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Ovarian Luteal Cell Toxicity of Ethylene Glycol Monomethyl Ether and Methoxy Acetic Acidin Vivoandin Vitro
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
142, 328–337 (1997)
TO968035
Ovarian Luteal Cell Toxicity of Ethylene Glycol Monomethyl Ether and Methoxy Acetic Acid in Vivo and in Vitro1 BARBARA J. DAVIS,*,2 JENNIFER L. ALMEKINDER,† NORRIS FLAGLER,† GREGORY TRAVLOS,† RALPH WILSON,† AND ROBERT R. MARONPOT† *Department of Microbiology, Parasitology, and Pathology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606; and †Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received July 10, 1996; accepted September 24, 1996
Ovarian Luteal Cell Toxicity of Ethylene Glycol Monomethyl Ether and Methoxy Acetic Acid in Vivo and in Vitro. DAVIS, B. J., ALMEKINDER, J. L., FLAGLER, N., TRAVLOS, G., WILSON, R., AND MARONPOT, R. R. (1997). Toxicol. Appl. Pharmacol. 142, 328–337. These studies define the site and mechanisms of reproductive toxicity of ethylene glycol monomethyl ether (EGME) in a nongravid female animal model using in vivo and in vitro methods. In vivo studies assessed vaginal cytology and histology, ovarian histology, and serum hormones in 80- to 90-day-old, adult, regularly cycling, female Sprague–Dawley rats treated daily with EGME or vehicle by oral gavage. Dose–response and time–course studies (four to nine rats per group per treatment) determined that 300 mg/kg EGME suppressed cyclicity without systemic toxicity within 3 to 8 days, and doses less than 100 mg/kg had no effect. Pathogenesis studies (six to nine rats per time and treatment) determined that 300 mg/kg EGME elevated serum progesterone within 32 hr after dosing, while serum estradiol, FSH, LH, and prolactin remained at baseline levels. In EGME-treated rats, cyclicity was suppressed, ovulation was inhibited, and corpora lutea were hypertrophied. Thus, EGME appeared to target the ovarian luteal cell. To further examine the toxicity in vitro, luteal cells were recovered from 23-day-old, hCG-primed Sprague–Dawley rats and treated with 0–10 mM methoxy acetic acid (MAA), the proximate toxic metabolite of EGME. MAA (1–10 mM) maintained elevated progesterone levels as production declined in untreated cells at 24 and 48 hr of culture. Progesterone production was maintained independent of LH-stimulated cAMP levels. MAA decreased ATP, but only at 48 hr and at 2.5 mM or greater concentrations. Thus, these studies establish that the ovarian luteal cell is a target of EGME and MAA in vivo and in vitro and that the effect on luteal cell progesterone production is likely independent of LH-stimulated cAMP pathways. q 1997 Academic Press
1
This report was made possible by Grant ES00233 from the National Institute of Environmental Health Sciences, NIH. 2 To whom correspondence should be addressed at NIEHS, MD B3-06, P.O. Box 12233, RTP, NC 27709. Fax: (919) 541-7666. E-mail: Davis1@ NIEHS.NIH.GOV.
0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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Human exposure to ethylene glycol monomethyl ether (EGME) continues to raise concern because EGME is a reproductive toxicant in laboratory animals (NIOSH, 1991). The source of human exposures is primarily occupational, but may also occur through use of products containing ethylene-based glycol ethers. Ethylene glycol ethers and their acetates are organic solvents commonly used in chemical, paint, photography, and food industries, in laboratories, in jet fuel and brake fluid as antiicing agents, in organic synthesis, and as components of varnishes, paints, inks, and cleaners (NIOSH, 1983, 1991). Ethylene glycol ethers are also used in the electronics industry, specifically in the manufacture of semiconductors, with an estimated 60,000 potentially exposed workers (Pastides et al., 1988). The estimated production of EGME and its acetate has been reported to be about 80 million pounds per year in 1983 with at least 140,000 workers potentially exposed to EGME between 1981 and 1983 (NIOSH, 1991). A number of epidemiological studies have incriminated the short-chain ethylene glycol ethers as human reproductive toxicants. For example, men working as shipyard painters exposed to EGME had about a fivefold increased prevalence of oligospermia and azoospermia, and about a 4% reduction in sperm count (Welch et al., 1988). A case-control study conducted at an infertility clinic found an odds ratio of 3.11 (p Å 0.004) for the presence of ethoxy acetic acid in the urine of subfertile or infertile male patients (Veulemans et al., 1993). Reproductive effects have also been seen among women. A series of studies has reported increased risks of spontaneous abortion, and menstrual and fertility problems among solvent-exposed women in the electronic semiconductor industry (Pastides et al., 1988; Gold et al., 1995; Correa et al., 1996). Using detailed industrial hygiene assessments to differentiate short-chain ethylene glycol ether exposures from other solvent exposures in the semiconductor industry, a recent study reported a 4.6-fold increase in risk of infertility (95% confidence interval 1.6–13.3) and a 2.8fold increase in risk of spontaneous abortion (95% confi-
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dence interval 1.4–5.6) (Correa et al., 1996). A dose–response relationship between estimated ethylene glycol exposure and each of these outcomes had also been observed. Animal studies have clearly demonstrated the reproductive toxicity of EGME in the male. In a number of species, by various routes of administration and at various doses, EGME causes testicular atrophy and decreased sperm number, and specifically kills pachytene spermatocytes (Miller et al., 1981, 1983, 1984; Foster et al., 1983; Chapin et al., 1984, 1985a,b; Nagano et al., 1984). The testicular toxicity has been attributed to the action of 2-methoxy acetic acid (MAA), the active metabolite of EGME (Miller et al., 1982; Moss et al., 1985; Foster et al., 1987), and the lesion can be inhibited by pyrazole, an inhibitor of alcohol dehydrogenase which converts the methoxyethanol to the methoxyacetate (Moss et al., 1985). The testicular toxicity can also be inhibited in vivo by coadministration of a calcium channel blocker, verapamil (Ghanayem and Chapin, 1990). In vitro, MAA-induced spermatocyte death can be inhibited by transmembrane calcium movement inhibitors (Li et al., 1996a,b). These data suggest that the methoxy acetate is responsible for the toxicity and that calcium movement is critical in the development of EGME- and MAA-induced testicular toxicity. Although the maternal, teratogenic, and embryolethal effects of EGME have been characterized in rodents (Nagano et al., 1981; Hanley et al., 1984; Brown et al., 1984) and nonhuman primates (Scott et al., 1989), the reproductive toxicity of EGME in the adult female rodent has not been as well characterized. Initial studies have reported no adverse female reproductive effects in Sprague–Dawley rats exposed to 300 ppm EGME for 13 weeks via inhalation (Rao et al., 1983; Hanley et al., 1984). However, EGME administered in drinking water significantly decreased fertility in Sprague–Dawley rats (Gulati et al., 1990), and in CD-1 (Gulati et al., 1988c), C57BI/6 (Gulati et al., 1988b), and C3H mice (Gulati et al., 1988a) in continuous breeding studies. Additionally, the EGME-treated C3H mice had significantly increased ovarian weights (Gulati et al., 1988a). Moreover, both EGME and MAA administration in continuous breeding studies caused decreased ovarian follicle counts in CD-1 mice (Heindel et al., 1989). These data suggest EGME does affect reproductive endpoints in the female and may target the ovary. The purpose of these studies is to establish the reproductive target site and define mechanisms of EGME toxicity in the nongravid adult, cycling, female rodent. The rat was chosen because of its well-characterized estrous cycle and ovarian morphology, and our previous experience using this model (Davis et al., 1994a). We demonstrate that at equivalent doses in vivo and in vitro, EGME specifically alters corpora luteal function and progesterone production.
