions in males exposed to 10 mg/kg DBA and the equimolar exposure of BCA (8 mg/kg) as well as males exposed to both the high dose (5 mg/kg DBA + 4 mg/kg BCA) and low dose (2 mg/kg DBA + 1.6 mg/kg BCA) mixtures. The derived ED50s were 7.15 mg/kg and 4.61 mg/kg for DBA and BCA, respectively (Fig. 2B). The equimolar equivalent of 7.15 mg/kg DBA is 5.72 mg/kg BCA. When the decrease in SP22 in extracts of sperm from males exposed to 2 mg/kg DBA or 1.6 mg/kg BCA was compared with the decrease observed for males exposed to the low dose mixture of 2 mg/kg DBA + 1.6 mg/kg BCA, additivity was indicated (Fig. 2C).



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FIG. 2. (A) SP22 in extracts of proximal cauda epididymal sperm in the dose finding study quantified following 2D SDS–PAGE. Values representing the background-corrected integrated optical density (I.O.D.) are presented as treatment means ± SEM. Significance (p < 0.05) relative to control is indicated *; significance between groups is indicated by difference in superscripts; n = 8 per group. Note: The lack of significant difference between 10 mg/kg DBA, 8 mg/kg BCA, and the high mixture of 5 mg/kg DBA + 4 mg/kg BCA indicated by (c) represents dose additivity. (B) Calculated ED50s reflecting the relative ability of DBA (left) and BCA (right) to result in decreased levels of SP22 on sperm. Note: 5.72 mg/kg BCA is the molar equivalent of 7.15 mg/kg DBA. (C) A graph illustrating the decreases in SP22 quantified by 2D SDS–PAGE attributed to 2 mg/kg DBA and 1.6 mg/kg BCA alone compared to the mixture (2 mg/kg DBA + 1.6 mg/kg BCA). A 2 x 2 factorial analysis indicated that the DBA and BCA-induced decrease in SP22 was additive.

 
Quantitation of SP22 by ELISA (Fig. 3A) also revealed significant diminutions in males exposed to 10 mg/kg DBA and the equimolar exposure of BCA (8 mg/kg) as well as males exposed to both the high dose (5 mg/kg DBA + 4 mg/kg BCA) and low dose (2 mg/kg DBA + 1.6 mg/kg BCA) mixtures. In addition, significant decreases were observed for males exposed to 5 mg/kg DBA and 4 mg/kg BCA. The derived ED50s were 8.10 mg/kg and 5.9 mg/kg for DBA and BCA, respectively (Fig. 3B). The equimolar equivalent of 8.1 mg/kg DBA is 6.5 mg/kg BCA. When the decrease in SP22 in extracts of sperm from males exposed to 2 mg/kg DBA or 1.6 mg/kg BCA was compared with the decrease observed for males exposed to the low dose mixture of 2 mg/kg DBA + 1.6 mg/kg BCA. additivity again was indicated. (Fig. 3C).



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FIG. 3. (A) SP22 in extracts of proximal cauda epididymal sperm in the dose finding study quantified by ELISA. Values representing the optical density (O.D.) are presented as treatment means ± SEM. Significance (p < 0.05) relative to control is indicated *; significance between groups is indicated by difference in superscripts; n = 8 per group. Note: The lack of significant difference between 10 mg/kg DBA, 8 mg/kg BCA, and the high mixture of 5 mg/kg DBA + 4 mg/kg BCA indicated by (c) represents dose additivity. (B) Calculated ED50s reflecting the relative ability of DBA (left) and BCA (right) to result in decreased levels of SP22 on sperm. Note: 6.5 mg/kg BCA is the molar equivalent of 8.1 mg/kg DBA. (C) A graph illustrating the decreases in SP22 quantified by ELISA attributed to 2 mg/kg DBA and 1.6 mg/kg BCA alone compared to the mixture (2 mg/kg DBA + 1.6 mg/kg BCA). A 2 x 2 factorial analysis indicated that the DBA and BCA-induced decrease in SP22 was additive.

 
Definitive Study
Based on the fact that exposure to 2 mg/kg DBA and 1.6 mg/kg BCA resulted in significant and additive changes in the sperm membrane proteome, the definitive study focused on these exposure levels and incorporated additional endpoints (e.g., fertility by IUI, testicular parenchyma SP22 evaluations). Again, there were no changes in the weights of the testes, epididymides, or seminal vesicles of males in any of the exposure groups (not shown).

