Sub-chronic Exposure to Dibromoacetic Acid, a Water Disinfection By-pr
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《毒物学科学杂志》
Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1683
ABSTRACT
Water disinfection by-products, such as dibromoacetic acid (DBA), are formed when drinking water is treated with chlorination, bromination, or ozonation. Epidemiological studies have linked these byproducts to adverse effects in humans such as cancer, developmental defects, and reproductive toxicities. DBA has been shown to produce reproductive toxicity in rodents at relatively high doses. The present study used a mouse model to determine the developmental and reproductive effects of sub-chronic, low-dose exposure to DBA. Pregnant mice (10/dose group) were exposed with DBA in drinking water at 0, 5, or 50 mg/kg/day from gestation day 15 though nursing. Upon weaning at 3 weeks, one group of pups (pre-pubertal group: 7–10 pups of each gender/treatment group) were euthanized and weights of liver, paired kidneys, testes, and ovaries were measured. In the 50 mg dose group, weights of testes and liver in males and weights of liver and kidneys in females were significantly higher (p < 0.05). The remaining pups (15–17 of each gender/dose group) continued to be dosed similarly through adulthood. At 7 weeks of age (neo-pubertal group), animals were euthanized and tissues weighed and processed for evaluation of reproductive organs and gametogenic potential. Except for decreased (p < 0.05) testes and kidney weights in 50 mg dose group males, there were no differences in organ weights. No significant differences were noted between control and dosed animals in daily sperm production, testicular sperm counts, epididymal sperm reserves, morphology of seminiferous epithelium, or ovarian follicle counts.
Key Words: dibromoacetic acid; ovary; follicles; testis; sperm; mouse.
INTRODUCTION
Chlorination, bromination, and ozonation are commonly used to disinfect drinking water. These types of water treatments have proven invaluable in decreasing the spread of water-borne illness (Faber, 1952; Mughal, 1992). However, several water disinfection by-products are formed when chlorine and bromine react with organic material in source waters (Bellar et al., 1974; Rook, 1974). Epidemiological studies have linked exposure to these by-products with cancer (Boorman et al., 1999; Morris, 1995; Nieuwenhuijsen et al., 2000) as well as developmental and reproductive toxicities (Graves et al., 2001; Nieuwenhuijsen et al., 2000; Reif et al., 1996). Haloacetic acids, including dibromoacetic acid (DBA), are one of the most prevalent classes of disinfection by-products found in treated drinking water and are thus possibly a major concern for public safety (Krasner et al., 1989; Richardson, 2002; Richardson and Thurston, 2003; Uden and Miller, 1983). To date, there are few epidemiological studies specifically involving haloacetic acids (Hinckley et al., 2005) but toxicological studies show that these chemicals have adverse effects in laboratory animals.
Several rodent studies involving DBA have focused on the effects of acute exposure to juvenile and adult rats. Testicular toxicity with adverse effects on sperm motility and morphological features following acute exposure to DBA (10 to 1250 mg/kg/day via oral gavage for 1 to 14 days) has been demonstrated in adult male rats (Linder et al., 1994a,b, 1997). There is a dose-related decrease in ability of adult male rats to sire multiple litters following oral gavage with 10, 50, or 250 mg/kg DBA (Linder et al., 1995). Five- and seven-week-old rats dosed with 5, 50, or 250 mg/kg DBA for two to four weeks were found to have retention of step 19 spermatids (Tsuchiya et al., 2000). Sperm membrane protein SP22 (a protein highly correlated with fertility) was significantly reduced in adult rats exposed to 10 mg/kg/day for 14 days (Kaydos et al., 2004). Changes in estrous cyclicity and alterations of steroid production by preovulatory follicles in vitro were found after dosing adult female rats with 90 and 270 mg/kg/day for 14 days (Balchak et al., 2000; Goldman and Murr, 2002).
Longer term, lower level exposure studies have also linked DBA with reproductive toxicities in rat models. Exposure to 400 ppm/day (76.3 mg/kg/day) DBA from gestation day 15 through adulthood in rats caused delays in preputial separation and vaginal opening as well as decreased fertility of cauda epididymal sperm and alterations in levels of sperm membrane protein SP22 (Klinefelter et al., 2004). In a multi-generational study involving rats, exposure to DBA altered sperm production and caused epididymal tubule changes at 250 and 650 ppm/day (22.4–55.6 and 52.4–132.0 mg/kg/day respectively) (Christian et al., 2002).
DBA studies involving rabbits have focused on chronic exposures from mid-gestation through adulthood. Male rabbits dosed from gestation day 15 through adulthood with 1, 5, or 50 mg/kg DBA via drinking water caused decreased sexual interest, increased ejaculatory failure, and decreased conception rates (Veeramachaneni et al., 2000). Female rabbits similarly treated were found to have decreased primordial follicle populations at 5 and 50 mg/kg/day DBA in animals examined at prepuberty and adulthood (Bodensteiner et al., 2004).
It is difficult to compare the effects of DBA on rabbits and rodents due to the observed differences in effects at similar doses. Toxicities are similar in both species but the rabbit seems to be more sensitive and adverse reproductive effects occur at lower doses. There is a paucity of data regarding the reproductive effects of DBA in species other than rats and rabbits. The purpose of this study was to determine the effects of sub-chronic gestational through pubertal exposure of DBA using a murine model.
