52, 45– 49 (1999) Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effect of Prenatal Exposure to TCDD on the Promotion of Endometriotic Lesion Growth by TCDD in Adult Female Rats and Mice Audrey M. Cummings,* ,1 Joan M. Hedge,* and Linda S. Birnbaum† *Reproductive Toxicology Division and †Experimental Toxicology Division, MD-72, NHEERL, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received August 31, 1998; accepted May 28, 1999
Exposure of pregnant dams to TCDD has been shown to produce adverse effects on the reproductive system or capacity of offspring. In a study conducted by Gray and Ostby (1995), the exposure of rat dams to 1 mg TCDD per kg on gestation day (GD) 8 resulted in female offspring in which there were increased numbers in constant estrus and a significantly greater incidence of cystic endometrial hyperplasia in the adults. In addition, fecundity was reduced in the offspring. Endometriosis in women and nonhuman primates consists of the presence of endometrial tissue at sites outside the uterine cavity. The disease can be extremely painful and often results in infertility (Haney, 1990). The etiology of endometriosis appears to involve a combination of the phenomena of retrograde menstruation and altered immunocompetence (D’Hooghe et al., 1994; Dmowski et al., 1991). Rats (Vernon and Wilson, 1985) and mice (Cummings and Metcalf, 1995) have been used in models of endometriosis in which endometrial tissue is transferred surgically to the peritoneal cavity. The growth of these surgically induced endometriotic lesions depends on a number of factors, including the presence of estrogen (Cummings and Metcalf, 1995). When rats and mice were exposed to TCDD prior to and at intervals following the induction of endometriosis, the chemical produced a significant dose-related increase in lesion diameter in mice and an increase in lesion diameter in rats when all time points were pooled (Cummings et al., 1996). Those experiments in rats and mice were pursued when previous work showed that TCDD exposure produced a dose-dependent increase in endometriosis in monkeys (Rier et al., 1993). Based on previous findings of (1) prenatal effects of TCDD on reproduction (Gray and Ostby, 1995) and (2) the promotion of endometriotic lesion growth by TCDD in adult rodents (Cummings et al., 1996), our hypothesis was that exposure of rodent dams to TCDD during fetal organogenesis, prior to reproductive tract development, may alter the prenatal and/or postnatal development of the offspring such that the sensitivity of the adults to TCDD treatment would be altered with respect to the degree of the TCDD-induced promotion of endometriotic lesion growth. To test this hypothesis, we treated rat and mouse dams with TCDD or vehicle on GD8, permitted the female offspring to reach adulthood, and evaluated the effect of
Several lines of research led to our hypothesis that perinatal exposure to TCDD may alter the sensitivity of adult rodents to the promotional effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on endometriosis. Pregnant rats and mice were treated on gestation day (GD) 8 with either 1 (rats) or 3 (mice) mg TCDD/kg or vehicle. Female offspring were reared to adulthood, and endometriosis was induced surgically. All animals received 0, 3, or 10 mg TCDD/kg 3 weeks prior to surgery, at the time of surgery, and 3, 6, and 9 weeks after surgery. Necropsies were performed 12 weeks after surgery. Measurements at necropsy included the diameter of endometriotic lesions and body, uterine, ovarian and liver weights. While no effect of treatment on lesion diameter was found in rats, analyses revealed that perinatal plus adult exposure to TCDD can increase the size of endometriotic lesions surgically induced in mice. These and additional data on body and organ weights are consistent with previous work. These data confirm the sensitivity of mice to the promotion of endometriotic lesion growth by TCDD and indicate a perinatal effect of TCDD on this parameter when perinatal exposure on GD8 is supplemented with adult exposure to TCDD of female mice. Key Words: TCDD; 2,3,7,8-tetrachlorodibenzo-p-dioxin; developmental effects; endometriosis; reproductive tract; rats; mice.
The chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental contaminant derived from sources that include industrial combustion and the commercial synthesis of 2,4,5-trichlorophenol (Birnbaum, 1994; Landers and Bunce, 1991). TCDD is often considered to act as an antiestrogen (Safe et al., 1991). It also appears to suppress cell-mediated and humoral immunity in animals (Holsapple et al., 1991). The aryl hydrocarbon (Ah) receptor is believed to mediate the toxic effects of TCDD (see Birnbaum, 1994 for review). The information in this article has been funded wholly by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use. 1 To whom correspondence should be addressed. Fax: (919) 541–5138. E-mail:
[email protected]. 45
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additional doses of TCDD on the promotion of surgically induced endometriotic growth. METHODS Animals. Female Sprague-Dawley rats were purchased as timed pregnant and received from Charles River (Raleigh, NC) on GD2. The same source was used to obtain C57BL/6 female mice bred with C3H males. These timed pregnant females were received on GD2 and delivered B6C3F1 offspring. Animals were housed individually in clear plastic cages containing heat-treated laboratory grade pine shavings (Northeastern Products Corp., Warrensburg, NY). All animals received feed (Pro-lab Rat, Mouse, or Hamster 3000; Agway, Syracuse, NY) and water ad libitum. The light cycle for rats was 14:10 (lights on at 0500 h EST) and for mice was 12:12 (lights on at 0600 hr). Both animal rooms were maintained at a temperature of 20 –24 °C and a relative humidity of 40 –50%. Chemicals. TCDD was obtained from Cambridge Isotope Laboratories, Inc. (Cambridge, MA). Corn oil was purchased from Sigma Chemical Co. (St. Louis, MO). Experimental Design. On GD8, groups of pregnant rats were weighed and received 0 mg (25 dams) or 1 mg (50 dams) TCDD/kg by gavage in corn oil. Also on GD8, pregnant mice similarly received 0 mg (18 dams) or 3 mg (40 dams) TCDD/kg by gavage in corn oil. Dosing volumes were 2 ml/kg for rats and 10 ml/kg for mice. The selection of these dosages was based on the work of Gray et al. (1995) for rats and Couture et al. (1990) for mice. In mice, the rationale for dose selection included an intention to avoid the cleft palate induced by higher doses on GD8. Day 8 was selected as an early time during a period of organogenesis for both species. A higher dosage was used in mice due to the lower sensitivity of mice to the prenatal effects of TCDD. After the pups were born, rat litters were culled to 8 per litter, predominantly females. Mice were not culled as their litters were small. At weaning, females were randomly assigned to postnatal treatment groups. Animals that had received 0 mg TCDD/kg perinatally received 0 or 3 mg TCDD/kg as adults, and animals that had received TCDD perinatally received 0, 3, or 10 mg TCDD/kg as adults. The same dosing volumes were used for dosing adult offspring as for dosing of the dams. Each treatment group initially contained 12 mice or rats; this number was reduced slightly by mortality largely occurring around the time of surgery. The timing of the TCDD dosing regimen for perinatally treated, adult rats and mice was similar to that used in previous studies for nonperinatally treated animals (Cummings et al., 1996). Animals from each perinatal dose group were randomly assigned to adult dose groups described above. Rats and mice were initially weighed and dosed with TCDD on postnatal day 77. Twenty-one days later, endometriosis was induced in rats and mice by a surgical procedure (Cummings and Metcalf, 1995; Vernon and Wilson, 1985), and an additional dose of TCDD was administered at that time according to body weight. All animals were weighed and received additional identical, doses of TCDD at 3-week intervals: 3, 6, and 9 weeks after surgery. During week 12 postsurgery, animals were necropsied; evaluations included the diameter of the endometriotic lesions (measured to the nearest 0.05 mm using calipers) and body, liver, thymus, ovarian, and liver weights. At 12 weeks, the lesion is a fluid-filled, spherical structure that has increased in size approximately 2-fold. Effects of perinatal exposure to TCDD on litter size and pup weight at birth were not evaluated in either species because