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MATERIALS AND METHODS Chemicals For in vivo studies, EGME (CAS No. 109-86-4) was obtained from Research Triangle Institute (RTP, NC). Chemical purity was determined to be greater than 99.9% pure by capillary gas chromatography. Dosage solutions were made in distilled water and delivered by gavage at a volume of 5 ml/kg. For in vitro studies, 98% pure MAA (CAS No. 625-45-6) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Luteinizing hormone (LH) was generously provided by the National Hormone and Pituitary Distribution Program, NIH. DMEM/H-12 with penicillin and streptomycin and phenol red were from Gibco (Grand Island, NY). Other reagents were from Sigma (St. Louis, MO) unless specified in text. Animals All animal work was performed in accordance with the NIH, NIEHS Guidelines for the Humane Use of Animals in Research. For in vivo studies, 80- to 90-day-old virgin, female, Sprague–Dawley rats (Charles River Breeding Laboratory, Raleigh, NC) were housed two or three per polyethylene cage with hardwood bedding maintained under 12-hr light:12-hr dark cycle at 707F. NIH-31 pelleted diet and distilled water were available ad libitum. Estrous cycle was assessed by cytology from daily vaginal lavage and continued throughout the study. Only rats with two consecutive 4-day estrous cycles were used in studies and rats were assigned to treatment groups according to their estrous cycle stage as previously described (Davis et al., 1994a). For in vitro studies, 23-day-old Sprague–Dawley rats (Charles River Breeding Laboratory) were obtained and housed as described. Rats were treated subcutaneously with 0.25 IU human chorionic gonadotropin (hCG), twice a day for 2 days to stimulate follicular growth, followed by a single dose of 25 IU hCG on Day 3 to induce ovulation. Corpora lutea were allowed to mature for 3–4 days before cells were harvested. All rats were killed by CO2 asphyxiation. In Vivo Study Design To determine the in vivo effect of EGME in the cycling female rat, a series of studies were conducted first to determine a dose (dose-finding study), then to determine the earliest time-point an effect could be delineated (time-to-effect study), and to determine the pathogenesis of the lesion (pathogenesis study) (Davis, 1993; Davis et al., 1994a). To determine the dose of EGME to use in vivo, rats (n Å 5 per group per time point) were dosed with 0, 100, 300, or 600 mg/kg EGME per day for 7 or 14 days and complete necropsies performed on Day 8 or 15. Doses were chosen based on known testicular toxicity in male rats (Foster et al., 1984). For the timeto-effect study, groups of six EGME-treated and four vehicle-treated control rats per time-point were dosed daily beginning at 0800 hr during vaginal proestrus and terminated 6, 12, 24 hr (concurrent proestrus and estrus) or 102, 108, and 132 hr (subsequent proestrus and estrus) after initial dosing. Based on the preliminary dose-finding and time-finding studies, the pathogenesis of the lesion was determined in two separate experiments where groups of six to nine EGME (300 mg/kg)-treated and six to nine control rats per time-point were dosed daily at 0800 hr starting on vaginal metestrus and terminated 4, 8, 28, 32, 56, 80, 104, 128, 152, or 176 hr after initial dosing. Finally, vaginal cytology was assessed in six rats per group dosed for 7 days with 300, 100, or 10 mg/kg EGME to determine a no-effect level. Histopathology. Both ovaries were fixed in 10% buffered formalin, step-sectioned every 100 mm (total of 12–16 sections per ovary) and stained with hematoxylin and eosin (HE). One section of formalin-fixed vagina and uterus was stained with HE. Morphological assessment of ovaries consisted of qualitative evaluation of all ovarian components in all sections prepared
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as previously described (Davis, 1993; Davis et al., 1994a). Ovulation was assessed in ovaries by the presence of ruptured follicles, corpora hemorrhagica, and/or oocytes within the oviducts. The point count fraction of corpora lutea was determined using a staggered grid (Russ, 1986) available on NIH Image software (V. 1.5) as previously described (Davis et al., 1994a). Hormones. Immediately prior to necropsy, serum was obtained from blood collected via cardiac puncture after light CO2 anesthesia. Serum estradiol and progesterone were measured using radioimmunoassay kits from Diagnostic Products, Inc. (Los Angeles, CA); LH, FSH, and prolactin were measured using kits from Amersham Corp. (Arlington Heights, IL). The interassay and intraassay coefficients of variation of the assays were less than 10%. In Vitro Study Design Once the target cell was identified, the in vitro effect of MAA was determined in luteal cells. Luteal cells were prepared from mechanically dispersed, hCG-stimulated rat ovaries as previously described for rat granulosa cells (Davis et al., 1994b; Treinen et al., 1990). Briefly, ovaries were removed aseptically and placed in DMEM/Ham’s F-12 medium (1:1) and washed. Mechanical dispersion was performed by gently puncturing ovaries with a 25-gauge needle in petri dishes containing fresh DMEM/H-12 media. Cells were then centrifuged and cell viability was determined to be about 60% by trypan blue exclusion. Subsequently, 100,000 viable cells per 500 ml DMEM/H-12 media were plated in eight-well Lab-Tek Chamber Slides (Nunc Inc., Naperville, IL). LH was added to cells at a maximally stimulating dose of 1000 ng, as determined in preliminary studies (data not shown). MAA was prepared as a stock solution of 500 mM in media by adjusting the pH to 7.2 with NaOH. Dilutions were made from this stock solution, and various concentrations of MAA (0 to 10 mM) were added to the cells in a 10-ml volume; exposure was for 3 to 48 hr. Concentrations were chosen based on previous studies in the male and correspond to concentrations found in vivo after EGME exposure at dose levels that cause testicular toxicity (Foster et al., 1987). Progesterone and cAMP were measured by standardized radioimmunoassay (Diagnostic Products, Inc.) from media stored at 0707C. Neither EGME nor MAA (5 mM) cross-reacted with the progesterone assay (data not shown). Protein concentrations were determined on individual cell pellets dissolved in 0.01 N NaOH using the Pierce BCA protein assay reagent (Rockville, IL) as previously described (Davis et al., 1994b). Cellular ATP was extracted and measured by a luciferase assay as previously described (Treinen et al., 1990). Statistical Analysis For in vitro studies, progesterone, cAMP, and ATP were normalized to cell protein before analysis. Treatment (in vivo and in vitro) and time effects on hormones, cAMP, or ATP and morphometric ratios were assessed using analysis of variance (ANOVA) methods and least significant difference (JMP, SAS Institute Inc., Cary, NC). Hormones were normalized by logarithmic transformation prior to applying ANOVA. Differences in cycle days were analyzed by the Pearson x2 test (JMP, SAS). For all statistical tests, a was set at p õ 0.05.
RESULTS
In Vivo Studies Dose-finding study. No significant differences were found between body and organ weights after 8 days in EGME-treated vs control groups (data not shown). At 15 days, body weight was significantly increased and kidney weights were decreased in the 600 and 300 mg/kg dose
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groups compared to control (data not shown). The number of females with 4-day cycles in the control, 100, 300, and 600 mg EGME/kg/day groups, each with 10 rats per group, was 9, 6, 2, and 0, respectively, indicating that 300 and 600 mg EGME/kg/day significantly suppressed cyclicity (Pearson x2 test, p õ 0.05). No significant organ histopathology was found at any dose except in the ovary where corpora lutea were greatly enlarged and there was no histomorphological evidence of ovulation in either the 600 or 300 mg/ kg EGME dose groups. At 100 mg/kg EGME, 4 of 10 rats had hypertrophied corpora lutea and no evidence of ovulation. Since the 300 mg/kg dose had clear effects on the reproductive tract but no other significant deleterious effects within an 8-day period, this was the dose chosen for subsequent studies. Time-finding study. All EGME-treated rats (300 mg/kg) ovulated the first estrus after dosing was initiated on the morning of proestrus, as determined by histomorphological evaluation of the ovaries. However, none of six EGMEtreated rats ovulated the subsequent estrus with continuous daily dosing, while four of four control rats had ovulated. Additionally, corpora lutea of EGME-treated rats appeared enlarged compared to control rats. Thus, the chemical effect could be determined after a proestrus-initiated dosing and prior to the subsequent proestrus. Consequently for the pathogenesis studies, rats were treated with EGME (300 mg/ kg) starting during vaginal metestrus, that is, 3 days prior to proestrus and ovulation. Pathogenesis studies. During 8 days of gavage dosing with 300 mg EGME/kg/day, vaginal cytology was characterized as diestrus, whereas control rats had two proestrus-toproestrus cycles during the 8 days (data not shown). Vaginal histology of rats treated with EGME for 5 to 8 days was characterized by an epithelial cell layer of tall, vacuolated, columnar cells comparable to histology of pregnant rats. In contrast, control rats exhibited vaginal epithelial changes consistent with various cycle days of proestrus, estrus, metestrus, and diestrus. Thus, EGME treatment suppressed vaginal cyclicity and produced prolonged days in diestrus. Ovarian histomorphology revealed the presence of normal follicular maturation and preovulatory follicles, but showed no evidence of ruptured follicles or corpora hemorrhagica in EGME-treated rats at any time-point. In contrast, all controls had ruptured follicles, corpora hemorrhagica, and oocytes in oviducts consistent with ovulation by the first estrus after metestrus-initiated dosing, and five of six controls ovulated the second estrus after metestrus-initiated dosing. Moreover, corpora lutea were hypertrophied in EGMEtreated rats compared to controls by Day 8 (Figs. 1–3). The increase in size was attributed to a paucity of luteal cell death compared to controls (Figs. 4, 5). Thus, EGME treatment inhibited ovulation and caused corpora lutea hypertrophy.