MATERIALS AND METHODS
Mouse husbandry.
Six- to eight-week-old C57Bl/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and allowed to acclimate for two weeks. Mice were housed in plastic tubs with cedar chip bedding and kept under a 12:12 h light:dark cycle. Females were housed 4/tub and males were housed individually. Mice were fed Teklad 8640 mouse chow (Harlan, Madison, WI) and deionized water ad libitum. Males were placed with females at 6:00 P.M. and removed at 6:00 A.M. the following morning. Presence of a vaginal plug was designated day 0 of gestation. Pregnant females were separated and housed individually. Males and un-mated females were given a day of rest between successive breedings.
Chemicals and dosing.
DBA (CAS#631-64-1, lot#03807JS, purity 97%) was obtained from Aldrich (Milwaukee, WI). Dosing solutions were prepared by adding DBA to deionized water and bringing the pH to 6.8–7.4 using 1N NaOH. Stock solutions were made twice weekly.
Three groups (n = 10) of pregnant mice were dosed via drinking water with 0 (deionized water control), 5, or 50 mg/kg/day DBA. Dosing of dams began on gestation day 15 and continued through parturition and weaning of pups at three weeks postpartum. At parturition, litters were examined for any terata and the number of pups recorded. At weaning, 15–17 pups of each gender/treatment group were housed individually and continued to receive their respective treatments until necropsy at 7 weeks (neo-puberty). The remaining pups, 7–10 of each gender/treatment group, were used for evaluation at 3 weeks (pre-puberty). DBA concentration in dosing solutions was adjusted weekly according to animal weights and average water consumption. Water changes were made with fresh dosing solution twice weekly.
Pre-pubertal (3-week-old) evaluation.
Animals were euthanized with CO2-asphyxiation. Body weight, length, and ano-genital distance (AGD) were measured. Viscera and reproductive organs were examined for any abnormalities. Weights were taken for liver, kidneys, testes, and ovaries. Right testes and ovaries were fixed in 4% glutaraldehyde for 24 h then stored in 2% cacodylate while left testes and ovaries were fixed in Bouin's solution for 48 h then stored in 70% alcohol until further processing for histopathology.
Neo-pubertal (7-week-old) evaluation.
Procedures for necropsy and tissue collection were similar to those performed at 3 weeks except that for a group of 10–11 males/treatment, half of the right testis was fixed in Bouin's solution and the remaining half and the epididymis were fixed in glutaraldehyde. The left testis and epididymis were weighed separately and frozen in liquid nitrogen for determination of daily sperm production and epididymal sperm reserves. Frozen tissues were maintained at –80°C until processing for determination of sperm counts.
Tissue processing.
Testes of neo-pubertal males fixed in glutaraldehyde were embedded in plastic and sectioned for light microscopy (Veeramachaneni et al., 1986). Briefly, testes were washed in 0.1 M cacodylate then placed in a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 90 min. Tissues were then washed in cacodylate and dehydrated through a graded series of ethanol (15 min each in 50%, 70%, 95% x 3, and 100% x 3), rinsed in propylene oxide (15 min x 3), and embedded in Poly/Bed 812 (Polysciences Inc. Warrington, PA)(50:50 propylene oxide:Poly/Bed on turn table at room temperature overnight; 100% Poly/Bed in vacuum for 8 h; 100% Poly/Bed in vacuum for 16 h; 45°C oven for 8 h; 60°C oven for 16 h). One-μm-thick sections were cut and mounted on glass slides for light microscopy. Slides were placed on a heating block and stained with 0.5% toluidine blue in 1% borate for 30 s. Ovaries from neo-pubertal animals fixed in Bouin's solution were embedded in paraffin, serially sectioned at 6 μm and stained with hematoxylin and eosin. To ensure that tissue orientation was maintained during paraffin embedding ovaries were first embedded in Histogel (Richard Allen Scientific, Kalamazoo, MI).
Morphometry and histopathological evaluation.
Evaluations were performed in a treatment-blinded manner. Normalcy of seminiferous epithelium was assessed using criteria established for evaluation of bull (Veeramachaneni et al., 1986) and rabbit (Higuchi et al., 2003) testes. A testis section from each mouse was examined and all seminiferous tubule profiles were graded as follows: grade 0 (normal seminiferous epithelium); grade 1 (seminiferous epithelium with pyknosis and desquamation of spermatids and/or focal vacuolation); grade 2 (seminiferous epithelium with intermediate changes between grades 1 and 3); grade 3 (seminiferous epithelium with mostly spermatogonia and Sertoli cells); grade 4 (epithelium with mostly vacuolated Sertoli cells); grade 4a (hypoplastic tubules with Sertoli cells only and without any vacuolation); grade 5 (no epithelium, leaving only the basement membrane); grade 6 (tubules with sperm stasis, sperm granuloma, or mineralization); and grade 7 (tubules with only fibrous tissue elements). Whereas grades 1 to 3 reflect increasing severity of loss of differentiating germ cells, grades 4 to 7 reflect complete loss of germ cells including stem spermatogonia. A weight between 0 and 1 was assigned to each grade—0, 1/4, 1/2, and 3/4 for grades 0, 1, 2, and 3 respectively and 4/4 to grades 4 through 7—to indicate relative degree of germinal epithelial loss (DGEL). A percentage of seminiferous tubules classified into 1 of the 9 grades was calculated from the total number of tubules counted for each mouse. The percentage of tubules in each grade was multiplied by the respective assigned weight, and the products were summed. The resulting sum estimated the DGEL per 100 tubule cross sections for each animal. Tissue sections were also examined for atypical residual bodies and retained step 16 spermatids as outlined by Linder and colleagues (1994a,b, 1995, 1997).
