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Toxicology |
Division of Pharmacology, Drug Safety and Metabolism,2
Department of Research and Development,3 Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan
Study Department,4 Nihon Bioresearch, Inc., Fukuju-cho, Hashima, Gifu 501-6251, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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embryo, early development, ovary, ovum, toxicology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have investigated the toxicity of NAC. For example, Bonanomi and Gazzaniga [4] reported the median lethal doses obtained in mice or rats by oral or i.v. administration. They also administered NAC to rats and dogs and reported that NAC did not affect hematological parameters, hepatic or renal function, prothrombin or bleeding time, or findings at necropsy or histopathology. Johnston et al. [5] also described the acute, subacute, and chronic toxicity of NAC administered to various animals by various routes and concluded that NAC was a safe compound.
With regard to the reproductive and developmental toxicity of orally administered NAC, Bonanomi and Gazzaniga [4] reported that no effects were observed in a male rat fertility study at daily doses of 250–1000 mg/kg, in a rat teratogenicity study at daily doses of 500–2000 mg/kg, in a rabbit teratogenicity study at daily doses of 250–1000 mg/kg, and in a rat peri- and postnatal study at daily doses of 250–1000 mg/kg. Johnston et al. [5] also reported that oral administration at a dose of 500 mg kg-1 day-1 from Gestational Day (GD) 6 to GD16 showed no teratogenic effects in the rabbit. Thus, a number of studies have examined the reproductive and developmental toxicity of orally administered NAC. To our knowledge, however, no reports concerning reproductive developmental toxicity of i.v. NAC have appeared.
The present study was conducted to investigate the fertility and embryonic developmental toxicity of NAC administered i.v. in female rats at daily doses of 100, 300, and 1000 mg/kg from before mating to male and female rats and during the period from before mating to embryogenesis. In addition, unfertilized oocytes (UO) after ovulation and fertilized embryos on GD1 and GD2 were assessed morphologically to clarify the cause of the infertility observed in female rats that received 1000 mg/kg of NAC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female Sprague-Dawley rats (Charles River Japan, Kanagawa, Japan) were used at 8 and 10 wk of age, respectively, in the fertility and embryonic developmental toxicity study and at 12 and 10 wk of age, respectively, in morphological assessment. All animals were maintained under controlled conditions at a temperature of 23 ± 3°C and a relative humidity of 55% ± 15% in a room with a 12L:12D photoperiod. The animals were allowed free access to food and water.
The rats received NAC solutions by i.v. infusion via the tail vein using an infusion pump at a rate of 5 ml kg-1 min-1 to a volume of 20 ml/kg.
The female rats were caged 1:1 with male rats overnight, and the morning on which spermatozoa were observed in the vaginal smear was designated as GD0. The female rat that did not copulate with the male rat during 14 days (first mating period) was caged again with the other male rat that was successfully copulated at the first mating in the same dose group for an additional 14 days (second mating period). The longest period before copulation was 19 days.
The use and care of experimental animals was conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, published by the Consortium for Developing a Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching, First Edition, 1988. This study was approved by the Institutional Committee on the Care and Use of Laboratory Animals of Otsuka Pharmaceutical Factory, Inc., and Nihon Bioresearch, Inc.
Test Substance
The NAC used in the present study was purchased from Ajinomoto Co., Inc. (Tokyo, Japan). The NAC solutions were prepared at 0.5% (100 mg/kg), 1.5% (300 mg/kg), and 5% (1000 mg/kg) for use in the fertility and embryonic developmental toxicity study and at 5% for use in morphological assessment. In the preparation of NAC solutions that have neutral pH, distilled water (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) containing 61.3 ml of sodium hydroxide solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 1 mol/L was used to dissolve 10 g of NAC powder.