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In Vitro Studies
FIG. 1. Quantitative comparisons of corpora lutea size in EGME-treated vs control rats. The point count fraction of corpora lutea and total ovary was determined from a computer-generated staggered grid placed over electronically captured images of 12–16 sections each of both ovaries from six EGME-treated and six control rats at selected time points. The point count fraction of corpora lutea is expressed as a percentage of total ovary. Variation of CL volume fraction in control ovaries is attributed to changes in the cycle. Data represent means { SE (*p õ 0.05).
EGME treatment also significantly altered serum hormone levels. Serum progesterone was elevated in EGME-treated rats beginning 32 hr (late diestrus) after treatment initiation, and it remained elevated over the 176 hr (8 days) of treatment (Fig. 6). In contrast, serum estradiol, FSH, LH, and prolactin levels were suppressed in EGME-treated rats compared to controls (Figs. 6–7).
The elevated progesterone levels combined with the hypertrophied corpora lutea indicated that the ovarian luteal cell was a primary target for EGME in the cycling female rat. To confirm the in vivo findings and determine potential mechanisms of EGME toxicity, isolated rat luteal cells were exposed to 0–10 mM MAA, the active metabolite of EGME, in vitro. MAA elevated progesterone production at §1 mM compared to controls after 48 hr of culture (Fig. 8). Temporal evaluation suggested that 5 mM MAA caused continued production of progesterone at 24 and 48 hr of culture while progesterone production declined in controls (Fig. 9). Since luteal cell progesterone production is stimulated by LH and cAMP pathways (Richards et al., 1983), we tested the hypothesis that MAA maintained progesterone production through LH-stimulated cAMP pathways. However, MAA increased progesterone whether cells were or were not stimulated with LH (Fig. 8). Moreover, levels of cAMP were not significantly altered by any concentration of MAA at 48 hr (Fig. 10) or by 5 mM MAA at 1, 3, 24, or 48 hr (data not shown). In contrast, levels of ATP were significantly decreased in MAA-treated cells at concentrations of 2.5 mM and greater after 48 hr (Fig. 11), and the decreases were seen only at 48 hr of culture (data not shown). DISCUSSION
These studies establish that the ovarian luteal cell is a target of EGME and its active metabolite MAA, and that the reproductive toxicity in the female rat is manifested morphologi-
FIG. 2. Photomicrograph of an ovary from a rat treated with vehicle for 8 days. The ovary contains a corpora hemmorhagica (arrow) and numerous vascularized corpora albicans (arrowheads). Bar represents 250 mm.
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FIG. 3. Photomicrograph of an ovary from a rat treated with 300 mg/kg EGME for 8 days. The ovary contains numerous, well-organized, greatly hypertrophied corpora lutea (arrows). Bar represents 250 mm, the same magnification as Fig. 2.
cally as luteal cell hypertrophy and functionally as progesterone hypersecretion both in vivo and in cultured rat luteal cells. Corpora lutea only transiently produce progesterone in the cycling rat, and must be stimulated to maintain progesterone secretion during pregnancy (or pseudopregnancy) by a postovulatory, secondary surge of LH and/or prolactin that is triggered by cervical stimulation during mating (Everett, 1961; Smith et al., 1975). Consequently, EGME could act
indirectly on the ovary and increase corpora luteal progesterone secretion by triggering pituitary hormone surges. However, the serum hormone levels in the EGME-treated rats suggest that progesterone was increased and corpora lutea hypertrophied independent of any pituitary hormone surges. MAA also effected progesterone production in isolated rat luteal cells in vitro, clearly indicating that EGME/MAA directly alters luteal cell progesterone production.
FIG. 4. Photomicrograph of ovarian luteal cells from a rat treated with vehicle for 8 days. There are numerous pyknotic nuclei, nuclear debris, and fragmented cells with shrunken cytoplasmic borders, characteristic of luteolysis. Bar represents 100 mm.
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FIG. 5. Photomicrograph of ovarian luteal cells from a rat treated with 300 mg/kg EGME for 8 days. The cells are arranged in a glandular pattern separated by a fine vascular stroma. Luteal cells are homogeneously round to polygonal with abundant vacuolated cytoplasm and distinct cells borders. There is a paucity of cellular debris. Bar represents 100 mm.
The morphological and physiological manifestations of EGME treatment mimic a pregnant or pseudopregnant state in the rat in which high levels of progesterone are produced by hypertrophied corpora lutea and cyclicity is suppressed through progesterone-induced inhibition of FSH and LH surges (Everett, 1961). Vaginal mucification also occurs in response to the prolonged high levels of progesterone. Because progesterone was elevated within 32 hr of EGME treatment and prior to alterations in estradiol or pituitary hormones, we conclude that the elevated progesterone induced by EGME is the primary mediator of the subsequent reproductive alterations including the suppressed cyclicity, suppressed estradiol and pituitary hormones, and vaginal mucification similar to that which occurs in pregnant or pseudopregnant rats. The conversion of corpus lutea of the cycle to corpus lutea of pregnancy is dependent on the ability of luteal cells to maintain progesterone secretion and resist luteolytic signals. Despite the potential differences in the physiology of the luteal cell from a cycling rat compared to a superovulated/ pseudopregnant rat, both our in vivo and in vitro data support the conclusion that EGME/MAA acts to maintain elevated progesterone secretion. In luteal cells, progesterone is produced through LH-stimulated cAMP-dependent and -independent pathways (Oonk et al., 1989). Our data suggest that MAA maintains progesterone production independent of LH and cAMP pathways. MAA also affects progesterone independent of cellular ATP levels because ATP was decreased only at §2.5 mM MAA and only at 48 hr, while progesterone was elevated by 1 mM MAA at 24 hr. However, the declining
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ATP levels may also suggest that MAA affects cell viability. Thus, the question remains whether the apparent luteotrophic effects of EGME and MAA are due to a direct action of the chemicals or whether the chemicals directly affect progesterone production which in turn mediates the apparent luteotrophic effect. While additional studies are needed to determine the mechanisms of EGME toxicity in the luteal cell, we have demonstrated that EGME and MAA have comparable noeffect levels both in vivo and in vitro, and are comparable in the female and male systems. For example, in the female in vivo, 100 mg EGME/kg/day for 6 days had caused corpora lutea hypertrophy and suppressed cyclicity in 4 of 10 rats, while 10 mg EGME/kg/day for 6 days had no effect. In vitro, concentrations of MAA lower than 1 mM had no effect on luteal cell progesterone, and 1 mM MAA would be expected in peak blood levels after a 100 mg/kg EGME dose in vivo (Foster et al., 1984, 1987). Similarly, in the male, in vivo doses less than 100 mg EGME/kg/day for 3 to 5 days had no effect on testis morphology (Chapin et al., 1985a,b). Although EGME and MAA have many comparable effects in female and male rodents, we were surprised by the distinct manifestation of the toxicity in the cycling female rat. For example, in the male, EGME and MAA induce germ cell death (Miller et al., 1981, 1983, 1984; Foster et al., 1983; Chapin et al., 1984, 1985a; Nagano et al., 1984). Additionally, EGME and MAA decrease ovarian follicle counts in CD-1 mice in continuous breeding studies (Heindel et al., 1989). Accordingly, we had hypothesized that EGME and MAA would target the female germ cell, thus accounting
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estrous cyclicity does decrease preantral follicle counts in mice (Telfer et al., 1991). Our results also differ from two early studies which reported no adverse female reproductive effects in Sprague– Dawley rats exposed to 300 ppm EGME via inhalation (Rao et al., 1983; Hanley et al., 1984). However, these initial assessments had evaluated pregnancy outcome after exposure was stopped. In contrast, we addressed EGME effects on ovarian histomorphology and serum hormones during concurrent exposure. Continuous breeding studies have
FIG. 6. Effect of EGME on serum progesterone and estradiol in vivo. Serum progesterone was measured from rats (six to nine treated and six to nine control) gavaged daily with 300 mg EGME/kg/day or vehicle beginning on metestrus and terminated from 4 to 176 hr after initial treatment. Met, Di, Pro, and Est indicate expected cycle stage in control rats. Data represent means { SE (*p õ 0.05).
for the decreased follicle counts and having an effect comparable to the male. Instead, EGME and MAA apparently inhibit luteal cell death and maintain progesterone secretion in the cycling female rat and in the cultured luteal cell, but did not cause follicular pathology or decrease follicle numbers (data not shown). However, subtle changes in follicle numbers may be difficult to detect (Heindel et al., 1989), particularly in a short-term study such as ours. Moreover, the reported decrease in follicle counts occurred in EGMEtreated offspring of EGME-treated adults (J. J. Heindel, personal communication), suggesting that length of exposure and perhaps stage of exposure are important factors in the manifestation of follicle toxicity. Finally, an apparent female germ cell or follicle toxicity of EGME could be a secondary effect of the EGME-mediated increase in progesterone, since progesterone treatment in doses high enough to suppress
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FIG. 7. Effect of EGME on serum, FSH, prolactin, and LH. Serum hormones were measured and analyzed as described in the legend to Fig. 6.