those data have been reported in earlier studies (Gray et al., 1995). Postnatal survival was comparable for all groups. Surgery. Surgery to induce endometriosis in rats and mice was performed aseptically and in a manner identical to that described by Vernon and Wilson (1985) for rats, and by Cummings and Metcalf (1995) for mice. Briefly, a portion (approximately 1 cm) of each rat’s uterus or an entire mouse uterine horn was ligated, excised, and placed in warm Ham’s F-12 medium. Each segment of uterus was slit longitudinally and cut into 6 pieces (rat) or 3 pieces (mouse) measuring approximately 2.5 mm 2 each. Squares of uterine tissue were sutured, using 4 – 0 nylon sutures, to vessels of the intestinal mesentery
in the same animal from which the uterine tissue was taken. The abdominal muscle layer was sutured with gut, and the skin was closed with wound clips. Statistics. Within each animal, the diameters of the 6 (rat) or 3 (mouse) endometriotic lesions in each animal were averaged. This animal mean was then used as the unit of analysis. All data for each species, including lesion diameter, body weight, body weight change, organ weights standardized by body weight (organ weight/body weight), were analyzed by a one way analysis of variance (General Linear Models; SAS, 1985). Body weight change was calculated as the body weight at necropsy minus the body weight on the first day of dosing of the adult offspring. When the overall p value for a parameter was found to be less than 0.10, the data for that parameter were analyzed by least squares means (SAS, 1985) for individual preplanned comparisons in which p , 0.05 was used for significance.
RESULTS
As shown in Figure 1, neither perinatal nor adult dosing had an effect on lesion diameter in rats when data were collected at 12 weeks after surgery. Figure 1 also shows a trend toward decreased ovarian weight (p . 0.1) as a result of perinatal exposure and a statistically significant, dose-dependent decline in thymus weight as a consequence of adult exposure to TCDD. Data on uterine horn weights indicated no clear effect of perinatal or adult dosing with TCDD (Fig. 1). In Table 1, increased liver weight is shown to be a function of adult dosing alone. Both final body weight and body weight change showed an adverse effect of TCDD administration to adults. Statistical analysis of lesion diameter in mice revealed that while neither prenatal (0 – 0 vs 3– 0) or postnatal (0 – 0 vs 0 –3) exposure to TCDD had a significant effect, the combination of prenatal and postnatal dosing (0 – 0 vs 3–3) resulted in a significant increase in lesion diameter (Fig. 2). Ovarian weight (standardized to body weight) was decreased due to adult exposure, and thymus weight (standardized) was decreased by prenatal exposure to TCDD (Fig. 2). The effect of TCDD on uterine horn weight in mice appears complex. While a decrease in uterine weight due to perinatal dosing is apparent (0 – 0 vs 3– 0), this observation is not borne out by the remaining data (0 –3 vs 3–3; Fig. 2). In mice, there was no effect of prenatal or adult TCDD exposure on body weight gain between the time of the first dose to offspring of TCDD and the time of necropsy (Table 2). Liver weight was clearly increased as a result of adult exposure (Table 2). DISCUSSION
While perinatal exposure to TCDD does not affect the diameter of endometriotic lesions induced surgically in the offspring of treated rat dams, a combination of perinatal and adult exposure to TCDD produced an increase in lesion diameter in mice. Such a species difference is not surprising given similar differences seen in previous work (Cummings et al., 1996). Thus, exposure of mice to TCDD on GD8 augments the growth of surgically induced endometriotic lesions only if the animals are also exposed to additional TCDD as adults. This finding of a TCDD-induced developmental effect in adult mice is con-
PRENATAL TCDD AND ENDOMETRIOSIS
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FIG. 1. Effect of prenatal and/or postnatal (adult) exposure of rats to TCDD. Rats were treated as described in Methods. Pairs of numbers on the X axes represent the prenatal and adult dosages of TCDD. Data are plotted as the means 6 SE for all parameters. Data with differing superscripts are significantly different from each other, p , 0.05.