Ovarian function was estimated using quantification of follicular development. Briefly, beginning with an ovarian section that contained a follicle, all sections were counted at a consecutive interval of 66 μm. Area of each ovarian section that was evaluated was determined using ImagePro Plus version 3.0 (Media Cybernetics). Due to variation in ovary size and thus sections counted per animal, follicle counts are expressed as numbers per mm2 of ovary. A classification system described by Pedersen and Peters (1968) as modified by Bodensteiner and associates (2004) was used to categorize ovarian follicles. The criteria were based on number and state of maturity of granulosa cell layers and included the following categories: primordial (1 layer of squamous granulosa cells), primary (1–<2 layers of cuboidal granulosa cells), small preantral (2–3 layers of cuboidal granulosa cells), large preantral (4–6 layers of cuboidal granulosa cells), and small antral (>6 layers of granulosa cells with the presence of an antral cavity) follicles.
Daily sperm production and epididymal sperm reserves.
Daily sperm production (DSP) and epididymal sperm reserve were determined using the procedure of Blazak and associates (1993) with slight modification. Briefly, frozen testes and epididymides were placed in 4 ml homogenization solution (10 ml 5% Triton-x-100, 9.0 g NaCl) and homogenized using a Polytron PT 10/35 (Brinkman Instruments, Westbury, NY) at setting 7 for 10 s. Two hemocytometers per sample were loaded with homogenate and homogenization-resistant sperm heads were counted using a phase contrast microscope with a 40x objective lens. Numbers of sperm for testis and epididymis were expressed as numbers of sperm per tissue. A time divisor of 4.84 days was used to calculate DSP. The efficiency of sperm production was calculated by expressing DSP on a testicular parenchyma basis.
Statistical analysis.
Statview (version 5.0, SAS Institute Inc., Cary, NC) was used for all statistical analyses. All parameters were analyzed using ANOVA with a Tukey/Kramer post-hoc test. Body weight was used as a covariate for weights of liver, kidney, testis, and ovary. Body length was used as a covariate for ano-genital distance. A level of significance of p 0.05 was used for all tests.
RESULTS
Due to variations in water consumption, delivered dose differed from the intended dose. The intended doses were: control = 0 mg/kg/day, low dose = 5 mg/kg/day, and high dose = 50 mg/kg/day. The average delivered doses for dams from gestation day 15 through weaning were: control = 0 mg/kg/day, low dose = 6.61 mg/kg/day, high dose = 65.86 mg/kg/day. Delivered doses for pups after weaning were: control = 0 mg/kg/day, low dose = 5.01 mg/kg/day, high dose = 46.80 mg/kg/day.
Gestation length did not vary between treatment groups and ranged 18–20 days in each of the three groups. Litter size ranged 6–11 in controls, 5–9 in low dose group, and 2–10 in high dose group. Mean litter size in DBA treatment groups was significantly lower (p < 0.05) than in controls (mean ± SEM; low-dose: 6.9 ± 0.31, high-dose: 6.6 ± 0.61 vs. control: 9.0 ± 0.45). No terata were found in any treatment group.
At the pre-pubertal evaluation control and dosed male mice had similar body measurements (body weights and lengths). There was a significant increase in testis and liver weights in the highest dose group (Table 1). High dose female mice at the same age also had increased liver and kidney weights. All other parameters measured were similar between dosed and control females (Table 2). At seven weeks of age, males in the high dose group had decreased testis and kidney weights. Remaining parameters were similar between control and dosed males (Table 1). In 7-week-old females, no differences were observed in any parameters measured between control and dosed animals (Table 2).
No significant difference was noted between control and dosed males in sperm, seminiferous epithelial and endocrine parameters (Tables 3 and 4). There was no difference between control and dosed mice in testis sperm counts or DSP. DGEL did not differ significantly between control and dosed animals. In two animals of the high dose group, incidence of grades 2 and 4 tubules was noted, resulting in DGEL scores of 15.63 and 7.04. Degenerative changes in seminiferous epithelium beyond grade 4 were not noted in any animal. Retention of step 16 spermatids was not seen in any dose group indicating that spermiation was not affected.
Control and dosed females had similar numbers of primordial, primary, small preantral, large preantral, and small antral follicles. One high-dose female had a noticeably elevated number (2–3x that of the group average) of every type from primordial through large preantral follicles and was not included in statistical analyses (Table 5).
DISCUSSION
Mice were dosed from gestation day 15 through postpartum at 3 weeks (weaning) or 7 weeks (puberty). This time period is relevant in that it encompasses final development and functional differentiation of the reproductive tract. A chemical toxic to the developing reproductive tract would be detected in this dosing regimen. Few adverse effects were noted at the dose levels tested in this study. The only significantly altered reproductive endpoints were decreased litter sizes in DBA-treated dams and decreased testes weight in high dose neo-pubertal males.