Figure 4 depicts representative silver stained 2-D gels of proteins in sperm extracts from the various exposure groups. The 28 kD SP22 protein was diminished by both DBA and BCA exposures in a dose-related fashion. The magnitude of the decrease was greater for the 2 mg/kg DBA + 1.6 mg/kg BCA mixture than for the individual exposures supporting the synergy results depicted in Figure 5. Quantitative analysis (Fig. 5A) indicated that SP22 was significantly decreased in extracts of males exposed to 4 mg/kg DBA, as well as in the extracts of males exposed to the 2 mg/kg DBA + 1.6 mg/kg BCA mixture. When the decrease associated with the 2 mg/kg DBA exposure or the 1.6 mg/kg BCA exposure was compared to the decrease observed for the mixture of DBA and BCA, synergy was indicated (Fig. 5B). Quantitation of SP22 by ELISA (Fig. 5C) followed a pattern that was similar to that observed for the 2-D gel analysis, but significance was only attained for the 2 mg/kg DBA + 1.6 mg/kg BCA mixture. Once again, when the decrease associated with the 2 mg/kg DBA exposure or the 1.6 mg/kg BCA exposure was compared to the decrease observed for the mixture of DBA and BCA, synergy was evident (Fig. 5D).



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FIG. 4. Silver stained 2-D gel profiles of proteins in detergent extracts of proximal cauda epididymal sperm from animals in the Definitive Study. The SP22 protein (spot) is indicated with an arrow ({uparrow}). Note that the intensity of this spot decreases in a treatment-related fashion, with the greatest diminution in extracts from animals exposed to the 2 mg/kg DBA + 1.6 mg/kg BCA mixture. These differences are represented graphically in Figure 5.

 


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FIG. 5. (A) SP22 in extracts of proximal cauda epididymal sperm in the definitive study quantified by 2D SDS–PAGE. Values represent I.O.D. treatment means ± SEM. Significance (p < 0.05) relative to control is indicated *; significance between groups is indicated by difference in superscripts; n = 8 per group. Note: The significant difference between 4 mg/kg DBA and 3.2 mg/kg BCA (b) compared to the mixture of 2 mg/kg DBA + 1.6 mg/kg BCA (c) indicates dose synergy. (B) A graph illustrating the decreases in SP22 quantified by 2D SDS–PAGE attributed to 2 mg/kg DBA and 1.6 mg/kg BCA alone compared to the mixture (2 mg/kg DBA + 1.6 mg/kg BCA). A 2 x 2 factorial analysis indicated that the DBA and BCA-induced decrease in SP22 was synergistic. (C) SP22 in extracts of proximal cauda epididymal sperm quantified by ELISA. Values represent O.D. treatment means ± SEM. Significance (p < 0.05) relative to control is indicated *; significance between groups is indicated by difference in superscripts; n = 8 per group. Note: The significant difference between 4 mg/kg DBA and 3.2 mg/kg BCA (b) compared to the mixture of 2 mg/kg DBA + 1.6 mg/kg BCA (c) indicates dose synergy. (D) A graph illustrating the decreases in SP22 quantified by ELISA attributed to DBA and BCA alone compared to the mixture. A 2 x 2 factorial analysis indicated that the DBA and BCA-induced decrease in SP22 was synergistic.

 
Insemination of 5 x 106 sperm from control males into each uterine horn of receptive females resulted in 65 % fertility. By contrast, fertility was decreased in all exposure groups (Fig. 6A), with significance attained for all but the 2 mg/kg DBA exposure. The fertility of sperm from males exposed to the 2 mg/kg DBA + 1.6 mg/kg BCA mixture was only 15 %. The derived ED50s were 3.5 mg/kg and 2.7 mg/kg for DBA and BCA, respectively (Fig. 6B). The equimolar equivalent of 3.5 mg/kg DBA is 2.8 mg/kg BCA. The decreased fertility observed for males exposed to the haloacid mixture was additive relative to the decreases observed for individual haloacid exposures. (Fig. 6C).