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FIG. 8. Effects of various concentrations of MAA on LH-stimulated rat luteal cell progesterone production. Rat luteal cells were cultured for 48 hr with or without 1000 ng LH and 0–10 mM MAA. Progesterone was measured in media by RIA and standardized to cell protein. Each value represents the mean { SE of quadruplicate determinations from three separate experiments. (a) Significantly different from LH-stimulated control at p õ 0.05; (b) significantly different from basal control at p õ 0.05.
shown that EGME is a female reproductive toxicant (Gulati et al., 1990, 1988a,b,c), which is consistent with our results. Given that concurrent treatment with EGME has adverse effects on hormones, breeding, and pregnancy, Rao’s and Hanley’s results may suggest that female rats can recover from EGME toxicity, which has been seen in male rats (Chapin et al., 1985a). We are currently examining the potential reversibility in the female rat.
FIG. 9. Temporal effect of MAA on rat luteal cell progesterone production. Luteal cells were cultured with 1000 ng LH without or with 5 mM MAA for 1, 3, 24, and 48 hr. Progesterone was measured in media by RIA and normalized to cell protein. Values represent means { SE of quadruplicate replicates from three separate experiments. *Significantly different from control at p õ 0.05 by ANOVA and LSD.
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FIG. 10. Absence of MAA dose-related effect on LH-stimulated rat luteal cell cAMP. Luteal cells were cultured for 48 hr with 1000 ng LH and 0–10 mM MAA. Cyclic AMP was measured in media by RIA and standardized to cell protein. Each value represents the mean { SE of quadruplicate determinations from three separate experiments. No significance by ANOVA.
To our knowledge, this is the first report demonstrating the site and a potential mechanism of action of EGME and MAA in the nongravid female rodent. This suggests EGME may cause menstrual cycle disruption and subfertility in women as well as spontaneous abortions and perhaps teratogenic effects. However, our data are limited in their direct applicability to predicting EGME reproductive toxicity in women not only because of the different routes of exposure, but also because of the distinct difference in the function of
FIG. 11. Dose-related effects of MAA on LH-stimulated rat luteal cell ATP levels. Luteal cells were cultured for 48 hr with 1000 ng LH and 0– 10 mM MAA. Intracellular ATP was measured by a luciferase assay and normalized to cell protein determined in representative wells. Values represent means { SE of quadruplicate replicates from three separate experiments. *Significantly different from control at p õ 0.05 by ANOVA and LSD.
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the corpora lutea in rats and women. Specifically, in the rat, EGME stimulates luteal cell progesterone during a cycle in which the luteal cell does not normally produce progesterone. Women naturally have a luteal phase of their cycle characterized by luteal cell progesterone secretion. Consequently, it is difficult to directly extrapolate how EGME may affect luteal function in women. However, our data in the rat clearly indicate that the luteal cell is a target of EGME and MAA, both in vivo and in vitro, and suggest that the luteal cell should be examined as a potential target in exposed women. ACKNOWLEDGMENTS This work was presented in abstract form at the 1995 Society for the Study of Reproduction 28th Annual Meeting and at the 1996 Society of Toxicology 35th Annual Meeting. The authors thank Drs. Andrew Rowland, Robert Chapin, and Ling-Hong Li and Mr. Robert Wine for valuable comments and help.
REFERENCES Brown, N. A., Holt, D., and Webb, M. (1984). The teratogenicity of methoxyacetic acid in the rat. Toxicol. Lett. 22, 93–100. Chapin, R. E., Dutton, S. L., Ross, M. D., Sumrell, B. M., and Lamb, J. C. IV (1984). The effects of ethylene glycol monomethyl ether on testicular histology in F344 rats. J. Androl. 5(5), 369–380. Chapin, R. E., Dutton, S. L., Ross, M. D., and Lamb, J. C., IV (1985a). Effects of ethylene glycol monomethyl ether (EGME) on mating performance and epididymal sperm parameters in F344 rats. Fundam. Appl. Toxicol. 5, 182–189. Chapin, R. E., Dutton, S. L., Ross, M. D., Swaisgood, R. R., and Lamb, J. C., IV (1985b). The recovery of the testes over 8 weeks after shortterm dosing with ethylene glycol monomethyl ether: Histology, cellspecific enzymes, and rete testes fluid protein. Fundam. Appl. Toxicol. 5, 515–525. Correa, A., Gray, R., Cohen, R., Rothman, N., Shah, F., Seacat, H., and Corn, M. (1996). Ethylene glycol ethers and risks of spontaneous abortion and subfertility. Am. J. Epidemiol. 143, 707–717. Davis, B. (1993). Ovarian target cell toxicity. In Female Reproductive Toxicology. (R. Chapin and J. Heindel, Eds.), Vol. 3B, pp. 69–78. Academic Press, San Diego. Davis, B., Maronpot, R., and Heindel, J. (1994a). Di-(2-ethylhexyl) phthalate suppresses estradiol and ovulation in cycling rats. Toxicol. Appl. Pharmacol. 128, 216–223. Davis, B., Weaver, R., Gaines, L., and Heindel, J. (1994b). Mono-(2-ethylhexyl phthalate) suppresses estradiol production independent of FSHcAMP stimulation in rat granulosa cells. Toxicol. Appl. Pharmacol. 128, 224–228. Everett, J. (1961). The mammalian female reproductive cycle and its controlling mechanisms. In Sex and Internal Secretions (W. Young, Ed.), Vol. 1, pp. 497–555. Williams and Wilkins, Baltimore. Foster, P., Creasy, D., Foster, J., Thomas, L., Cook, M., and Gongolli, S. (1983). Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol. Appl. Pharmacol. 69, 385–399. Foster, P. M. D., Creasy, D. M., Foster, J. R., and Gray, T. J. B. (1984). Testicular toxicity produced by ethylene glycol monomethyl and monoethyl ethers in the rat. Environ. Health Perspect. 57, 207–217.
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Foster, P. M. D., Lloyd, S. C., and Blackburn, D. M. (1987). Comparison of the in vivo and in vitro testicular effects produced by methoxy-, ethoxy-, and N-butoxy acetic acids in the rat. Toxicology 43, 17–30. Ghanayem, B. I., and Chapin, R. E. (1990). Calcium channel blockers protect against ethylene glycol monomethyl ether (2-methoxyethanol)-induced testicular toxicity. Exp. Mol. Pathol. 52, 279–290. Gold, E. B., Eskenazi, B., Hammond, S. K., Lasley, B. L., Samuels, S. J., Rasor, M. O., Hines, C. J., Overstreet, J. W., and Schenker, M. B. (1995). Prospectively assessed menstrual cycle characteristics in female waterfabrication and nonfabrication semiconductor employees. Am. J. Ind. Med. 28, 799–815. Gulati, D., Hope, E., Barnes, L., Russell, S., Poonacha, K., and Chapin, R. (1990). Development of reproduction and fertility assessment protocol in CD Sprague-Dawley rats: Litter 5 design. National Toxicology Program, Washington, DC. Gulati, D., Hope, E., Barnes, L., Russell, S., Poonacha, K., Chapin, R., and Morrissey, R. (1988a). Ethylene glycol monomethyl ether: Reproduction and fertility assessment in C3H mice when administered in drinking water. Final Study Report, National Toxicology Program, Washington, DC. Gulati, D., Hope, E., Barnes, L., Russell, S., Poonacha, K., Chapin, R., and Morrissey, R. (1988b). Ethylene glycol monomethyl ether: Reproduction and fertility assessment in C57BL/6 mice when administered in drinking water. Final Study Report, National Toxicology Program, Washington, DC. Gulati, D., Hope, E., Barnes, L., Russell, S., Poonacha, K., Chapin, R., and Morrissey, R. (1988c). Ethylene glycol monomethyl ether: Reproduction and fertility assessment in CD-1 mice when administered in drinking water. Final Study Report, National Toxicology Program, Washington, DC. Hanley, T., Young, J., John, J., and Rao, K. (1984). Ethylene glycol monomethyl ether (EGME) and propylene glycol monomethyl ether (PGME): Inhalation fertility and teratogenicity studies in rats, mice and rabbits. Environ. Health Perspect. 57, 7–12. Heindel, J. J., Thomford, P. J., and Mattison, D. R. (1989). Histological assessment of ovarian follicle number in mice as a screen for ovarian toxicity. In Growth Factors and the Ovary (A. N. Hirshfield, Ed.), pp. 421–426. Plenum Press, New York. Li, L.-H., Wine, R. N., and Chapin, R. E. (1996a). 2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: An in vitro comparison. J. Androl., in press. Li, L.-H., Wine, R. N., and Chapin, R. E. (1996b). Sertoli cells mediate germ cell apoptosis induced by 2-methoxyethanol. Toxicologist 30, 625. Miller, R., Carreon, R., Young, J., and McKenna, M. (1982). Toxicity of methoxyacetic acid in rats. Fundam. Appl. Toxicol. 2, 158–160. Miller, R., Herman, E., Young, J., Landry, T., and Calhoun, L. (1984). Ethylene glycol monomethyl ether and propylene glycol monomethyl ether: Metabolism, disposition and subchronic inhalation toxicity studies. Environ. Health Perspect. 57, 233–239. Miller, R. R., Ayres, J. A., Calhoun, L. L., Young, J. T., and McKenna, M. J. (1981). Comparative short-term inhalation toxicity of ethylene glycol monomethyl ether and propylene glycol monomethyl ether in rats and mice. Toxicol. Appl. Pharmacol. 61, 368–377. Miller, R. R., Ayres, J. A., Young, J. T., and McKenna, M. J. (1983). Ethylene glycol monomethyl ether. I. Subchronic vapor inhalation study with rats and rabbits. Fundam. Appl. Toxicol. 3, 49–54. Moss, E. J., Thomas, L. V., Cook, M. W., Walters, D. G., Foster, P. M. D., Creasy, D. M., and Gray, T. J. B. (1985). The role of metabolism in 2methoxyethanol-induced testicular toxicity. Toxicol. Appl. Pharmacol. 79, 480–489.