sistent with a report by Theobald and Peterson (1997) where in utero (GD14) exposure of mice to TCDD resulted in a decrease in uterine weight in offspring. Such a decrease, found also in the current study, may reflect more occult changes in uterine physiology that could contribute to an increased vulnerability toward endometriotic growth. The data show a trend toward a stimulation of lesion growth due to adult exposure, i.e., comparing the 3– 0 group with the TABLE 1 Effect of Prenatal and/or Postnatal Exposure of Rats to TCDD Dose of TCDD (mg/kg) pre/postnatal
Final body weight (g)
Body weight change (g)
Liver weight (g)
0/0 0/3 1/0 1/3 1/10
357.9 6 15.3 a 342.4 6 11.9 a 353.4 6 7.9 a 347.3 6 7.1 a 303.8 6 16.6 b
78.8 6 8.3 a 55.8 6 5.3 b 81.5 6 6.7 a 68.7 6 5.5 a 29.5 6 11.4 b
11.53 6 0.5 a 13.77 6 0.5 b 10.95 6 0.3 a 13.57 6 0.4 b 13.50 6 1.1 b
Note. Data represent means 6 SE for all parameters. Statistics for liver weight are derived from the standardized (liver weight/body weight) data. Data with differing superscripts are significantly different from each other, p , 0.05.
3–3 or 3–10 group. The finding of no statistically significant effect of adult exposure, alone, to TCDD is a situation that differs from that found in previous reports (Cummings et al., 1996; Johnson et al., 1997), and several factors may have contributed to these results. The major one is that this rodent model of endometriosis involves a significant level of variability, and a large component of this variability lies in the potential for the reproductive cycle to affect results. For example, variations in the day of estrus on which each animal undergoes surgery and/or is necropsied may impact the size of the lesions, based on the known stimulation of lesion growth by estrogen in mice (Cummings and Metcalf, 1995). Variability due to the effect of cyclicity on lesion diameter has been ruled out in rats (Cummings, unpublished data) but not in mice. The best solution for this problem would be to control for the day of the cycle on which surgeries and necropsies are done. One way other investigators have done this has been to ovariectomize the animals, treat them with estrogen plus TCDD, and surgically induce the endometrial lesions (Yang and Foster, 1997). However, the intact and ovariectomized plus estrogen models are not directly comparable. The data in the literature, when taken together, suggest that the mechanism by which TCDD promotes endometriotic lesion diameter requires the presence of the ovary. There may be roles for not only ovarian steroid
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FIG. 2. Effect of prenatal and/or postnatal (adult) exposure of mice to TCDD. Mice were treated as described in MeETHODS. Pairs of numbers on the X axes represent the prenatal and adult dosages of TCDD. Data are plotted as the means 6 SE for all parameters. Data with differing superscripts are significantly different from each other, p , 0.05.
hormones but also unidentified factors that may potentially mediate the observed effects of TCDD on endometriotic lesions. As other workers have shown (Li et al., 1995), such factors may include, but are not limited to, ovarian and/or TCDD interactions with the hypothalamic-pituitary axis. Although previous work has demonstrated that similar dose levels of TCDD administered to pregnant rats (GD15) results in morphological alterations in the reproductive tract of the TABLE 2 Effect of Prenatal and/or Postnatal Exposure of Mice to TCDD Dose of TCDD (mg/kg) pre/postnatal
Final body weight (g)
Body weight change (g)
Liver weight (g)
0/0 0/3 3/0 3/3 3/10
30.4 6 1.1 a 31.6 6 1.0 a 32.6 6 1.4 a 34.4 6 1.0 a,b 33.2 6 0.8 a,b
6.6 6 1.0 a 7.0 6 0.8 a 7.1 6 1.6 a 9.9 6 0.9 a 7.8 6 1.1 a
1.21 6 0.06 a 1.49 6 0.06 b 1.34 6 0.06 a 1.55 6 0.05 b 1.55 6 0.04 b
Note. Data represent means 6 SE for all parameters. Statistics for liver weight are derived from the standardized (liver weight/body weight) data. Data with differing superscripts are significantly different from each other, p , 0.05.
