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
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ABSTRACT |
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Key Words: disinfection by-products; puberty; fertility; sperm biomarker.
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INTRODUCTION |
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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, with qualitative effects prevailing at lower exposures (Linder et al., 1994, 1995
, 1997
). For example, at 10 mg/kg, there was an increase in delayed spermiation as the total number of retained spermatids increased over three-fold in the stages IXXII of DBA-exposed testes. In a subsequent 70-day study (Linder et al., 1995
, 1997
), it became apparent that DBA exposure produced qualitative alterations in sperm function prior to quantitative alterations. For example, when fertility of males was evaluated by intrauterine insemination (IUI), only 20% of the males were able to produce a litter after 16 days of exposure to 250 mg/kg DBA. As the duration of exposure increased to 31 days the quantitative effects were manifest, as sperm for IUI could be obtained from only 20% of the males; cauda epididymal sperm numbers were reduced from 240 million to 33 million.
Haloacids and other DBPs are not currently considered endocrine-disruptive chemicals. However, given the accumulating data indicating that many endocrine-disrupting chemicals alter reproductive development (e.g., acquisition of puberty), and the lack of these data for DBPs, it seemed important to examine the possibility that a prevalent haloacid like DBA might alter reproductive development, specifically the acquisition of puberty (i.e., preputial separation and vaginal opening) upon exposure throughout gestation and lactation.
It is well accepted that laboratory rodents such as the rat are robust breeders, requiring a significant toxic insult to detect a significant reduction in fertilizing capacity by natural mating (Amann, 1986). In recent years, in hopes of identifying a biomarker capable of detecting subtle changes in sperm quality that might be applicable to human exposures and effects, we began utilizing a fertility assessment by IUI in which only a threshold number of sperm are inseminated. We determined that a novel sperm membrane protein (SP22) was correlated with the fertility of proximal cauda epididymal sperm following exposure to chemicals that disrupt epididymal sperm maturation in the rat (Klinefelter et al., 1997
).
Given that the disubstituted haloacid DBA alters spermiogenesis and the fertility of epididymal sperm (Linder et al., 1995), we reasoned that a 14-day exposure would suffice to detect changes in the fertility and membrane proteome of proximal cauda epididymal sperm. We tested another prevalent haloacid, BCA, and found that both the fertility and the membrane proteome of proximal cauda epididymal sperm were compromised even at the lowest dose tested (i.e., 8 mg/kg) (Klinefelter et al., 2002a
). Importantly, the diminutions of the SP22 protein were highly correlated with the reductions in fertility; the r2 = 0.84. The SP22 protein is present on sperm from all species examined to date, including human (Klinefelter et al., 2002b
). Thus, SP22 might be a suitable biomarker for detecting compromised sperm quality in men exposed to increased levels of haloacids.
In the present study, we sought to determine whether reproductive development (i.e., preputial separation and vaginal opening) is altered by exposure to DBA, and whether reproductive competence (i.e., fertility and sperm quality) is altered at adulthood.
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MATERIALS AND METHODS |
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Animals. For the dose-finding study, 12 timed pregnant Sprague Dawley rats (Harlan Sprague-Dawley Inc., Indianapolis, IN) were allowed to acclimate for a week to room conditions of 12 h light/dark, 22 ± 1°C, 50% ± 10% relative humidity. They were housed separately in cages on racks equipped with red polycarbonate water bottles with ballpoint sipper tubes. The U.S. EPA NHEERL Institutional Animal Care and Use Committee approved all procedures. Animals were ranked by weight and distributed into four treatment groups (Fig. 1). Three dams per treatment group (0, 400, 600, 800 ppm) were dosed from gestation day (GD) 15 through weaning on postnatal day (PND) 21. Pups were sexed on PND 1 and weighed on PND 1, 3, 6, 10, 13, 17, and 20. On PND 3 anogenital distance was measured on all pups. Males were evaluated for areolas and nipples on PND 13. At weaning, male pups from each litter were moved to separate cages and continued on the same dose as their dams.
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For the definitive study, pregnant Sprague Dawley rats were used to generate 48 litters, 12 per dose group (0, 4, 40, 400 ppm DBA), and pups were randomly assigned to one of four experimental groups (Fig. 1); one pup of each sex were selected per litter. For each of these groups, dosing began on GD 15 and continued through weaning (PND 21). Pups were sexed (PND 1) and weighed on PND 1, 5, 8, 12, 15, and 19. Each group comprised two experimental blocks to accommodate sacrifice schedules. For Groups I and II, males and females were dosed from GD 15 until weaning; Group II animals were then put on regular water. Males from both groups were killed on either PND 56 or 57; females were killed subsequent to VO. For Groups III and IV, males were dosed from GD 15 and either kept on treatment after weaning and killed on PND 120 (Group III) or treatment was discontinued at weaning and animals were killed on PND 120 (Group IV). Group III and IV females were killed on the first day of estrus after VO for measurement of in vitro progesterone (P) production.