Although the focus of this study was on reproductive effects of DBA, some differences were noted between non-gonadal tissues of control and dosed animals. At 3 weeks of age both male and female mice had increased liver weights. However, there was no difference in liver weights between dosed and control animals at 7 weeks of age. DBA has been reported to cause liver damage in mice (Parrish et al., 1996). Kidneys of pre-pubertal high dose females and neo-pubertal high dose males were significantly heavier than those of control animals. Dosing with dichloroacetic acid (DCA) is known to cause kidney enlargement in rats (Smith et al., 1992); oxalic acid, a metabolite of DBA and DCA, is a known renal toxin. Although 3-week-old females and 7-week-old males in this study could have suffered kidney damage, kidneys were not enlarged in 3-week-old males or 7-week-old females. Since histological evaluation of non-reproductive organs was not performed (as the main focus of this study was on gametogenic potential), it was not possible to determine the nature of possible toxicity to liver and kidney.
Testes of high dose pre-pubertal males were significantly larger than control males of the same age, but at 7 weeks of age high dose males had significantly smaller testes than control males. It is possible that an initial insult of DBA to the testes of pre-pubertal mice caused increased fluid secretion and/or impaired resorption in excurrent ducts leading to increased testis weight. Conversely, decreased testicular weights of the neo-pubertal mice could be indicative of a direct detrimental effect of DBA on the seminiferous epithelium as indicated by presence of grade 3 tubules in two high-dose males or an indirect progressive damage resulting from possible fluid retention earlier.
Several studies have shown that DBA causes decreased testis size in adult rats (Linder et al., 1994a,b; Tsuchiya et al., 2000); the doses used in these studies were considerably higher (250–1250 mg/kg/day) than those used in the present study. However, the 70-day-long studies by Linder et al. (1995, 1997) used doses ranging from 2 mg/kg/day to 250 mg/kg/day. In these studies, delayed spermiation was observed at 10, 50, and 250 mg/kg dose levels after 30 days of exposure. In the present study, delayed spermiation was not observed at 50 mg/kg/day suggesting that the mouse is less sensitive than rat. Exposure of male rabbits to DBA at a dose regimen similar to that used in the present study led to impaired sexual function and fertility (Veeramachaneni et al., 2000). Sexual behavior was altered in rabbits at doses as low as 5 mg/kg/day. When fertility of the treated male rabbits was tested by inseminating a known number of sperm into untreated females, doses as low as 1 mg/kg/day caused decreased fertility indicating that the quality of sperm was compromised. Since fertility and sexual behavior were not examined in the present study it is difficult to compare rabbit and mouse data, but dosed mice did not have reduced DSP, testicular sperm counts, or epididymal sperm reserves. Whether sperm quality remained unaffected was not determined.
With regard to folliculogenesis, no differences in follicle populations were found between dosed and control female mice. Bodensteiner et al. (2004) found that rabbits treated with similar doses of DBA for 26 weeks, beginning from mid-gestation through adulthood, had significantly fewer primordial follicles than controls. However, it is not clear whether the reduction in primordial follicle population in rabbits was due to a detrimental, stage-specific effect of DBA on primordial follicles, or because of enhanced recruitment of primordial follicles into subsequent growth-committed stages that in turn would lead to faster depletion of the pool of primordial follicles.
When a comparable dosing regimen (exposure in utero from gestation day 15 through puberty) as used in the current mouse study was used for rabbits (Bodensteiner et al., 2004), DBA-dosed rabbits had significant differences in ovarian follicle populations. This is in contrast to results obtained for mice in the present study, i.e., DBA-dosed mice had follicle numbers similar to control animals. It is possible that due to differences in the timing of specific events in folliculogenesis between mice and rabbits, a critical time period was missed in the present study. For example, entry of oocytes into meiotic prophase occurs on day 13 of gestation in mice but 2 days after birth in rabbits (Peters, 1978). If the mode of action of DBA is disruption of the initial phase of meiotic division then a dosing regimen beginning on gestation day 15 would disrupt folliculogenesis in rabbits but not in mice. Thus, an event in folliculogenesis that is vulnerable to DBA may occur after gestation day 15 in rabbits but not in mice.
Another explanation for the relative insensitivity of mice with respect to gametogenic potential may be related to the duration of exposure to DBA (e.g., 8 weeks in mice vs. 26 weeks in rabbits; Bodensteiner et al., 2004). Furthermore, animals with a short period of reproductive development relative to life span, such as mice, may exhibit less toxicity than animals with long periods of reproductive development, such as rabbits. Thus, mice would probably display less reproductive damage than rabbits when dosed under the same conditions.
Although the competency of gametes produced in these mice is unknown, DBA does not appear to be detrimental to the gametogenic potential of mice at the dose levels tested. However, since rabbits dosed under similar conditions did exhibit reproductive toxicity (e.g., diminished number of primordial follicles), the use of mice as an animal model may not be ideal to test chronic, long-term reproductive toxicity of chemicals such as DBA. Furthermore, it should be recognized that some laboratory animal models may be refractory to haloacetic acids as exemplified by Xenopus laevis in which developmental exposure to DBA at doses as high as 16000 ppm did not produce any detrimental effect (Weber et al., 2004). Thus for reproductive toxicology studies, it is important to choose an appropriate animal model and a relevant exposure paradigm so as to reflect the biological events and exposure possibilities in humans. As history has shown with such chemicals as thalidomide, testing of chemicals in several different species is imperative in determining different toxic sequelae and possible links to effects in humans.