offspring (Gray et al., 1997), the lack of effect of perinatal TCDD exposure on lesion diameter in rats is consistent with the lack of effect of TCDD on lesion diameter at 12 weeks after surgery alone, without prenatal exposure (Cummings et al., 1996). The finding of no effect of adult exposure to TCDD in rats is also consistent with the report from Cummings et al., (1996) where TCDD was shown to affect lesion diameter in rats at 10 mg TCDD/kg only if data from all time points (3, 6, 9, and 12 weeks) were pooled. In a previous report (Cummings et al., 1996), an increase in the occurrence of persistent estrus was shown for rats treated with 10 mg TCDD/kg at 3-week intervals for 12 weeks, but no relationship between the proportion of rats showing persistent estrus and the size of endometriotic lesions was shown. In a previous study, TCDD produced a decrease in ovarian weight in rats but not mice (Cummings et al., 1996), and in another report a TCDD-induced decrease in ovarian weight was seen in mice (Johnson et al., 1997). In this study, ovarian weight declined in mice and a trend toward smaller ovaries was evident in rats when the highest dose of TCDD was administered to adults. A decline in ovarian weight is consistent with a TCDD-induced disruption of cyclicity, possibly due to the antiestrogenic effects of TCDD on the hypothalamic–pituitary axis, as previously suggested for rats (Cummings et al., 1996).
PRENATAL TCDD AND ENDOMETRIOSIS
TCDD-induced ovulatory arrest could lead to a decrease in ovarian weight since corpora lutea are formed only as a result of ovulation, and corpora lutea comprise a significant portion of each ovary’s weight. As seen previously in mice (Theobald and Peterson, 1997), prenatal exposure to TCDD produced decreased uterine weights in both rats and mice in this study. The apparent modulation of uterine weight by adult exposure to TCDD of perinatally exposed mice may actually reflect the inherent variability in uterine weight that occurs on different days of the estrous cycle due to changes in serum levels of estrogen. Although TCDD had no adverse effect on body weight gain in mice, treated rats gained less weight than vehicle-treated controls, indicating a general toxic effect. In both rats and mice, the finding of increased liver weight is indicative of the well-known induction of hepatomegaly by TCDD. The patterns of results found for these two parameters were similar to those found in previous studies (Cummings et al., 1996). It is possible that exposure of dams to TCDD on a different day of gestation could have produced a different effect. GD8 exposure was chosen based on earlier work with rats by Gray et al. (1995). Although GD8 in the rat is not identical developmentally with GD8 in the mouse, in both cases the exposure occurs early in organogenesis. In any case, the long t 1/2 of TCDD ensures that exposure likely occurred through much of organogenesis. In summary, we have shown that perinatal plus adult exposure to TCDD promotes the growth of surgically induced endometriotic lesions in mice. TCDD had no effect on this parameter in rats. These data are not entirely consistent with previously published results, due most likely to variability inherent in the model. Ovarian weight appears to decline as a result of postnatal exposure to TCDD in both rats and mice. Prenatal exposure to TCDD produces a reduction in uterine weight in both species. Based on previous work, the mechanism by which TCDD promotes the growth of endometriotic lesions in adult rats and mice may involve both endocrine and immunologic characteristics. Current studies are directed toward elucidating such a mechanism. REFERENCES Birnbaum, L. S. (1994). The mechanism of dioxin toxicity: Relationship to risk assessment. Environ. Health Perspect. 102 (Suppl. 9), 157–167. Couture, L. A., Harris, M. W., and Birnbaum, L. S. (1990). Characterization of the peak period of sensitivity for the induction of hydronephrosis in C57BL/6N mice following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fund. Appl. Toxicol. 15, 142–150. Cummings, A. M., and Metcalf, J. L. (1995). Induction of endometriosis in mice: A new model sensitive to estrogen. Reprod. Toxicol. 9, 233–238.