For both studies, animals from the same litter were averaged for day and weight at VO and PPS so that n = litter. Animals were weighed twice each week throughout the study. Water consumption was measured by weighing water bottles when they were put on and taken off (three times per week). Consumed dosage was calculated weekly by using the average daily body weight of the animal and the average daily water (i.e., DBA) consumption.
Dose Finding Study
Male necropsy measures. The left testis and epididymis from these animals were excised prior to fixation, trimmed, and weighed. The right testis and epididymis from six animals per group were fixed in situ by vascular perfusion via the descending aorta with Dulbecco's PBS (DPBS), followed by 5% glutaraldehyde in a 0.05 M collidine buffer with 0.1 M sucrose. After 1015 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 glycol methacrylate (JB-4 Plus, Polysciences, Inc., Warrington, PA). Blocks were sent to Pathology Associates International (Frederick, MD) for sectioning at 2 µm and staining with hematoxylin and eosin or periodic acid Schiff counterstained with Gill's hematoxylin. Testis sections were examined by light microscopy for delayed spermiation and the presence of atypical residual bodies (Klinefelter et al., 2002a; Linder et al., 1997
). For this, 20 cross sections from each animal per treatment group were evaluated. Spermiation was considered delayed when at least three spermatids were retained beyond stage VIII of the spermatogenic cycle (Hess, 1990
). DBA-induced testicular lesions were evaluated as incidence (percentage of animals) within a treatment group.
Serum obtained following cardiac puncture, as well as testicular interstitial fluid, collected from the left testis as described previously (Holmes et al., 2001), were assayed for T 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.
The left epididymides were used for sperm morphology, sperm motility, sperm protein extraction for 2D gels, and fertility assessment by IUI. For this, the cauda epididymidis of each male was placed in a 35-mm culture dish containing 2 ml of Medium 199 (M199, Gibco, Grand Island, NY) buffered with 26 mM sodium bicarbonate and containing 3 mg/l (0.1x) DL-methionine, 0.2% protease-free bovine serum albumin (BSA, Sigma, St Louis, MO), 100 mM sodium pyruvate (Gibco, Grand Island, NY), 10 mM nonessential amino acids (Gibco, Grand Island, NY), 12 mg gentamicin sulfate (Gibco, Grand Island, NY), a insulin/transferrin/selenium mixture (Gibco, Grand Island, NY), and 200 nM T and 200 nM dihydrotestosterone, pH 7.2. On the day of IUI (see below) 0.25 mg/ml bovine lipoprotein (Sigma, St. Louis, MO) was added to the medium. The epididymal tubule was incised with a #11 scalpel blade, and sperm were allowed to diffuse out 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 to 30 x 106/ml. The remaining fixed sample was used for morphology evaluations as described previously (Klinefelter et al., 2002a). After removing the sample for insemination, aliquots were removed for sperm motion analysis using an Ivos (Hamilton Thorne, Beverly, MA) with Tox IVOS version 10.8 q software. Sperm were tracked for one second at 60 frames per second with a minimum track length of 30 frames (0.5 s) as described previously (Klinefelter et al., 2002a
).
The procedure for IUI of cauda epididymal sperm has been described previously (Klinefelter et al., 1997). 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 after dispersion of proximal cauda epididymal sperm, 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 (Klinefelter et al., 1994b
). 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.
The remaining sperm were processed for protein analysis (Klinefelter et al., 2002a). For this, sperm (1040 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 g/l NaHCO3 and containing 0.9 g/l D-glucose, 100 mM sodium pyruvate, and 0.025 g/l soybean trypsin inhibitor, pH 7.4 with freshly-added 0.2 mM phenylmethylsulphonyl fluoride (PMSF, Sigma, St. Louis, MO). 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 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, Bedford, MA). 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 (310 only; Serva, Heidelberg), 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 (310 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 silver staining kit (Amersham Biosciences, Upsula, Sweden).
A Kepler 2D 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.
Statistics. All data with the exception of the histopathology evaluation 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. Only treatment-related changes that differ significantly from control are discussed in the results.
Definitive Study
Groups I and II pubertal and necropsy measures. Females and males were checked for VO and PPS beginning on PND 26 and 37, respectively. For males necropsied on PND 56, the left and right testis and epididymis, seminal vesicles, prostate, kidney, and pituitary were trimmed and weighed. While our assessment of serum T in the dose-range study indicated no significant differences, we wanted to address the hypothesis that the steroidogenic potential of Leydig cells was altered by treatment in vivo. Therefore we measured ex vivo T production (Klinefelter et al., 1994a). 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 media. T production was assessed by incubating parenchyma (four 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 as described above.