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OVARIAN TOXICITY OF EGME Nagano, K., Nakyama, E., Oobayashi, H., Nishizawa, T., Okuda, H., and Yamada, T. (1984). Experimental studies on toxicity of ethylene glycol alkyl ethers in Japan. Environ. Health Perspect. 57, 75–84. Nagano, K., Nakyama, E., Oobayashi, H., Yamada, T., Adachi, H., et al. (1981). Embryotoxic effects of ethylene glycol monomethyl ether in mice. Toxicology 20, 335–343. National Institute for Occupational Safety and Health (1983). The glycol ethers, with particular reference to 2-methoxyethanol and 2-ethoxyethanol: Evidence of adverse reproductive effects. U.S. Department of Health and Human Services Public Health Service, Cincinnati, OH. National Institute for Occupational Safety and Health (1991). Occupational exposure to ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, and their acetates. U.S. Department of Health and Human Services Public Health Service, Cincinnati, OH. Oonk, R. B., Krasnow, J. S., Beattie, W. G., and Richards, J. S. (1989). Cyclic AMP-dependent and -independent regulation of cholesterol side chain cleavage cytochrome P-450 (P-450scc) in rat ovarian granulosa cells and corpora lutea. J. Biol. Chem. 264(36), 21934–21942. Pastides, H., Calabrese, E., Hosmer, D., and Harris, D. (1988). Spontaneous abortion and general illness symptoms among semiconductor manufacturers. J. Occup. Med. 30, 543–551. Rao, K., Cobel-Geard, S., Young, J., Hanley, T., Hayes, W., Honh, J., and Miller, R. (1983). Ethylene glycol monomethyl ether II. Reproductive and dominant lethal studies in rats. Fundam. Appl. Toxicol. 3, 80–85.
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Richards, J. S., Sehgal, N., and Tash, J. S. (1983). Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea. J. Biol. Chem. 258(8), 5227–5232. Russ, J. C. (1986). Practical Stereology. Plenum Press, New York. Scott, W., Fradkin, R., Wittfoht, W., and Nau, H. (1989). Teratologic potential of 2-methoxyethanol and transplacental distribution of its metabolite, 2-methoxyacetic acid, in non-human primates. Teratology 39, 363–373. Smith, M. S., Freeman, M. E., and Neil, J. D. (1975). The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: Prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96, 319. Telfer, E., Gosden, R. G., and Faddy, M. J. (1991). Impact of exogenous progesterone on ovarian follicular dynamics and function in mice. J. Reprod. Fertil. 93, 263–269. Treinen, K. A., Dodson, W. C., and Heindel, J. J. (1990). Inhibition of FSHstimulated cAMP accumulation and progesterone production by mono (2-ethylhexyl) phthalate in rat granulosa cell cultures. Toxicol. Appl. Pharmacol. 106, 334–340. Veulemans, H., Steeno, O., Masschelein, R., and Groeseneken, D. (1993). Exposure to ethylene glycol ethers and spermatogenic disorders in man: A case-control study. Br. J. Ind. Med. 50, 71–78. Welch, L. S., Schrader, S. M., Turner, T. W., and Cullen, M. R. (1988). Effects of exposure to ethylene glycol ethers on shipyard painters: II. Male reproduction. Am. J. Ind. Med. 14, 509–526.
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Ovarian Luteal Cell Toxicity of Ethylene Glycol Monomethyl Ether and Methoxy Acetic Acid in Vivo and in Vitro1 BARBARA J. DAVIS,*,2 JENNIFER L. ALMEKINDER,† NORRIS FLAGLER,† GREGORY TRAVLOS,† RALPH WILSON,† AND ROBERT R. MARONPOT† *Department of Microbiology, Parasitology, and Pathology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606; and †Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Received July 10, 1996; accepted September 24, 1996
Ovarian Luteal Cell Toxicity of Ethylene Glycol Monomethyl Ether and Methoxy Acetic Acid in Vivo and in Vitro. DAVIS, B. J., ALMEKINDER, J. L., FLAGLER, N., TRAVLOS, G., WILSON, R., AND MARONPOT, R. R. (1997). Toxicol. Appl. Pharmacol. 142, 328–337. These studies define the site and mechanisms of reproductive toxicity of ethylene glycol monomethyl ether (EGME) in a nongravid female animal model using in vivo and in vitro methods. In vivo studies assessed vaginal cytology and histology, ovarian histology, and serum hormones in 80- to 90-day-old, adult, regularly cycling, female Sprague–Dawley rats treated daily with EGME or vehicle by oral gavage. Dose–response and time–course studies (four to nine rats per group per treatment) determined that 300 mg/kg EGME suppressed cyclicity without systemic toxicity within 3 to 8 days, and doses less than 100 mg/kg had no effect. Pathogenesis studies (six to nine rats per time and treatment) determined that 300 mg/kg EGME elevated serum progesterone within 32 hr after dosing, while serum estradiol, FSH, LH, and prolactin remained at baseline levels. In EGME-treated rats, cyclicity was suppressed, ovulation was inhibited, and corpora lutea were hypertrophied. Thus, EGME appeared to target the ovarian luteal cell. To further examine the toxicity in vitro, luteal cells were recovered from 23-day-old, hCG-primed Sprague–Dawley rats and treated with 0–10 mM methoxy acetic acid (MAA), the proximate toxic metabolite of EGME. MAA (1–10 mM) maintained elevated progesterone levels as production declined in untreated cells at 24 and 48 hr of culture. Progesterone production was maintained independent of LH-stimulated cAMP levels. MAA decreased ATP, but only at 48 hr and at 2.5 mM or greater concentrations. Thus, these studies establish that the ovarian luteal cell is a target of EGME and MAA in vivo and in vitro and that the effect on luteal cell progesterone production is likely independent of LH-stimulated cAMP pathways. q 1997 Academic Press
1
This report was made possible by Grant ES00233 from the National Institute of Environmental Health Sciences, NIH. 2 To whom correspondence should be addressed at NIEHS, MD B3-06, P.O. Box 12233, RTP, NC 27709. Fax: (919) 541-7666. E-mail: Davis1@ NIEHS.NIH.GOV.