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Cummings, A. M., Metcalf, J. L., and Birnbaum, L. (1996). Promotion of endometriosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats and mice: Time-dose dependence and species comparison. Toxicol. Appl. Pharmacol. 138, 131–139. D’Hooghe, T. M., Bambra, C. S., Suleman, M. A., Dunselman, G. A., Evers, H. L., and Koninckx, P. R. (1994). Development of a model of retrograde menstruation in baboons (Papio anubis). Fertil. Steril. 62, 635– 638. Dmowski, W. P., Braun, D., and Gebel, H. (1991). The immune system in endometriosis. In Modern Approaches to Endometriosis (E.J.Thomas and J.A. Rock, Eds.), pp. 97–111. Kluwer Academic, Boston. Gray, L. E., Jr., Kelce, W. R., Monosson, E., Ostby, J. S., and Birnbaum, L. S. (1995). Exposure to TCDD during development permanently alters reproductive function in male Long-Evans rats and hamsters: Reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol. Appl. Pharmacol. 131, 108 –118. Gray, L. E., Jr. and Ostby, J. S. (1995). In utero 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) alters reproductive morphology and function in female rat offspring. Toxicol. Appl. Pharmacol. 133, 285–294. Gray, L. E., Wolf, C., Mann, P., and Ostby, J. S. (1997). In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters reproductive development of female Long Evans hooded rat offspring. Toxicol. Appl. Pharmacol. 146(2):237–244. Haney, A. F. (1990). Etiology and histogenesis of endometriosis. Prog. Clin. Biol. Res. 323, 1–14. Holsapple, M. P., Snyder, N. K., Wood, S. C., and Morris, D. L. (1991). A review of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence:1991 update. Toxicology 69, 219 –255. Johnson, K. L., Cummings, A. M., and Birnbaum, L. S. (1997). Promotion of endometriosis in mice by polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls. Environ. Health Perspect. 105(7), 750 –755. Landers, J. P. and Bunce, N. J. (1991). The Ah receptor and the mechanism of dioxin toxicity. Biochem. J. 276, 273–287. Li, X., Johnson, D. C., and Rozman, K. K. (1995). Reproductive effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) in female rats: Ovulation, hormonal regulation, and possible mechanisms. Toxicol. Appl. Pharmacol. 133, 321–327. Rier, S. E., Martin, D. C., Bowman, R. E., Dmowski, W. P., and Becker, J. L. (1993). Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam. Appl. Toxicol. 21, 433– 441. Safe, S., Astroff, B., Harris, M., Zacharewski, T., Dickerson, R., Romkes, M., and Biegel, L. (1991). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds as antioestrogens: Characterization and mechanism of action. Pharmacol. Toxicol. 69, 400 – 409. SAS Institute Inc. (1985). SAS User’s Guide: Statistics, 5th ed. SAS Institute, Cary, NC. Theobald, H. M., and Peterson, R. E. (1997). In utero and lactational exposure to 2,3,7,8- tetrachlorodibenzo-p-dioxin: effects on development of the male and female reproductive system of the mouse. Toxicol. Appl. Pharmacol. 145(1):124 –135. Vernon, M. W. and Wilson, E. A. (1985). Studies on the surgical induction of endometriosis in the rat. Fertil. Steril. 44, 684 – 694. Yang, J. Z., and Foster, W. G. (1997). Continuous exposure to 2,3,7,8tetrachlorodibenzo-p-dioxin inhibits the growth of surgically induced endometriosis in the ovariectomized mouse treated with high dose estradiol. Toxicol. Ind. Health 13(1), 15–25.