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FIG. 6. (A) Fertility of proximal cauda epididymal sperm in the definitive study was assessed by IUI. Values represent treatment means ± SEM. Significance (p < 0.05) relative to control is indicated *; significance between groups is indicated by difference in superscripts; n = 8 per group. Note: The lack of significant difference between 4 mg/kg DBA, 3.2 mg/kg BCA, and the mixture of 2 mg/kg DBA + 1.6 mg/kg BCA is indicated (b) and represents dose additivity. (B) Calculated ED50s reflecting the relative ability of DBA (left) and BCA (right) to result in decreased fertility of sperm. Note: 2.8 mg/kg BCA is the molar equivalent of 3.5 mg/kg DBA. (C) A graph illustrating the decreases in fertility attributed to 2 mg/kg DBA and 1.6 mg/kg BCA alone compared to the mixture (2 mg/kg DBA + 1.6 mg/kg BCA). A 2 x 2 factorial analysis indicated that the DBA and BCA-induced decrease in SP22 was additive.

 
We established how well SP22 quantitation by 2D SDS–PAGE correlated with SP22 quantitation by ELISA, and how each of these methods of SP22 quantitation correlated with fertility (Fig. 7). The ELISA values for SP22 were significantly correlated with the SP22 values determined by 2-D gel analysis, and the r2 was 0.43. Both the ELISA and 2-D gel values for SP22 were correlated with fertility. The r2 was 0.82 for the correlation between SP22 valued determined by 2D SDS–PAGE and fertility, and the r2 was 0.72 for the correlation between SP22 determined by ELISA and fertility.



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FIG. 7. Graphs depicting the relationships between various endpoints in the definitive study. (A) SP22 quantified by ELISA versus SP22 quantified by 2D SDS–PAGE; r2 = 0.43, n = 31. The equation for the regression line is y = 378x + 129.68. (B) Levels of SP22 quantified by 2D SDS–PAGE versus fertility assessed by in IUI; r2 = 0.82, n = 31. The nonlinear equation for the graph is IODSP22 = .632 x exp((.032/.007) x (1 – e(–.007 x SP22 via 2D SDS-PAGE))), where IODSP22 is the integrated optical density at protein concentration of SP22. The dotted line represents the 95% confidence limits for the fitted line. (C) Levels of SP22 quantified by ELISA versus fertility assessed by IUI; r2 = 0.72, n = 31. The nonlinear equation is ASP22 = 1.44e-8 x exp((317.5/14.5) x (1 – e(–14.5 x SP22 via absorbance))) where ASP22 is the absorbance (O.D.) at protein concentration of SP22. The dotted lines represent the 95% confidence limits for the fitted line.

 
Previously, we established that SP22 is expressed in round and elongating spermatids in the seminiferous epithelium (Klinefelter et al., 2002bGo). After establishing that SP22 was diminished on proximal cauda epididymal sperm, we wished to determine whether we could detect decreased expression of SP22 in the testis. Immunostaining of SP22 in the testes of control animals revealed intense staining of pachytene spermatocytes and round and elongating spermatids within the seminiferous epithelium, as well as on the equatorial segment and cytoplasmic droplet of sperm in the tubule lumen (Fig. 8A). While the pattern of immunostaining was similar in the testes of males exposed to the 2 mg/kg DBA + 1.6 mg/kg BCA mixture, there was less staining in the cytoplasm of pachytene spermatocytes and round spermatids, and an absence of punctuate (i.e., cytoplasmic droplet) staining in the lumen (Fig. 8B). Quantitation of this immunostaining revealed that the observed decreases were not significant. To increase the sensitivity of our SP22 evaluation in the testis, we quantified SP22 in testis extracts by both immunoblotting and ELISA. Figures 9A and 9B illustrate the pattern of SP22 and its charged variants in both silver stained gels and immunoblots of testis extracts. Immunoblots of single dimension gels were quantified across treatments (Fig. 9C). The intensity of the SP22 band was decreased significantly in testis extracts of males exposed to 1.6 mg/kg BCA, as well as those that received the mixture (Fig. 10A). When SP22 in testicular extracts was evaluated by ELISA, similar results were obtained (Fig. 10B). For these data, however, the decrease in SP22 observed in the 2 mg/kg DBA group was significant. The correlation between SP22 in testis extracts quantified by immunoblotting versus by ELISA was significant (r2 = 0.50).



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FIG. 8. Immunocytochemical localization of SP22. (A) Confocal image of the seminiferous epithelium in a control testis with SP22 localization indicated in white. Staining is found in the cytoplasm of pachytene spermatocytes and round spermatids (arrows), elongated spermatids adjacent to the lumen (*), as well as the cytoplasmic droplets and equatorial segment of sperm in the lumen (arrows). (B) Comparable image of the seminiferous epithelium in a testis exposed to the 2 mg/kg DBA + 1.6 mg/kg BCA mixture. Staining in germ cells is less intense, with no staining of cytoplasmic droplets on sperm in the lumen. (C) Graph showing the relative difference in quantified FITC immunostaining. n = 3 for each treatment group.