Study of the Reproductive and Developmental Toxicity of NAC
Effects on fertility and embryonic development Male and female rats in the NAC group received NAC by i.v. infusion at doses of 100, 300, and 1000 mg kg-1 day-1 (n = 20 each group) from 2 wk before mating to GD17 in female rats or from 4 wk before mating to 22–25 days after mating in male rats. Male and female rats in the control group (n = 20 of each sex) received the same volume of saline solution by i.v. infusion. Body weights in female rats were measured at GD0–20. The copulation rates and the pregnancy rates were calculated from mating and pregnancy results.
Five male rats in each group were dissected under ether anesthesia on the day after the end of administration. Sperm obtained from the right cauda epididymis were evaluated for motility, morphology, viability, and survivability; sperm obtained from the left cauda epididymis were evaluated for number.
All female rats were killed under ether anesthesia on GD20 to confirm pregnancy. When nonpregnant rats were found at necropsy, implantation sites were identified by dyeing the uterus with 2% potassium hydroxide solution. When pregnancy was confirmed, the number of corpora lutea, the number of implantation sites, and the litter size were determined to calculate the implantation, preimplantation loss, and postimplantation loss rates. Deaths or resorptions were classified as implantation scars, early resorptions (no formation of a fetus), late resorptions (presence of a fetus without limb formation), macerated fetuses (presence of a fetus with limb formation, but macerated), or dead fetuses (presence of a fetus without maceration).
Evaluation of male fertility in the 1000-mg/kg group Whether the infertility observed in the 1000-mg/kg group was attributable to the male rats or the female rats was investigated. All male rats in the 1000-mg/kg group and in the control group used in the study of effects on fertility and embryonic development were used for this evaluation. The male rats were continuously treated with NAC and mated with nontreatment female rats overnight (n = 20 in each group), and the copulation rate and pregnancy rate were calculated. The copulated female rats were killed under ether anesthesia and underwent cesarean section on GD13 to confirm pregnancy.
Morphological Assessment of Oocytes and Embryos
Female rats received NAC by i.v. infusion for more than 1 wk until the day of vaginal proestrus for the examination of UO (n = 10) or until copulation was confirmed on GD1 (n = 6) or GD2 (n = 10) for the examination of embryos. The dose of NAC was 1000 mg kg-1 day-1, which was confirmed to induce infertility. Animals in the control group (same number of animals for UO, GD1, and GD2, respectively) were designated as nontreatment rats. Vaginal proestrus was determined by vaginal smear.
Morphological and morphometrical assessment of UO The rats were killed under ether anesthesia at 1000–2400 h on the day of vaginal estrus, and the ovaries were dissected out bilaterally to count the corpora lutea. The oviducts were isolated, and the ampullae of the uterine tubes were opened in a dish containing 0.1% hyaluronidase (Worthington Biochemical Corporation, NJ) to obtain the oocyte-cumulus complexes. These were denuded by hyaluronidase treatment, and the UO were assessed morphologically using a stereomicroscope. The collection rate was calculated by counting the number of female rats in which UO could be recovered.
For all UO collected from the right ovary, the diameter of the oocyte and the diameter of the oocyte including the zona pellucida (ZP) were measured using a micrometer under the microscope. The thickness of the ZP was calculated as the difference between these two measured values.
Morphological assessment of GD1 and GD2 embryos Copulated rats were killed under ether anesthesia at 1400–1700 h on GD1 or GD2, and the ovaries were dissected out bilaterally. The embryos were collected from the oviducts or uterus by flushing with saline solution. The collection rate was calculated and morphological assessment performed as described above. The incidence rates were calculated based on the stage of development of the embryos.
Statistical Analysis
The copulation, pregnancy, fertility, and collection rates were analyzed using the chi-square test. All other data in the NAC and control groups were calculated as mean ± SD for each litter. All data in the fertilization and embryonic developmental toxicity study were analyzed using the Dunnett multiple method, and all other data were analyzed using the Student t-test. A value of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects on fertility and embryonic development All male and female rats in both the NAC and control groups were copulated. The fertilization rates and pregnancy rates in the 100- and 300-mg/kg groups were comparable to those in the control group (90%–100%), but these rates were markedly decreased (10%) in the 1000-mg/kg group (Table 1).