0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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Human exposure to ethylene glycol monomethyl ether (EGME) continues to raise concern because EGME is a reproductive toxicant in laboratory animals (NIOSH, 1991). The source of human exposures is primarily occupational, but may also occur through use of products containing ethylene-based glycol ethers. Ethylene glycol ethers and their acetates are organic solvents commonly used in chemical, paint, photography, and food industries, in laboratories, in jet fuel and brake fluid as antiicing agents, in organic synthesis, and as components of varnishes, paints, inks, and cleaners (NIOSH, 1983, 1991). Ethylene glycol ethers are also used in the electronics industry, specifically in the manufacture of semiconductors, with an estimated 60,000 potentially exposed workers (Pastides et al., 1988). The estimated production of EGME and its acetate has been reported to be about 80 million pounds per year in 1983 with at least 140,000 workers potentially exposed to EGME between 1981 and 1983 (NIOSH, 1991). A number of epidemiological studies have incriminated the short-chain ethylene glycol ethers as human reproductive toxicants. For example, men working as shipyard painters exposed to EGME had about a fivefold increased prevalence of oligospermia and azoospermia, and about a 4% reduction in sperm count (Welch et al., 1988). A case-control study conducted at an infertility clinic found an odds ratio of 3.11 (p Å 0.004) for the presence of ethoxy acetic acid in the urine of subfertile or infertile male patients (Veulemans et al., 1993). Reproductive effects have also been seen among women. A series of studies has reported increased risks of spontaneous abortion, and menstrual and fertility problems among solvent-exposed women in the electronic semiconductor industry (Pastides et al., 1988; Gold et al., 1995; Correa et al., 1996). Using detailed industrial hygiene assessments to differentiate short-chain ethylene glycol ether exposures from other solvent exposures in the semiconductor industry, a recent study reported a 4.6-fold increase in risk of infertility (95% confidence interval 1.6–13.3) and a 2.8fold increase in risk of spontaneous abortion (95% confi-
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dence interval 1.4–5.6) (Correa et al., 1996). A dose–response relationship between estimated ethylene glycol exposure and each of these outcomes had also been observed. Animal studies have clearly demonstrated the reproductive toxicity of EGME in the male. In a number of species, by various routes of administration and at various doses, EGME causes testicular atrophy and decreased sperm number, and specifically kills pachytene spermatocytes (Miller et al., 1981, 1983, 1984; Foster et al., 1983; Chapin et al., 1984, 1985a,b; Nagano et al., 1984). The testicular toxicity has been attributed to the action of 2-methoxy acetic acid (MAA), the active metabolite of EGME (Miller et al., 1982; Moss et al., 1985; Foster et al., 1987), and the lesion can be inhibited by pyrazole, an inhibitor of alcohol dehydrogenase which converts the methoxyethanol to the methoxyacetate (Moss et al., 1985). The testicular toxicity can also be inhibited in vivo by coadministration of a calcium channel blocker, verapamil (Ghanayem and Chapin, 1990). In vitro, MAA-induced spermatocyte death can be inhibited by transmembrane calcium movement inhibitors (Li et al., 1996a,b). These data suggest that the methoxy acetate is responsible for the toxicity and that calcium movement is critical in the development of EGME- and MAA-induced testicular toxicity. Although the maternal, teratogenic, and embryolethal effects of EGME have been characterized in rodents (Nagano et al., 1981; Hanley et al., 1984; Brown et al., 1984) and nonhuman primates (Scott et al., 1989), the reproductive toxicity of EGME in the adult female rodent has not been as well characterized. Initial studies have reported no adverse female reproductive effects in Sprague–Dawley rats exposed to 300 ppm EGME for 13 weeks via inhalation (Rao et al., 1983; Hanley et al., 1984). However, EGME administered in drinking water significantly decreased fertility in Sprague–Dawley rats (Gulati et al., 1990), and in CD-1 (Gulati et al., 1988c), C57BI/6 (Gulati et al., 1988b), and C3H mice (Gulati et al., 1988a) in continuous breeding studies. Additionally, the EGME-treated C3H mice had significantly increased ovarian weights (Gulati et al., 1988a). Moreover, both EGME and MAA administration in continuous breeding studies caused decreased ovarian follicle counts in CD-1 mice (Heindel et al., 1989). These data suggest EGME does affect reproductive endpoints in the female and may target the ovary. The purpose of these studies is to establish the reproductive target site and define mechanisms of EGME toxicity in the nongravid adult, cycling, female rodent. The rat was chosen because of its well-characterized estrous cycle and ovarian morphology, and our previous experience using this model (Davis et al., 1994a). We demonstrate that at equivalent doses in vivo and in vitro, EGME specifically alters corpora luteal function and progesterone production.
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MATERIALS AND METHODS Chemicals For in vivo studies, EGME (CAS No. 109-86-4) was obtained from Research Triangle Institute (RTP, NC). Chemical purity was determined to be greater than 99.9% pure by capillary gas chromatography. Dosage solutions were made in distilled water and delivered by gavage at a volume of 5 ml/kg. For in vitro studies, 98% pure MAA (CAS No. 625-45-6) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Luteinizing hormone (LH) was generously provided by the National Hormone and Pituitary Distribution Program, NIH. DMEM/H-12 with penicillin and streptomycin and phenol red were from Gibco (Grand Island, NY). Other reagents were from Sigma (St. Louis, MO) unless specified in text. Animals All animal work was performed in accordance with the NIH, NIEHS Guidelines for the Humane Use of Animals in Research. For in vivo studies, 80- to 90-day-old virgin, female, Sprague–Dawley rats (Charles River Breeding Laboratory, Raleigh, NC) were housed two or three per polyethylene cage with hardwood bedding maintained under 12-hr light:12-hr dark cycle at 707F. NIH-31 pelleted diet and distilled water were available ad libitum. Estrous cycle was assessed by cytology from daily vaginal lavage and continued throughout the study. Only rats with two consecutive 4-day estrous cycles were used in studies and rats were assigned to treatment groups according to their estrous cycle stage as previously described (Davis et al., 1994a). For in vitro studies, 23-day-old Sprague–Dawley rats (Charles River Breeding Laboratory) were obtained and housed as described. Rats were treated subcutaneously with 0.25 IU human chorionic gonadotropin (hCG), twice a day for 2 days to stimulate follicular growth, followed by a single dose of 25 IU hCG on Day 3 to induce ovulation. Corpora lutea were allowed to mature for 3–4 days before cells were harvested. All rats were killed by CO2 asphyxiation. In Vivo Study Design To determine the in vivo effect of EGME in the cycling female rat, a series of studies were conducted first to determine a dose (dose-finding study), then to determine the earliest time-point an effect could be delineated (time-to-effect study), and to determine the pathogenesis of the lesion (pathogenesis study) (Davis, 1993; Davis et al., 1994a). To determine the dose of EGME to use in vivo, rats (n Å 5 per group per time point) were dosed with 0, 100, 300, or 600 mg/kg EGME per day for 7 or 14 days and complete necropsies performed on Day 8 or 15. Doses were chosen based on known testicular toxicity in male rats (Foster et al., 1984). For the timeto-effect study, groups of six EGME-treated and four vehicle-treated control rats per time-point were dosed daily beginning at 0800 hr during vaginal proestrus and terminated 6, 12, 24 hr (concurrent proestrus and estrus) or 102, 108, and 132 hr (subsequent proestrus and estrus) after initial dosing. Based on the preliminary dose-finding and time-finding studies, the pathogenesis of the lesion was determined in two separate experiments where groups of six to nine EGME (300 mg/kg)-treated and six to nine control rats per time-point were dosed daily at 0800 hr starting on vaginal metestrus and terminated 4, 8, 28, 32, 56, 80, 104, 128, 152, or 176 hr after initial dosing. Finally, vaginal cytology was assessed in six rats per group dosed for 7 days with 300, 100, or 10 mg/kg EGME to determine a no-effect level. Histopathology. Both ovaries were fixed in 10% buffered formalin, step-sectioned every 100 mm (total of 12–16 sections per ovary) and stained with hematoxylin and eosin (HE). One section of formalin-fixed vagina and uterus was stained with HE. Morphological assessment of ovaries consisted of qualitative evaluation of all ovarian components in all sections prepared
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as previously described (Davis, 1993; Davis et al., 1994a). Ovulation was assessed in ovaries by the presence of ruptured follicles, corpora hemorrhagica, and/or oocytes within the oviducts. The point count fraction of corpora lutea was determined using a staggered grid (Russ, 1986) available on NIH Image software (V. 1.5) as previously described (Davis et al., 1994a). Hormones. Immediately prior to necropsy, serum was obtained from blood collected via cardiac puncture after light CO2 anesthesia. Serum estradiol and progesterone were measured using radioimmunoassay kits from Diagnostic Products, Inc. (Los Angeles, CA); LH, FSH, and prolactin were measured using kits from Amersham Corp. (Arlington Heights, IL). The interassay and intraassay coefficients of variation of the assays were less than 10%. In Vitro Study Design Once the target cell was identified, the in vitro effect of MAA was determined in luteal cells. Luteal cells were prepared from mechanically dispersed, hCG-stimulated rat ovaries as previously described for rat granulosa cells (Davis et al., 1994b; Treinen et al., 1990). Briefly, ovaries were removed aseptically and placed in DMEM/Ham’s F-12 medium (1:1) and washed. Mechanical dispersion was performed by gently puncturing ovaries with a 25-gauge needle in petri dishes containing fresh DMEM/H-12 media. Cells were then centrifuged and cell viability was determined to be about 60% by trypan blue exclusion. Subsequently, 100,000 viable cells per 500 ml DMEM/H-12 media were plated in eight-well Lab-Tek Chamber Slides (Nunc Inc., Naperville, IL). LH was added to cells at a maximally stimulating dose of 1000 ng, as determined in preliminary studies (data not shown). MAA was prepared as a stock solution of 500 mM in media by adjusting the pH to 7.2 with NaOH. Dilutions were made from this stock solution, and various concentrations of MAA (0 to 10 mM) were added to the cells in a 10-ml volume; exposure was for 3 to 48 hr. Concentrations were chosen based on previous studies in the male and correspond to concentrations found in vivo after EGME exposure at dose levels that cause testicular toxicity (Foster et al., 1987). Progesterone and cAMP were measured by standardized radioimmunoassay (Diagnostic Products, Inc.) from media stored at 0707C. Neither EGME nor MAA (5 mM) cross-reacted with the progesterone assay (data not shown). Protein concentrations were determined on individual cell pellets dissolved in 0.01 N NaOH using the Pierce BCA protein assay reagent (Rockville, IL) as previously described (Davis et al., 1994b). Cellular ATP was extracted and measured by a luciferase assay as previously described (Treinen et al., 1990). Statistical Analysis For in vitro studies, progesterone, cAMP, and ATP were normalized to cell protein before analysis. Treatment (in vivo and in vitro) and time effects on hormones, cAMP, or ATP and morphometric ratios were assessed using analysis of variance (ANOVA) methods and least significant difference (JMP, SAS Institute Inc., Cary, NC). Hormones were normalized by logarithmic transformation prior to applying ANOVA. Differences in cycle days were analyzed by the Pearson x2 test (JMP, SAS). For all statistical tests, a was set at p õ 0.05.