 


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FIG. 9. (A) Silver stained 2-D gel profile of proteins in a homogenate of testicular parenchyma from control males. The SP22 protein and its charged variants are indicated (arrow). (B) An immunoblot of a gel comparable to that shown in (A) demonstrating antibody specificity for the SP22 protein and its variants. (C) A typical pattern of immunoreactivity observed for SP22 expression in testicular parenchyma across four treatments: control, 2 mg/kg DBA, 1.6 mg/kg BCA, and the 2 mg/kgDBA + 1.6 mg/kg BCA mixture. Notice the decrease in immunoreactivity with all haloacid exposures.

 


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FIG. 10. Graphs depicting haloacid-induced decreases in SP22 in homogenates of testicular parenchyma from males in four treatments: control, 2 mg/kg DBA, 1.6 mg/kg BCA, and the 2 mg/kgDBA + 1.6 mg/kg BCA mixture. (A) SP22 quantified by immunoblot analysis. Significance (p < 0.05) relative to control is indicated *; n = 8 per group. (B) SP22 quantified by ELISA. Significance (p < 0.05) relative to control is indicated *; n = 3 per group. (C) The correlation between SP22 quantified by ELISA and immunoblotting, r2 = 0.502; n = 12.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There have been numerous toxicological studies in recent years on the reproductive effects attributed to DBP exposure (Beilmeier et al., 2004Go; Bodensteiner et al., 2004Go; Goldman and Murr, 2002Go, 2003Go; Holmes et al., 2001Go; Klinefelter et al., 2002aGo; Linder et al., 1994Go, 1995Go, 1997Go). To date, however, all such studies have focused on individual chemical exposures rather than a mixture of DBPs. For DBPs that have been shown to elicit similar reproductive and/or developmental alterations it is important to determine how the effect levels, and the magnitude of effects, change when similarly acting DBPs are administered as a mixture. After all, humans are exposed to a complex mixture of DBPs in drinking water. Haloacids and their well-documented effects on spermatogenesis provide an excellent opportunity to investigate the possibility that these DBPs act in an additive and/or synergistic manner, enabling the prediction of lower effect levels.

The reasons we selected DBA and BCA for a mixture study were twofold. First, these two haloacids are prevalent DBPs in finished drinking water. Second, each of these by-products elicits comparable effects on the male reproductive system (i.e., alterations in spermatogenesis and sperm quality) (Klinefelter et al., 2002aGo; Linder et al., 1994Go, 1995Go and 1997Go).

The data presented herein demonstrate that the ED50s for the haloacid-induced diminution in SP22 recovered from the membrane of proximal cauda epididymal sperm, as well as the ED50s for the decreased fertility of these sperm, are lower for BCA than DBA. This was true whether the data were quantified by ELISA or 2D SDS–PAGE. While the ED50s were lower for BCA than DBA, the ED50s for BCA were consistently similar to the equimolar equivalents of DBA, indicating comparable potentcy on an equimolar basis. In the dose-finding study the decreases in SP22 observed for animals exposed to a mixture of 2 mg/kg DBA and 1.6 mg/kg BCA were additive, but in the definitive study the magnitude of the observed decreases were clearly synergistic. This is attributed to the fact that individual exposures to DBA or BCA resulted in decreased levels of SP22 in the dose finding study, whereas neither 2 mg/kg DBA nor 1.6 mg/kg BCA alone were capable of diminishing SP22 levels in sperm extracts in the definitive study. The fertility of proximal cauda epididymal sperm was compromised by exposure to 2 mg/kg DBA or 1.6 mg/kg BCA alone, but the magnitude of the decrease was greater for the mixture than for the individual exposures, indicating additivity.

The correlations between fertility and decreases in SP22 measured by either ELISA or 2D SDS–PAGE were significant. When fit to the nonlinear threshold equation previously used to define the relationship between levels of SP22 in detergent extracts of sperm and the fertility of these sperm, the r2 between fertility and SP22 quantified by 2D SDS–PAGE was 0.82, while the r2 between fertility and SP22 quantified by ELISA was 0.72. Thus, levels of SP22 recovered from the plasma membrane of epididymal sperm were again well correlated with the fertility of these sperm.