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Body weights in the 100- and 300-mg/kg groups in female rats were not significantly different from those in the control group during pregnancy. Because few pregnant animals were found in the 1000-mg/kg group, statistical analysis for body weight could not be performed. However, these values tended to be low during the middle-to-late stage of pregnancy.
No changes were observed at necropsy in male rats in any of the groups. No abnormal findings were observed in the sperm examinations in male rats in any of the groups.
The results for cesarean section examination on GD20 are shown in Table 1. When cesarean section was performed on GD20, NAC was found to have no effects on the dams. The numbers of corpora lutea, implantation sites, and live fetuses as well as the implantation, preimplantation loss, and postimplantation loss rates in the 100- and 300-mg/kg groups were not significantly different from those in the control group. With regard to deaths or resorptions in the 100- and 300-mg/kg groups, the numbers of implantation scars and early resorptions were similar to those in the control group. Macerated or dead fetuses were not observed in any of the groups. Moreover, the number of live fetuses, sex ratio, fetal body weight, and number of anomalies in the 100- and 300-mg/kg groups were not significantly different from those in the control group.
Because only two pregnant animals were found in the 1000-mg/kg group, statistical analysis could not be performed in this group. The two pregnant animals had a total of four implantations, and three of the four were implantation scars or early resorptions. Therefore, only one live fetus was obtained in the 1000-mg/kg group.
Examination of male fertility in the 1000-mg/kg group These results are shown in Table 2. The copulation and fertilization rates were the same as those in the control group. When cesarean section was performed on GD13, all findings except for the postimplantation loss rate were similar to those in the control group.
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Morphological Assessment of Oocytes and Embryos
Morphological and morphometrical assessment of UO The collection rates of UO in the NAC and control groups were 7 of 10 (70%) and 10 of 10 (100%), respectively, with no significant difference observed between the two groups.
The results for the morphological assessment of UO are shown in Table 3. Despite the fact that processing by hyaluronidase treatment was performed in the same manner in the NAC group as in the control group, it was not possible to remove the cumulus cells. Many husk-like structures with large numbers of cumulus cells attached to the surrounding material, considered to be the ZP, were observed. The number of corpora lutea in the NAC group was decreased compared with the control group. The number of UO obtained from each female rat in the NAC group was only half that in the control group. Of the UO examined (Fig. 1, A and B), 97.4% were normal oocytes in the control group, compared with 9.4% in the NAC group. Moreover, ZP-lacking and partially ZP-lacking UO accounted for 69.6% and 21.0%, respectively, in the NAC group, both of which were significantly higher than in the control group (0.7% and 0.0%, respectively). Stereomicroscopic examination showed no abnormalities other than those related to the ZP.
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Morphometric analysis of the UO in the NAC group (87.2 ± 2.9 µm; n = 20 embryos) showed diameters similar to those in the control group (86.4 ± 3.4 µm; n = 49 embryos). However, the calculated thicknesses of the ZP were 3.7 ± 2.4 µm and 8.3 ± 1.1 µm in the NAC and control groups, respectively, indicating thinning of the ZP in the NAC group.
Morphological assessment of GD1 and GD2 embryos The copulation rates on both GD1 and GD2 in the NAC group were 100%. The number of corpora lutea on both GD1 and GD2 in the NAC group was similar to that in the control group (Table 3).