RESULTS
In Vivo Studies Dose-finding study. No significant differences were found between body and organ weights after 8 days in EGME-treated vs control groups (data not shown). At 15 days, body weight was significantly increased and kidney weights were decreased in the 600 and 300 mg/kg dose
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groups compared to control (data not shown). The number of females with 4-day cycles in the control, 100, 300, and 600 mg EGME/kg/day groups, each with 10 rats per group, was 9, 6, 2, and 0, respectively, indicating that 300 and 600 mg EGME/kg/day significantly suppressed cyclicity (Pearson x2 test, p õ 0.05). No significant organ histopathology was found at any dose except in the ovary where corpora lutea were greatly enlarged and there was no histomorphological evidence of ovulation in either the 600 or 300 mg/ kg EGME dose groups. At 100 mg/kg EGME, 4 of 10 rats had hypertrophied corpora lutea and no evidence of ovulation. Since the 300 mg/kg dose had clear effects on the reproductive tract but no other significant deleterious effects within an 8-day period, this was the dose chosen for subsequent studies. Time-finding study. All EGME-treated rats (300 mg/kg) ovulated the first estrus after dosing was initiated on the morning of proestrus, as determined by histomorphological evaluation of the ovaries. However, none of six EGMEtreated rats ovulated the subsequent estrus with continuous daily dosing, while four of four control rats had ovulated. Additionally, corpora lutea of EGME-treated rats appeared enlarged compared to control rats. Thus, the chemical effect could be determined after a proestrus-initiated dosing and prior to the subsequent proestrus. Consequently for the pathogenesis studies, rats were treated with EGME (300 mg/ kg) starting during vaginal metestrus, that is, 3 days prior to proestrus and ovulation. Pathogenesis studies. During 8 days of gavage dosing with 300 mg EGME/kg/day, vaginal cytology was characterized as diestrus, whereas control rats had two proestrus-toproestrus cycles during the 8 days (data not shown). Vaginal histology of rats treated with EGME for 5 to 8 days was characterized by an epithelial cell layer of tall, vacuolated, columnar cells comparable to histology of pregnant rats. In contrast, control rats exhibited vaginal epithelial changes consistent with various cycle days of proestrus, estrus, metestrus, and diestrus. Thus, EGME treatment suppressed vaginal cyclicity and produced prolonged days in diestrus. Ovarian histomorphology revealed the presence of normal follicular maturation and preovulatory follicles, but showed no evidence of ruptured follicles or corpora hemorrhagica in EGME-treated rats at any time-point. In contrast, all controls had ruptured follicles, corpora hemorrhagica, and oocytes in oviducts consistent with ovulation by the first estrus after metestrus-initiated dosing, and five of six controls ovulated the second estrus after metestrus-initiated dosing. Moreover, corpora lutea were hypertrophied in EGMEtreated rats compared to controls by Day 8 (Figs. 1–3). The increase in size was attributed to a paucity of luteal cell death compared to controls (Figs. 4, 5). Thus, EGME treatment inhibited ovulation and caused corpora lutea hypertrophy.
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In Vitro Studies
FIG. 1. Quantitative comparisons of corpora lutea size in EGME-treated vs control rats. The point count fraction of corpora lutea and total ovary was determined from a computer-generated staggered grid placed over electronically captured images of 12–16 sections each of both ovaries from six EGME-treated and six control rats at selected time points. The point count fraction of corpora lutea is expressed as a percentage of total ovary. Variation of CL volume fraction in control ovaries is attributed to changes in the cycle. Data represent means { SE (*p õ 0.05).
EGME treatment also significantly altered serum hormone levels. Serum progesterone was elevated in EGME-treated rats beginning 32 hr (late diestrus) after treatment initiation, and it remained elevated over the 176 hr (8 days) of treatment (Fig. 6). In contrast, serum estradiol, FSH, LH, and prolactin levels were suppressed in EGME-treated rats compared to controls (Figs. 6–7).
The elevated progesterone levels combined with the hypertrophied corpora lutea indicated that the ovarian luteal cell was a primary target for EGME in the cycling female rat. To confirm the in vivo findings and determine potential mechanisms of EGME toxicity, isolated rat luteal cells were exposed to 0–10 mM MAA, the active metabolite of EGME, in vitro. MAA elevated progesterone production at §1 mM compared to controls after 48 hr of culture (Fig. 8). Temporal evaluation suggested that 5 mM MAA caused continued production of progesterone at 24 and 48 hr of culture while progesterone production declined in controls (Fig. 9). Since luteal cell progesterone production is stimulated by LH and cAMP pathways (Richards et al., 1983), we tested the hypothesis that MAA maintained progesterone production through LH-stimulated cAMP pathways. However, MAA increased progesterone whether cells were or were not stimulated with LH (Fig. 8). Moreover, levels of cAMP were not significantly altered by any concentration of MAA at 48 hr (Fig. 10) or by 5 mM MAA at 1, 3, 24, or 48 hr (data not shown). In contrast, levels of ATP were significantly decreased in MAA-treated cells at concentrations of 2.5 mM and greater after 48 hr (Fig. 11), and the decreases were seen only at 48 hr of culture (data not shown). DISCUSSION
These studies establish that the ovarian luteal cell is a target of EGME and its active metabolite MAA, and that the reproductive toxicity in the female rat is manifested morphologi-
FIG. 2. Photomicrograph of an ovary from a rat treated with vehicle for 8 days. The ovary contains a corpora hemmorhagica (arrow) and numerous vascularized corpora albicans (arrowheads). Bar represents 250 mm.
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FIG. 3. Photomicrograph of an ovary from a rat treated with 300 mg/kg EGME for 8 days. The ovary contains numerous, well-organized, greatly hypertrophied corpora lutea (arrows). Bar represents 250 mm, the same magnification as Fig. 2.
cally as luteal cell hypertrophy and functionally as progesterone hypersecretion both in vivo and in cultured rat luteal cells. Corpora lutea only transiently produce progesterone in the cycling rat, and must be stimulated to maintain progesterone secretion during pregnancy (or pseudopregnancy) by a postovulatory, secondary surge of LH and/or prolactin that is triggered by cervical stimulation during mating (Everett, 1961; Smith et al., 1975). Consequently, EGME could act
indirectly on the ovary and increase corpora luteal progesterone secretion by triggering pituitary hormone surges. However, the serum hormone levels in the EGME-treated rats suggest that progesterone was increased and corpora lutea hypertrophied independent of any pituitary hormone surges. MAA also effected progesterone production in isolated rat luteal cells in vitro, clearly indicating that EGME/MAA directly alters luteal cell progesterone production.