Groups III and IV pubertal and necropsy measures. Males in both Groups III and IV were checked for PPS beginning on PND 37. Females in Group III (i.e., those exposed continuously) were checked for VO beginning on PND 26. Animals from the same litter were averaged for day and weight at VO and PPS so that n = litter. For Group III females receiving 0 and 400 ppm DBA, vaginal lavages were taken between 0800 and 0900 h on the day of VO. If vaginal smears contained cornified epithelial cells, the animal was killed under CO2, blood was taken for serum P, and newly formed corpora hemorrhagica (CH) were removed and incubated for 5 or 24 h ex vivo to evaluate P production. Briefly, two randomly selected CH were placed in small Teflon screw cap vials containing 2.7 ml of oxygenated phenol red free-M199 containing Earles salts, buffered with 10 mM HEPES and 4.4 mM sodium bicarbonate, and containing 0.1% BSA. Cultures were maintained at 37°C, without or with maximal (100 mIU) hCG stimulation, and recovered media was held at 70°C until P assay.
For males necropsied on PND 120, the testis and epididymis were excised, trimmed, and weighed. The cauda epididymidis of each animal was used for assessments of sperm morphology and motility, a quantitative evaluation of the sperm membrane protein, and a fertility assessment by IUI. Procedures for these measures are described above.
Statistics. All 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. Only treatment-related changes that differ significantly from control are discussed in the results. 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. A Pearson's correlation coefficient was determined for the relationship between VO and P production.
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RESULTS |
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DISCUSSION |
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The DBA-induced delays in puberty were confirmed in three distinct groups of animals. In the pilot study, VO was delayed until PND 44 in females exposed to 800 ppm. While control males in this study displayed PPS on PND 42, PPS in exposed male offspring was delayed until PND 4850. The delay in PPS was equivalent in all treatment groups, but the observed delays at 400 and 600 ppm were independent of body weight. In the definitive study, body-weight-independent delays in the onset of puberty were observed in male and female offspring in both Groups I and III exposed to 400 ppm DBA from GD 15 through acquisition of PPS and VO.
The ability of testicular parenchyma from exposed males to produce T under both unstimulated and hCG stimulated conditions was evaluated in Groups III and IV. hCG-stimulated T production was significantly increased for parenchyma from males exposed to 4 and 400 ppm in Group III, in which exposure was continuous until PND 120. The increased T production by males exposed to 4 ppm was two times greater than that by parenchyma from control males. Ex vivo T production was not increased by parenchyma in Group IV, in which DBA exposure was ceased at weaning. Interestingly, the ability of corpora hemorrhagica (CH) to produce P ex vivo was similarly altered by DBA exposure in Group III females exposed from gestation through acquisition of VO. CH from females exposed to 400 ppm DBA produced 2.25 times more P in response to hCG than CH from control females. While the DBA-induced delay in VO was not well correlated with the increase in P production, the correlation was greater for DBA-exposed females than in control females (i.e., R = 0.3 vs. 0.02). Thus, altered steroidogenesis may play a minor role in the observed pubertal delays.
Our previous studies failed to demonstrate any haloacid-induced decreases in LH, so our current hypothesis that haloacids, and other water soluble disinfection by-products, disrupt steroidogenesis directly stems from several lines of evidence. For example, in vitro DBA-exposed preovulatory follicles secrete less P (Balchak et al., 2000). Recently, Bielmeier et al., (2004)
demonstrated that, while in vitro exposure to bromodichloromethane (BDCM) decreased P production by corpora lutea, P production was increased by ex vivo incubation. The ex vivo steroidogenic rebounds associated with both DBA and BDCM exposures suggest a disinhibition of steroid production as the toxicant is diluted away from the tissue during in vitro incubation. While this notion needs to be investigated further, it is reasonable to assume that if steroidogenesis by the testis and ovary is inhibited in vivo by DBA, these inhibitions could account at least in part for the observed delays in PPS and VO despite the fact that decreases in serum hormones are not observed.