ACKNOWLEDGMENTS
We thank Carol Moeller and Jennifer Palmer for technical assistance.
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Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1683
ABSTRACT
Water disinfection by-products, such as dibromoacetic acid (DBA), are formed when drinking water is treated with chlorination, bromination, or ozonation. Epidemiological studies have linked these byproducts to adverse effects in humans such as cancer, developmental defects, and reproductive toxicities. DBA has been shown to produce reproductive toxicity in rodents at relatively high doses. The present study used a mouse model to determine the developmental and reproductive effects of sub-chronic, low-dose exposure to DBA. Pregnant mice (10/dose group) were exposed with DBA in drinking water at 0, 5, or 50 mg/kg/day from gestation day 15 though nursing. Upon weaning at 3 weeks, one group of pups (pre-pubertal group: 7–10 pups of each gender/treatment group) were euthanized and weights of liver, paired kidneys, testes, and ovaries were measured. In the 50 mg dose group, weights of testes and liver in males and weights of liver and kidneys in females were significantly higher (p < 0.05). The remaining pups (15–17 of each gender/dose group) continued to be dosed similarly through adulthood. At 7 weeks of age (neo-pubertal group), animals were euthanized and tissues weighed and processed for evaluation of reproductive organs and gametogenic potential. Except for decreased (p < 0.05) testes and kidney weights in 50 mg dose group males, there were no differences in organ weights. No significant differences were noted between control and dosed animals in daily sperm production, testicular sperm counts, epididymal sperm reserves, morphology of seminiferous epithelium, or ovarian follicle counts.
Key Words: dibromoacetic acid; ovary; follicles; testis; sperm; mouse.
INTRODUCTION
Chlorination, bromination, and ozonation are commonly used to disinfect drinking water. These types of water treatments have proven invaluable in decreasing the spread of water-borne illness (Faber, 1952; Mughal, 1992). However, several water disinfection by-products are formed when chlorine and bromine react with organic material in source waters (Bellar et al., 1974; Rook, 1974). Epidemiological studies have linked exposure to these by-products with cancer (Boorman et al., 1999; Morris, 1995; Nieuwenhuijsen et al., 2000) as well as developmental and reproductive toxicities (Graves et al., 2001; Nieuwenhuijsen et al., 2000; Reif et al., 1996). Haloacetic acids, including dibromoacetic acid (DBA), are one of the most prevalent classes of disinfection by-products found in treated drinking water and are thus possibly a major concern for public safety (Krasner et al., 1989; Richardson, 2002; Richardson and Thurston, 2003; Uden and Miller, 1983). To date, there are few epidemiological studies specifically involving haloacetic acids (Hinckley et al., 2005) but toxicological studies show that these chemicals have adverse effects in laboratory animals.
Several rodent studies involving DBA have focused on the effects of acute exposure to juvenile and adult rats. Testicular toxicity with adverse effects on sperm motility and morphological features following acute exposure to DBA (10 to 1250 mg/kg/day via oral gavage for 1 to 14 days) has been demonstrated in adult male rats (Linder et al., 1994a,b, 1997). There is a dose-related decrease in ability of adult male rats to sire multiple litters following oral gavage with 10, 50, or 250 mg/kg DBA (Linder et al., 1995). Five- and seven-week-old rats dosed with 5, 50, or 250 mg/kg DBA for two to four weeks were found to have retention of step 19 spermatids (Tsuchiya et al., 2000). Sperm membrane protein SP22 (a protein highly correlated with fertility) was significantly reduced in adult rats exposed to 10 mg/kg/day for 14 days (Kaydos et al., 2004). Changes in estrous cyclicity and alterations of steroid production by preovulatory follicles in vitro were found after dosing adult female rats with 90 and 270 mg/kg/day for 14 days (Balchak et al., 2000; Goldman and Murr, 2002).
Longer term, lower level exposure studies have also linked DBA with reproductive toxicities in rat models. Exposure to 400 ppm/day (76.3 mg/kg/day) DBA from gestation day 15 through adulthood in rats caused delays in preputial separation and vaginal opening as well as decreased fertility of cauda epididymal sperm and alterations in levels of sperm membrane protein SP22 (Klinefelter et al., 2004). In a multi-generational study involving rats, exposure to DBA altered sperm production and caused epididymal tubule changes at 250 and 650 ppm/day (22.4–55.6 and 52.4–132.0 mg/kg/day respectively) (Christian et al., 2002).
DBA studies involving rabbits have focused on chronic exposures from mid-gestation through adulthood. Male rabbits dosed from gestation day 15 through adulthood with 1, 5, or 50 mg/kg DBA via drinking water caused decreased sexual interest, increased ejaculatory failure, and decreased conception rates (Veeramachaneni et al., 2000). Female rabbits similarly treated were found to have decreased primordial follicle populations at 5 and 50 mg/kg/day DBA in animals examined at prepuberty and adulthood (Bodensteiner et al., 2004).
It is difficult to compare the effects of DBA on rabbits and rodents due to the observed differences in effects at similar doses. Toxicities are similar in both species but the rabbit seems to be more sensitive and adverse reproductive effects occur at lower doses. There is a paucity of data regarding the reproductive effects of DBA in species other than rats and rabbits. The purpose of this study was to determine the effects of sub-chronic gestational through pubertal exposure of DBA using a murine model.
MATERIALS AND METHODS
Mouse husbandry.