The collection rate in the NAC group on GD1 was decreased to one of six (16.7%) compared with that in the control group (six of six; 100.0%). This female animal had a total of only three GD1 embryos, including two normal 2-cell embryos, but with a thin ZP (Fig. 1D and Table 3). Because collection was possible in only one animal in the 1000-mg/kg group, statistical analysis was not possible in this group. On GD2, no embryos were collected from any of the animals in the present study. The collection rate in the NAC group was therefore 0%.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several studies have investigated the causes of infertility, such as a decrease in the pregnancy rate, litter size, or number of offspring, induced by various agents, and it has been reported that various factors are responsible for these decreases, such as insufficient folliculogenesis [6, 7], anovulation [8], abnormal embryogenesis [9], and anti-implantation effects [10, 11]. The results of the present study suggest that NAC had no effects on the reproductive behavior of female rats, because the copulation rate in the 1000-mg/kg group was similar to that in the control group. Because the number of corpora lutea in the 1000-mg/kg group was decreased in the observation of UO but was similar to that in the control group in the observation of GD1 and GD2 embryos, NAC may be considered not to have affected ovulation.
Morphological assessment was performed for the UO or the embryos to clarify the reasons for the infertility in the female rats that received high doses of NAC. Based on the results of our preliminary studies regarding the period and timing of NAC administration, the infertility observed in female animals receiving NAC clearly is related to the administration during the 4-day period before mating. Therefore, we decided that the period of NAC administration should begin more than 1 wk before mating for the morphological assessment of oocytes or embryos. In addition, we also decided that the NAC dose should be 1000 mg kg-1 day-1, which was confirmed to induce infertility.
The number of UO observed in the female rats in the NAC group was half that in the animals in the control group. Interestingly, many ZP-lacking oocytes and partially ZP-lacking oocytes in which the cytoplasm was extruded from the defect in the ZP were observed. In mammalian species, the diameter of the oocyte is 70–85 µm [12–14], and the thickness of the ZP is 6–10 µm [15]. The morphometric assessment of the UO in the present study showed no effects on the diameter of the oocyte but did show that the thickness of the ZP in the NAC group was reduced to 3.7 µm, compared with 8.3 µm in the control group. These findings indicate that NAC administration results in the production of oocytes with an abnormal ZP (e.g., a lacking, partially lacking, or thin ZP) and that such oocytes can be found in the ampulla of the uterine tube after ovulation.
Regarding the assessment of GD1 and GD2 embryos, it was difficult to recover both GD1 and GD2 embryos. In particular, GD2 embryos could not be collected from any of the animals. When we attempted to perform morphological assessment of GD4 embryos collected from the uterus at other times, we were able to collect only two degenerative embryos from only 1 of 15 dams. The number of embryos that could be collected decreased rapidly with increasing time after ovulation.
The principal finding in the present study was that the administration of NAC induced changes in the ZP of oocytes and embryos. The physiological roles of the ZP include preventing polyspermy, protecting the oocyte from harmful effects in the oviductal environment, promoting free movement along the oviduct, and ensuring the species-specific binding of sperm [14, 16, 17]. Liu et al. [18] reported that female mice homozygous for the mutated mZP3 allele (mZP3-/-) had eggs lacking a ZP despite the presence of growing and fully grown oocytes and that mZP3-/- females mated with wild-type males failed to become pregnant. Rankin et al. [19] created mouse lines (Zp1tm/tm) lacking a ZP around the growing oocyte. In their report, a ZP formed around growing Zp1tm/tm oocytes, but the matrix was more loosely organized than in the ZP around normal oocytes. They also reported that fewer 2-cell embryos were collected from ZP1-null females and that their litters were smaller than those of normal mice. Thus, it has been demonstrated that the ZP plays several important roles during fertilization and embryogenesis and that structural abnormalities or absence of ZP results in infertility or decreased litter size. The results of the present study indicate that NAC administration results in the production of ZP-lacking UO or partially ZP-lacking UO, suggesting that the decrease in the number of oocytes and embryos collected in the NAC group was caused by death after polyspermy, adherence to the oviduct, and damage related to physical and/or chemical effects during embryogenesis. It is suggested that the marked decrease of pregnancy rate observed in female rats that received NAC in the fertility and embryonic developmental toxicity study was related to abnormal embryogenesis resulting from abnormalities of the ZP.