The quality of sperm from the cauda epididymidis was clearly diminished by continuous DBA exposure via drinking water. In the dose finding study, a body-weight-independent decrease in the weight of the epididymis was observed, along with changes in several qualitative sperm parameters. At 600 and 800 ppm DBA, but not 400 ppm DBA, multiple motion parameters were compromised, including progressive motility, straight line velocity, straightness, and linear index of the proximal cauda sperm. Moreover, significant treatment-related decreases in the percentage of morphological normal sperm were observed at all DBA exposures in the dose finding study (not shown), with the percentage of normal sperm decreasing from 95 to 78% in males exposed to 400 ppm. However, neither sperm motion parameters nor sperm morphology were significantly correlated with fertility (not shown). Both the fertility and the sperm membrane proteome were compromised in the dose finding study. While fertility of proximal cauda epididymal sperm was decreased in a treatment-related fashion, the decreases were only significant at 600 and 800 ppm. Treatment-related decreases in the sperm membrane protein SP22 were significant at all exposures. The correlation between the decrease in SP22 and the decrease in fertility was significant (p < 0.05) and positive (r2 = 0.68) in this study.
DBA-induced alterations in sperm quality were comparable in the definitive study. While sperm motion parameters were unaffected (not shown), the percentage of morphologically normal sperm was decreased in males exposed to 400 ppm; sperm with misshapen heads and normal tails accounted for the predominant defect. With an increased incidence of sperm with misshapen heads, decreased sperm membrane proteins localized to the head of sperm (e.g., SP22; Klinefelter et al., 2002b) might be anticipated. Indeed, SP22 and two other proteins were diminished in the membrane extracts of proximal cauda sperm. These proteins (SP22, SP38, and SP8) were decreased in Group III males exposed continuously from GD 15 through adulthood, but not in Group IV males, in which exposure ceased at weaning. While the decrease in SP8 was only significant in males exposed to 400 ppm DBA, the decreases in both SP22 and SP38 were significant in males exposed to 40 and 400 ppm DBA. Treatment-related decreases in SP22 and SP38 are anticipated, as SP38 is a more basic charge variant of the SP22 protein (Klinefelter et al. 2002b
; Welch et al., 1998
). Historically though, only the expression of the SP22 variant has been correlated with fertility (Klinefelter et al., 1997
, 2002a
).
That the sperm membrane protein SP22 and its variant were only diminished in males exposed to DBA until adulthood, and not in males exposed only until weaning, is consistent with expression of the mRNA transcript and its translation in the testis. The 1.5 kB mRNA testis-specific transcript encoding SP22 first appears in the PND 30 testis, and expression increases until adulthood, as it is first expressed in pachytene spermatocytes, and later in spermatids (Welch et al., 1998). Likewise, the SP22 protein is first expressed in pachytene spermatocytes, with expression increasing in intensity in the round and elongating spermatids (Klinefelter et al., 2002b
). Pachytene spermatocytes and spermatids were not present during exposure of animals exposed only until weaning, accounting for the lack of diminished SP22 in sperm extracts of these animals at adulthood.
In contrast with our previous studies, including the dose finding study herein, the levels of SP22 on sperm from males in Group III that were exposed continuously to DBA were not significantly correlated with the fertility of sperm from these males. The only explanation we can offer is that control fertility in this study was much higher (85%) than in our previous studies (6575%). Thus in Group III, even though sperm from exposed males had diminished SP22 levels, the numbers of sperm inseminated were high enough to overcome this compromise.
Our results are comparable to results of a multigenerational study of DBA in rats (Christian et al., 2002). In this study, the no observed adverse effect level (NOAEL) and lowest observed adverse effect level (LOAEL) were 50 ppm (4.4 mg/kg) and 250 ppm (22.4 mg/kg), respectively. The LOAEL was set based on an increased incidence of delayed spermiation and atypical residual bodies in testes of P and F1 males exposed to 250 ppm DBA. We observed an increase in delayed spermiation and atypical residual bodies in males exposed to 400 ppm DBA (37.3 mg/kg; Table 4) in the dose finding study. Similarly, 2- and 3-day delays in PPS and VO were observed in male and female offspring exposed to 650 ppm DBA (83.4 mg/kg average from PND 22 to PND 70) in the Christian et al. study. These differences were not significant when body weight the day after weaning (PND 22) was used as a covariant. Statistical significance for the 4- and 3-day delays in PPS and VO observed herein at 400 ppm (76.3 mg/kg on PND 47) is likely due to the fact that body weight the day of acquisition was used as the covariant.
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Future research is needed to establish the biological relevance of these data to humans exposed to increased levels of haloacids, especially those in areas where bromide levels are high. The modes of action underlying the haloacid-induced alterations in both the pubertal delay and sperm quality also warrant further investigation. We are now incorporating evaluations of sperm quality, including SP22 quantified by ELISA, in an epidemiology study of men exposed to drinking water containing various levels of haloacid by-products.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (919) 541-4017. Email: klinefelter.gary{at}epa.gov.
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REFERENCES |
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