Six- to eight-week-old C57Bl/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and allowed to acclimate for two weeks. Mice were housed in plastic tubs with cedar chip bedding and kept under a 12:12 h light:dark cycle. Females were housed 4/tub and males were housed individually. Mice were fed Teklad 8640 mouse chow (Harlan, Madison, WI) and deionized water ad libitum. Males were placed with females at 6:00 P.M. and removed at 6:00 A.M. the following morning. Presence of a vaginal plug was designated day 0 of gestation. Pregnant females were separated and housed individually. Males and un-mated females were given a day of rest between successive breedings.
Chemicals and dosing.
DBA (CAS#631-64-1, lot#03807JS, purity 97%) was obtained from Aldrich (Milwaukee, WI). Dosing solutions were prepared by adding DBA to deionized water and bringing the pH to 6.8–7.4 using 1N NaOH. Stock solutions were made twice weekly.
Three groups (n = 10) of pregnant mice were dosed via drinking water with 0 (deionized water control), 5, or 50 mg/kg/day DBA. Dosing of dams began on gestation day 15 and continued through parturition and weaning of pups at three weeks postpartum. At parturition, litters were examined for any terata and the number of pups recorded. At weaning, 15–17 pups of each gender/treatment group were housed individually and continued to receive their respective treatments until necropsy at 7 weeks (neo-puberty). The remaining pups, 7–10 of each gender/treatment group, were used for evaluation at 3 weeks (pre-puberty). DBA concentration in dosing solutions was adjusted weekly according to animal weights and average water consumption. Water changes were made with fresh dosing solution twice weekly.
Pre-pubertal (3-week-old) evaluation.
Animals were euthanized with CO2-asphyxiation. Body weight, length, and ano-genital distance (AGD) were measured. Viscera and reproductive organs were examined for any abnormalities. Weights were taken for liver, kidneys, testes, and ovaries. Right testes and ovaries were fixed in 4% glutaraldehyde for 24 h then stored in 2% cacodylate while left testes and ovaries were fixed in Bouin's solution for 48 h then stored in 70% alcohol until further processing for histopathology.
Neo-pubertal (7-week-old) evaluation.
Procedures for necropsy and tissue collection were similar to those performed at 3 weeks except that for a group of 10–11 males/treatment, half of the right testis was fixed in Bouin's solution and the remaining half and the epididymis were fixed in glutaraldehyde. The left testis and epididymis were weighed separately and frozen in liquid nitrogen for determination of daily sperm production and epididymal sperm reserves. Frozen tissues were maintained at –80°C until processing for determination of sperm counts.
Tissue processing.
Testes of neo-pubertal males fixed in glutaraldehyde were embedded in plastic and sectioned for light microscopy (Veeramachaneni et al., 1986). Briefly, testes were washed in 0.1 M cacodylate then placed in a solution of 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) for 90 min. Tissues were then washed in cacodylate and dehydrated through a graded series of ethanol (15 min each in 50%, 70%, 95% x 3, and 100% x 3), rinsed in propylene oxide (15 min x 3), and embedded in Poly/Bed 812 (Polysciences Inc. Warrington, PA)(50:50 propylene oxide:Poly/Bed on turn table at room temperature overnight; 100% Poly/Bed in vacuum for 8 h; 100% Poly/Bed in vacuum for 16 h; 45°C oven for 8 h; 60°C oven for 16 h). One-μm-thick sections were cut and mounted on glass slides for light microscopy. Slides were placed on a heating block and stained with 0.5% toluidine blue in 1% borate for 30 s. Ovaries from neo-pubertal animals fixed in Bouin's solution were embedded in paraffin, serially sectioned at 6 μm and stained with hematoxylin and eosin. To ensure that tissue orientation was maintained during paraffin embedding ovaries were first embedded in Histogel (Richard Allen Scientific, Kalamazoo, MI).
Morphometry and histopathological evaluation.
Evaluations were performed in a treatment-blinded manner. Normalcy of seminiferous epithelium was assessed using criteria established for evaluation of bull (Veeramachaneni et al., 1986) and rabbit (Higuchi et al., 2003) testes. A testis section from each mouse was examined and all seminiferous tubule profiles were graded as follows: grade 0 (normal seminiferous epithelium); grade 1 (seminiferous epithelium with pyknosis and desquamation of spermatids and/or focal vacuolation); grade 2 (seminiferous epithelium with intermediate changes between grades 1 and 3); grade 3 (seminiferous epithelium with mostly spermatogonia and Sertoli cells); grade 4 (epithelium with mostly vacuolated Sertoli cells); grade 4a (hypoplastic tubules with Sertoli cells only and without any vacuolation); grade 5 (no epithelium, leaving only the basement membrane); grade 6 (tubules with sperm stasis, sperm granuloma, or mineralization); and grade 7 (tubules with only fibrous tissue elements). Whereas grades 1 to 3 reflect increasing severity of loss of differentiating germ cells, grades 4 to 7 reflect complete loss of germ cells including stem spermatogonia. A weight between 0 and 1 was assigned to each grade—0, 1/4, 1/2, and 3/4 for grades 0, 1, 2, and 3 respectively and 4/4 to grades 4 through 7—to indicate relative degree of germinal epithelial loss (DGEL). A percentage of seminiferous tubules classified into 1 of the 9 grades was calculated from the total number of tubules counted for each mouse. The percentage of tubules in each grade was multiplied by the respective assigned weight, and the products were summed. The resulting sum estimated the DGEL per 100 tubule cross sections for each animal. Tissue sections were also examined for atypical residual bodies and retained step 16 spermatids as outlined by Linder and colleagues (1994a,b, 1995, 1997).