In the present study, the effect of NAC on both the pregnancy rate and the ZP thickness was observed in the 1000-mg/kg group but not in the 300-mg/kg group. Generally, NAC is administered at 3–40 mg kg-1 day-1 as nutrition infusion solutions and at 20–67 mg kg-1 day-1 as an inhalation expectorant, and we consider that the effect of NAC shown in the present study may not be seen in humans.
The mechanism of the effect of NAC on the ZP is not clear. However, ZP in mice is composed of ZP1, ZP2, and ZP3 glycoproteins. Wassarman [14] reported that a unit of ZP2 and ZP3 is repeated and becomes like a filament and that ZP1 linked these filament sheets to keep ZP protein structure. The ZP1 is a dimer of polypeptide chains held together by intermolecular disulfide bonds. The ZP2 and ZP3 have intramolecular disulfide bonds. The NAC has a thiol group. The low-molecular-weight thiol group attacks the disulfide bonds existing in protein through a thiol-disulfide exchange reaction, and the disulfide bond is consequently cleaved [20]. In fact, NAC as an expectorant is an applied mechanism of this reaction. We consider that ZP protein structure may be fragmented by the disulfide-thiol exchange reaction. The reason that NAC did not affect the male reproductive organs is not clear. We consider that the distribution of NAC may differ in organs such as the ovary, testis, and cauda epididymis.
Few studies have examined the effects of drugs on the ZP. Sarma and Mahanta [21] reported that the administration of an ethanolic crude extract of composite roots, used by the folk women of Assam to prevent pregnancy, to albino rats at a dose of 1000 mg kg-1 day-1 for 12 days could modulate histological changes in the structures of the ovary and uterus. They also reported that the ZP surrounding normal oocytes was not observed in the treated ovarian follicle and that this dose was previously found to induce sterility in the albino rat. Al-Qarawi et al. [22] reported that the administration of aqueous extracts of Cynomorium coccineum and Withania somnifera, used in folk medicines as an aphrodisiac, tonic, and narcotic, to 25-day-old Wistar rats at a dose of 47 mg per 100 g for 6 days induced the formation of an unclear ZP and frequent detachment of oocytes. Although many primary, secondary, tertiary, and antral follicles were present in animals treated with both extracts, a distinct ZP was not observed. In the present study, on the other hand, we investigated the effects of NAC, which is actually used as a therapeutic agent. To our knowledge, there have been no reports of infertility or decreased litter size induced by a general amino acid.
Regarding UO, a few oocytes that were normal except for thinning of the ZP were observed in the NAC group. On GD1, only a few normal 2-cell embryos were observed. Wassarman et al. [23] reported that although the width of the ZP (2.7 µm) in mZP3+/- females was less than half that in wild-type mice (6.2 µm), no differences were found in the pregnancy rate or litter size between mZP3+/- mice and wild-type mice. Their results suggest that UO with a thin ZP (3.7 µm) should be fertile and develop normally. Moreover, it should be assumed that such UO could develop into normal GD1 embryos. Finally, we considered that such an embryo should have developed into the single live fetus observed in the fertility and embryonic developmental toxicity study.
The site of ZP glycoprotein synthesis in mammals is a controversial issue. Several researchers have suggested that the ZP is expressed by oocytes [24, 25] or both oocytes and granulosa cells [26]. It is thought that ZP proteins originate from the oocyte in mice [14], but the evidence is not conclusive. If NAC affects oocytes and/or granulosa cells, the effects on the ZP observed in the present study may have been caused by suppression of the production of ZP proteins by oocytes and/or granulosa cells. Whether NAC has direct effects on the ZP is unclear, but the histopathological examination of oocytes and granulosa cells in the follicles in the ovary should provide a definitive answer to this question. In the present study, the effects on the ZP were observed by examination of UO after ovulation, but the ZP around growing oocytes may have been affected in the follicles in the ovary before ovulation.
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FOOTNOTES |
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Received: 27 November 2002.
First decision: 24 December 2002.
Accepted: 26 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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