This is the first study to associate the decreased SP22 in epididymal sperm extracts with decreased SP22 in the testis. While it is possible for SP22 levels to be decreased on epididymal sperm during a 14-day exposure due to a direct insult to the epididymis, we found no change in SP22 levels on epididymal sperm when the exposure period was decreased to 4 days (unpublished observations). Therefore, we speculated that diminished SP22 on epididymal sperm resulted from decreased expression in the testis. Indeed, SP22 was decreased in extracts of testicular parenchyma by both ELISA and immunoblotting.

The qualitative haloacid-induced decreased immunostaining of SP22 within the seminiferous epithelium and lumen of the seminiferous tubule suggested a greater degree of SP22 loss in the lumen of the seminiferous tubule. If this is correct, the decrease in SP22 in the parenchyma extracts primarily reflects decreased association of SP22 with newly released step 19 spermatids. At the present time we do not understand how expression of this protein changes from the cytoplasm in round and elongating spermatids, to the cytoplasmic droplet of rete testis sperm, and ultimately to the equatorial segment of epididymal sperm (Klinefelter et al., 2002bGo). We speculate that haloacid insult to the testis results in compromised protein interactions, which disrupt the targeting of SP22 to the cytoplasmic droplet in the lumen of the seminiferous tubule.

We validated the use of ELISA as an efficacious, less tedious, and more rapid method for quantifying SP22 in sperm or tissue extracts. Correlations for SP22 quantitation by ELISA versus 2D SDS–PAGE were 0.43 and 0.50 for SP22 measured in epididymal sperm extracts and testis parenchyma extracts, respectively. Given these data, we now plan to quantify SP22 by ELISA in sperm extracts on a larger scale, including men from both epidemiology and clinical studies.

We failed to identify a no observed adverse effect level (NOAEL) for either DBA or BCA exposure. Based on decreases in SP22 in the testis (DBA and BCA) and fertility of cauda epididymal sperm (BCA), 2 mg/kg DBA and 1.6 mg/kg BCA represent the low observed adverse effect levels (LOAELs). Using estimated NOAELs of 0.2 mg/kg and 0.16 mg/kg (LOAEL/10), and the assumption that a 60-kg adult male consumes 2 liters of water a day containing 20 µg/l DBA and 20 µg/l BCA (Krasner et al., 1989Go, Krasner, 2000Go, Richardson and Thurston, 2003Go), individual margins of exposure of 298 mg/kg and 238 mg/kg can be calculated for DBA and BCA : 0.2 mg/kg/([2 l x 0.02 mg/l]/60 kg) = 298 and 0.16 mg/kg/([2 l x 0.02 mg/l]/60 kg) = 238. Since the combination of these two haloacids is at least additive with respect to the decrease the mixture would elicit on fertility of sperm, it is possible to "predict" lower effect levels. For example, a mixture expected to achieve an effect equivalent to the effective individual exposure of 1.6 mg/kg BCA would be 0.80 mg/kg BCA + 1.0 mg/kg DBA (the equimolar equivalent of 0.80 mg/kg BCA). In this predicted case, the margins of exposure would be 149 mg/kg and 119 mg/kg for DBA and BCA, respectively. However, finished drinking water contains many haloacids, regulated and unregulated. Assuming that each acts on spermatogenesis in similar additive fashion, the potential exists for margins of exposure that are considerably lower than these.

In summary, this is the first defined binary mixture study of the effects of the disubstituted haloacids on the male reproductive system. We focused on the most sensitive adult endpoints identified to date (i.e., fertility by IUI and the sperm fertility biomarker SP22). We were unable to identify a NOAEL in this study as compromised fertility and/or diminutions in SP22 in epididymal sperm extracts and/or testis parenchyma extracts were observed at the lowest exposures. Both dose and effect additivity were observed. Data were at least additive, and sometimes synergistic, for haloacid-induced decreases in SP22 and fertility. Our data validate the use of an ELISA for quantitation of SP22 to replace the preexisting 2D SDS–PAGE analysis. Finally, this is the first demonstration that diminished levels of SP22 on cauda epididymal sperm reflect haloacid-induced decreases in SP22 expression in the testis. Ongoing proteomic work is focused on elucidating a mode of action for this compromise.


    ACKNOWLEDGMENTS
 
E.H.K was funded by NCSU/U.S. EPA Cooperative Training Agreement CT826512010.


    NOTES
 
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.

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


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