Ovarian function was estimated using quantification of follicular development. Briefly, beginning with an ovarian section that contained a follicle, all sections were counted at a consecutive interval of 66 μm. Area of each ovarian section that was evaluated was determined using ImagePro Plus version 3.0 (Media Cybernetics). Due to variation in ovary size and thus sections counted per animal, follicle counts are expressed as numbers per mm2 of ovary. A classification system described by Pedersen and Peters (1968) as modified by Bodensteiner and associates (2004) was used to categorize ovarian follicles. The criteria were based on number and state of maturity of granulosa cell layers and included the following categories: primordial (1 layer of squamous granulosa cells), primary (1–<2 layers of cuboidal granulosa cells), small preantral (2–3 layers of cuboidal granulosa cells), large preantral (4–6 layers of cuboidal granulosa cells), and small antral (>6 layers of granulosa cells with the presence of an antral cavity) follicles.
Daily sperm production and epididymal sperm reserves.
Daily sperm production (DSP) and epididymal sperm reserve were determined using the procedure of Blazak and associates (1993) with slight modification. Briefly, frozen testes and epididymides were placed in 4 ml homogenization solution (10 ml 5% Triton-x-100, 9.0 g NaCl) and homogenized using a Polytron PT 10/35 (Brinkman Instruments, Westbury, NY) at setting 7 for 10 s. Two hemocytometers per sample were loaded with homogenate and homogenization-resistant sperm heads were counted using a phase contrast microscope with a 40x objective lens. Numbers of sperm for testis and epididymis were expressed as numbers of sperm per tissue. A time divisor of 4.84 days was used to calculate DSP. The efficiency of sperm production was calculated by expressing DSP on a testicular parenchyma basis.
Statistical analysis.
Statview (version 5.0, SAS Institute Inc., Cary, NC) was used for all statistical analyses. All parameters were analyzed using ANOVA with a Tukey/Kramer post-hoc test. Body weight was used as a covariate for weights of liver, kidney, testis, and ovary. Body length was used as a covariate for ano-genital distance. A level of significance of p 0.05 was used for all tests.
RESULTS
Due to variations in water consumption, delivered dose differed from the intended dose. The intended doses were: control = 0 mg/kg/day, low dose = 5 mg/kg/day, and high dose = 50 mg/kg/day. The average delivered doses for dams from gestation day 15 through weaning were: control = 0 mg/kg/day, low dose = 6.61 mg/kg/day, high dose = 65.86 mg/kg/day. Delivered doses for pups after weaning were: control = 0 mg/kg/day, low dose = 5.01 mg/kg/day, high dose = 46.80 mg/kg/day.
Gestation length did not vary between treatment groups and ranged 18–20 days in each of the three groups. Litter size ranged 6–11 in controls, 5–9 in low dose group, and 2–10 in high dose group. Mean litter size in DBA treatment groups was significantly lower (p < 0.05) than in controls (mean ± SEM; low-dose: 6.9 ± 0.31, high-dose: 6.6 ± 0.61 vs. control: 9.0 ± 0.45). No terata were found in any treatment group.
At the pre-pubertal evaluation control and dosed male mice had similar body measurements (body weights and lengths). There was a significant increase in testis and liver weights in the highest dose group (Table 1). High dose female mice at the same age also had increased liver and kidney weights. All other parameters measured were similar between dosed and control females (Table 2). At seven weeks of age, males in the high dose group had decreased testis and kidney weights. Remaining parameters were similar between control and dosed males (Table 1). In 7-week-old females, no differences were observed in any parameters measured between control and dosed animals (Table 2).
No significant difference was noted between control and dosed males in sperm, seminiferous epithelial and endocrine parameters (Tables 3 and 4). There was no difference between control and dosed mice in testis sperm counts or DSP. DGEL did not differ significantly between control and dosed animals. In two animals of the high dose group, incidence of grades 2 and 4 tubules was noted, resulting in DGEL scores of 15.63 and 7.04. Degenerative changes in seminiferous epithelium beyond grade 4 were not noted in any animal. Retention of step 16 spermatids was not seen in any dose group indicating that spermiation was not affected.
Control and dosed females had similar numbers of primordial, primary, small preantral, large preantral, and small antral follicles. One high-dose female had a noticeably elevated number (2–3x that of the group average) of every type from primordial through large preantral follicles and was not included in statistical analyses (Table 5).
DISCUSSION
Mice were dosed from gestation day 15 through postpartum at 3 weeks (weaning) or 7 weeks (puberty). This time period is relevant in that it encompasses final development and functional differentiation of the reproductive tract. A chemical toxic to the developing reproductive tract would be detected in this dosing regimen. Few adverse effects were noted at the dose levels tested in this study. The only significantly altered reproductive endpoints were decreased litter sizes in DBA-treated dams and decreased testes weight in high dose neo-pubertal males.