FIG. 4. Photomicrograph of ovarian luteal cells from a rat treated with vehicle for 8 days. There are numerous pyknotic nuclei, nuclear debris, and fragmented cells with shrunken cytoplasmic borders, characteristic of luteolysis. Bar represents 100 mm.
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FIG. 5. Photomicrograph of ovarian luteal cells from a rat treated with 300 mg/kg EGME for 8 days. The cells are arranged in a glandular pattern separated by a fine vascular stroma. Luteal cells are homogeneously round to polygonal with abundant vacuolated cytoplasm and distinct cells borders. There is a paucity of cellular debris. Bar represents 100 mm.
The morphological and physiological manifestations of EGME treatment mimic a pregnant or pseudopregnant state in the rat in which high levels of progesterone are produced by hypertrophied corpora lutea and cyclicity is suppressed through progesterone-induced inhibition of FSH and LH surges (Everett, 1961). Vaginal mucification also occurs in response to the prolonged high levels of progesterone. Because progesterone was elevated within 32 hr of EGME treatment and prior to alterations in estradiol or pituitary hormones, we conclude that the elevated progesterone induced by EGME is the primary mediator of the subsequent reproductive alterations including the suppressed cyclicity, suppressed estradiol and pituitary hormones, and vaginal mucification similar to that which occurs in pregnant or pseudopregnant rats. The conversion of corpus lutea of the cycle to corpus lutea of pregnancy is dependent on the ability of luteal cells to maintain progesterone secretion and resist luteolytic signals. Despite the potential differences in the physiology of the luteal cell from a cycling rat compared to a superovulated/ pseudopregnant rat, both our in vivo and in vitro data support the conclusion that EGME/MAA acts to maintain elevated progesterone secretion. In luteal cells, progesterone is produced through LH-stimulated cAMP-dependent and -independent pathways (Oonk et al., 1989). Our data suggest that MAA maintains progesterone production independent of LH and cAMP pathways. MAA also affects progesterone independent of cellular ATP levels because ATP was decreased only at §2.5 mM MAA and only at 48 hr, while progesterone was elevated by 1 mM MAA at 24 hr. However, the declining
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ATP levels may also suggest that MAA affects cell viability. Thus, the question remains whether the apparent luteotrophic effects of EGME and MAA are due to a direct action of the chemicals or whether the chemicals directly affect progesterone production which in turn mediates the apparent luteotrophic effect. While additional studies are needed to determine the mechanisms of EGME toxicity in the luteal cell, we have demonstrated that EGME and MAA have comparable noeffect levels both in vivo and in vitro, and are comparable in the female and male systems. For example, in the female in vivo, 100 mg EGME/kg/day for 6 days had caused corpora lutea hypertrophy and suppressed cyclicity in 4 of 10 rats, while 10 mg EGME/kg/day for 6 days had no effect. In vitro, concentrations of MAA lower than 1 mM had no effect on luteal cell progesterone, and 1 mM MAA would be expected in peak blood levels after a 100 mg/kg EGME dose in vivo (Foster et al., 1984, 1987). Similarly, in the male, in vivo doses less than 100 mg EGME/kg/day for 3 to 5 days had no effect on testis morphology (Chapin et al., 1985a,b). Although EGME and MAA have many comparable effects in female and male rodents, we were surprised by the distinct manifestation of the toxicity in the cycling female rat. For example, in the male, EGME and MAA induce germ cell death (Miller et al., 1981, 1983, 1984; Foster et al., 1983; Chapin et al., 1984, 1985a; Nagano et al., 1984). Additionally, EGME and MAA decrease ovarian follicle counts in CD-1 mice in continuous breeding studies (Heindel et al., 1989). Accordingly, we had hypothesized that EGME and MAA would target the female germ cell, thus accounting
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estrous cyclicity does decrease preantral follicle counts in mice (Telfer et al., 1991). Our results also differ from two early studies which reported no adverse female reproductive effects in Sprague– Dawley rats exposed to 300 ppm EGME via inhalation (Rao et al., 1983; Hanley et al., 1984). However, these initial assessments had evaluated pregnancy outcome after exposure was stopped. In contrast, we addressed EGME effects on ovarian histomorphology and serum hormones during concurrent exposure. Continuous breeding studies have
FIG. 6. Effect of EGME on serum progesterone and estradiol in vivo. Serum progesterone was measured from rats (six to nine treated and six to nine control) gavaged daily with 300 mg EGME/kg/day or vehicle beginning on metestrus and terminated from 4 to 176 hr after initial treatment. Met, Di, Pro, and Est indicate expected cycle stage in control rats. Data represent means { SE (*p õ 0.05).
for the decreased follicle counts and having an effect comparable to the male. Instead, EGME and MAA apparently inhibit luteal cell death and maintain progesterone secretion in the cycling female rat and in the cultured luteal cell, but did not cause follicular pathology or decrease follicle numbers (data not shown). However, subtle changes in follicle numbers may be difficult to detect (Heindel et al., 1989), particularly in a short-term study such as ours. Moreover, the reported decrease in follicle counts occurred in EGMEtreated offspring of EGME-treated adults (J. J. Heindel, personal communication), suggesting that length of exposure and perhaps stage of exposure are important factors in the manifestation of follicle toxicity. Finally, an apparent female germ cell or follicle toxicity of EGME could be a secondary effect of the EGME-mediated increase in progesterone, since progesterone treatment in doses high enough to suppress
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FIG. 7. Effect of EGME on serum, FSH, prolactin, and LH. Serum hormones were measured and analyzed as described in the legend to Fig. 6.
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FIG. 8. Effects of various concentrations of MAA on LH-stimulated rat luteal cell progesterone production. Rat luteal cells were cultured for 48 hr with or without 1000 ng LH and 0–10 mM MAA. Progesterone was measured in media by RIA and standardized to cell protein. Each value represents the mean { SE of quadruplicate determinations from three separate experiments. (a) Significantly different from LH-stimulated control at p õ 0.05; (b) significantly different from basal control at p õ 0.05.
shown that EGME is a female reproductive toxicant (Gulati et al., 1990, 1988a,b,c), which is consistent with our results. Given that concurrent treatment with EGME has adverse effects on hormones, breeding, and pregnancy, Rao’s and Hanley’s results may suggest that female rats can recover from EGME toxicity, which has been seen in male rats (Chapin et al., 1985a). We are currently examining the potential reversibility in the female rat.
FIG. 9. Temporal effect of MAA on rat luteal cell progesterone production. Luteal cells were cultured with 1000 ng LH without or with 5 mM MAA for 1, 3, 24, and 48 hr. Progesterone was measured in media by RIA and normalized to cell protein. Values represent means { SE of quadruplicate replicates from three separate experiments. *Significantly different from control at p õ 0.05 by ANOVA and LSD.
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FIG. 10. Absence of MAA dose-related effect on LH-stimulated rat luteal cell cAMP. Luteal cells were cultured for 48 hr with 1000 ng LH and 0–10 mM MAA. Cyclic AMP was measured in media by RIA and standardized to cell protein. Each value represents the mean { SE of quadruplicate determinations from three separate experiments. No significance by ANOVA.
To our knowledge, this is the first report demonstrating the site and a potential mechanism of action of EGME and MAA in the nongravid female rodent. This suggests EGME may cause menstrual cycle disruption and subfertility in women as well as spontaneous abortions and perhaps teratogenic effects. However, our data are limited in their direct applicability to predicting EGME reproductive toxicity in women not only because of the different routes of exposure, but also because of the distinct difference in the function of
FIG. 11. Dose-related effects of MAA on LH-stimulated rat luteal cell ATP levels. Luteal cells were cultured for 48 hr with 1000 ng LH and 0– 10 mM MAA. Intracellular ATP was measured by a luciferase assay and normalized to cell protein determined in representative wells. Values represent means { SE of quadruplicate replicates from three separate experiments. *Significantly different from control at p õ 0.05 by ANOVA and LSD.
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the corpora lutea in rats and women. Specifically, in the rat, EGME stimulates luteal cell progesterone during a cycle in which the luteal cell does not normally produce progesterone. Women naturally have a luteal phase of their cycle characterized by luteal cell progesterone secretion. Consequently, it is difficult to directly extrapolate how EGME may affect luteal function in women. However, our data in the rat clearly indicate that the luteal cell is a target of EGME and MAA, both in vivo and in vitro, and suggest that the luteal cell should be examined as a potential target in exposed women. ACKNOWLEDGMENTS This work was presented in abstract form at the 1995 Society for the Study of Reproduction 28th Annual Meeting and at the 1996 Society of Toxicology 35th Annual Meeting. The authors thank Drs. Andrew Rowland, Robert Chapin, and Ling-Hong Li and Mr. Robert Wine for valuable comments and help.
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