Although the focus of this study was on reproductive effects of DBA, some differences were noted between non-gonadal tissues of control and dosed animals. At 3 weeks of age both male and female mice had increased liver weights. However, there was no difference in liver weights between dosed and control animals at 7 weeks of age. DBA has been reported to cause liver damage in mice (Parrish et al., 1996). Kidneys of pre-pubertal high dose females and neo-pubertal high dose males were significantly heavier than those of control animals. Dosing with dichloroacetic acid (DCA) is known to cause kidney enlargement in rats (Smith et al., 1992); oxalic acid, a metabolite of DBA and DCA, is a known renal toxin. Although 3-week-old females and 7-week-old males in this study could have suffered kidney damage, kidneys were not enlarged in 3-week-old males or 7-week-old females. Since histological evaluation of non-reproductive organs was not performed (as the main focus of this study was on gametogenic potential), it was not possible to determine the nature of possible toxicity to liver and kidney.
Testes of high dose pre-pubertal males were significantly larger than control males of the same age, but at 7 weeks of age high dose males had significantly smaller testes than control males. It is possible that an initial insult of DBA to the testes of pre-pubertal mice caused increased fluid secretion and/or impaired resorption in excurrent ducts leading to increased testis weight. Conversely, decreased testicular weights of the neo-pubertal mice could be indicative of a direct detrimental effect of DBA on the seminiferous epithelium as indicated by presence of grade 3 tubules in two high-dose males or an indirect progressive damage resulting from possible fluid retention earlier.
Several studies have shown that DBA causes decreased testis size in adult rats (Linder et al., 1994a,b; Tsuchiya et al., 2000); the doses used in these studies were considerably higher (250–1250 mg/kg/day) than those used in the present study. However, the 70-day-long studies by Linder et al. (1995, 1997) used doses ranging from 2 mg/kg/day to 250 mg/kg/day. In these studies, delayed spermiation was observed at 10, 50, and 250 mg/kg dose levels after 30 days of exposure. In the present study, delayed spermiation was not observed at 50 mg/kg/day suggesting that the mouse is less sensitive than rat. Exposure of male rabbits to DBA at a dose regimen similar to that used in the present study led to impaired sexual function and fertility (Veeramachaneni et al., 2000). Sexual behavior was altered in rabbits at doses as low as 5 mg/kg/day. When fertility of the treated male rabbits was tested by inseminating a known number of sperm into untreated females, doses as low as 1 mg/kg/day caused decreased fertility indicating that the quality of sperm was compromised. Since fertility and sexual behavior were not examined in the present study it is difficult to compare rabbit and mouse data, but dosed mice did not have reduced DSP, testicular sperm counts, or epididymal sperm reserves. Whether sperm quality remained unaffected was not determined.
With regard to folliculogenesis, no differences in follicle populations were found between dosed and control female mice. Bodensteiner et al. (2004) found that rabbits treated with similar doses of DBA for 26 weeks, beginning from mid-gestation through adulthood, had significantly fewer primordial follicles than controls. However, it is not clear whether the reduction in primordial follicle population in rabbits was due to a detrimental, stage-specific effect of DBA on primordial follicles, or because of enhanced recruitment of primordial follicles into subsequent growth-committed stages that in turn would lead to faster depletion of the pool of primordial follicles.
When a comparable dosing regimen (exposure in utero from gestation day 15 through puberty) as used in the current mouse study was used for rabbits (Bodensteiner et al., 2004), DBA-dosed rabbits had significant differences in ovarian follicle populations. This is in contrast to results obtained for mice in the present study, i.e., DBA-dosed mice had follicle numbers similar to control animals. It is possible that due to differences in the timing of specific events in folliculogenesis between mice and rabbits, a critical time period was missed in the present study. For example, entry of oocytes into meiotic prophase occurs on day 13 of gestation in mice but 2 days after birth in rabbits (Peters, 1978). If the mode of action of DBA is disruption of the initial phase of meiotic division then a dosing regimen beginning on gestation day 15 would disrupt folliculogenesis in rabbits but not in mice. Thus, an event in folliculogenesis that is vulnerable to DBA may occur after gestation day 15 in rabbits but not in mice.
Another explanation for the relative insensitivity of mice with respect to gametogenic potential may be related to the duration of exposure to DBA (e.g., 8 weeks in mice vs. 26 weeks in rabbits; Bodensteiner et al., 2004). Furthermore, animals with a short period of reproductive development relative to life span, such as mice, may exhibit less toxicity than animals with long periods of reproductive development, such as rabbits. Thus, mice would probably display less reproductive damage than rabbits when dosed under the same conditions.
Although the competency of gametes produced in these mice is unknown, DBA does not appear to be detrimental to the gametogenic potential of mice at the dose levels tested. However, since rabbits dosed under similar conditions did exhibit reproductive toxicity (e.g., diminished number of primordial follicles), the use of mice as an animal model may not be ideal to test chronic, long-term reproductive toxicity of chemicals such as DBA. Furthermore, it should be recognized that some laboratory animal models may be refractory to haloacetic acids as exemplified by Xenopus laevis in which developmental exposure to DBA at doses as high as 16000 ppm did not produce any detrimental effect (Weber et al., 2004). Thus for reproductive toxicology studies, it is important to choose an appropriate animal model and a relevant exposure paradigm so as to reflect the biological events and exposure possibilities in humans. As history has shown with such chemicals as thalidomide, testing of chemicals in several different species is imperative in determining different toxic sequelae and possible links to effects in humans.
ACKNOWLEDGMENTS
We thank Carol Moeller and Jennifer Palmer for technical assistance.
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