Role of Strain-Specific Reproductive Patterns in the Appearance of

Chapter 31

Role of Strain-Specific Reproductive Patterns in the Appearance of M a m m a r y Tumors in Atrazine-

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Treated Rats

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James W. Simpkins, J. Charles Eldridge, and Lawrence T. Wetzel 1

Department of Pharmacodynamics, University of Florida, Gainesville, FL 32610 Department of Physiology and Pharmacology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC 27157 Departmentof Toxicology, Novartis Crop Protection, Inc., P.O. Box 18300 Greensboro, NC 27419

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Atrazine has been a major agricultural herbicide in the U.S. for more than 25 years. It is used for the control of broadleaf and grass weeds in corn and sorghum crops. Because of its common use, the toxicity of atrazine has been the subject of many studies. Atrazine is not toxic with acute administration, with an oral and dermal LD of greater than 3,000 mg/kg. In tests of mutagenicity, atrazine have been negative in more than 50 tests (1). Atrazine is not a teratogen or a reproductive toxin, and lacks carcinogenic activity in male and female mice and Fischer 344 (F344) rats, as well as in male Sprague-Dawley (SD) rats. Five tests of the tumorogenicity of atrazine in SD rats have been conducted since the 1960s. Two of these tests, which assessed atrazine at doses up to 500 ppm, produced negative results, while 3 other studies have shown an earlier time of onset and/or an increased incidence of mammary tumors (2-4). With the exception of one study (4), the earlier onset of mammary tumors occurred at doses ≥a maximum tolerated dose (2,3). A no-observed-effect-level (NOEL) for tumorogenicity was established in all studies. Atrazine is not a mutagen (1), a direct acting carcinogen and it has no intrinsic estrogenic activity (5,6). The increased incidence and/or earlier age of appearance of mammary tumors in female SD, but not Fischer 344 rats warrants an evaluation of the strainspecificity of this response. The results discussed here present strong evidence that the specificity of the tumor-enhancing effects of atrazine in the female SD rat are the result of a treatment-related earlier appearance of persistent estrus in that strain. 50

©1998 American Chemical Society In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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400 Rodent Strains and Tumor Incidence


The background incidence of mammary tumors varies greatly among rodent strains (7,8). The background incidence of spontaneous mammary tumors in 2 year old female SD rats ranges as high as 70 to 80% (7,8) which is in marked contrast to the very low incidence of spontaneous mammary tumors in F-344 rats (2,3). Clearly, the SD female rat is very susceptible to factors which promote occurrence of mammary tumors, while the F-344 rat is comparatively resistant. Endocrine Factors that Enhance the Growth of Mammary Tumors It has been clearly established that the ovarian steroid hormone, estrogen, and the anterior pituitary peptide hormone, prolactin which plays a role in the normal development and physiology of the breast, are also involved in the rate of mammary tumor growth (7). Chronic elevation of estrogens (9-11) or prolactin (12,13) increases the incidence and decrease the onset time of mammary tumors in either SD or F-344 rats. As such, treatment with agents that increase chronic secretion of estrogens and/or prolactin would be expected to increase the occurrence of mammary tumors in these strains. Comparison of the SD and F-344 Rats as Surrogate Models for Human Assessment of Mammary Tumors In women, the passage from normal menstrual cycles into reproductive senescence results from exhaustion of ovarian follicles, and is accompanied by a precipitous decline in ovarian steroid hormones, particularly, estradiol (14). At the menopause, and for a considerable time thereafter, the hypothalmic-pituitary axis still has the capacity to regulate anterior pituitary hormone secretion. With the decline in estrogens, serum L H and FSH secretion increases markedly and can be suppressed by hormone replacement therapy (14). In addition, regimens of estrogen treatment that induce an L H surge are also able to induce L H surges in postmenopausal women (15). Therefore, the mechanism for transduction of the estrogen signal to an L H surge appears to be intact in postmenopausal women. By contrast, in the aging SD and related strains of rats, the ovary retains a substantial number of follicles (16,17). Reproductive senescence in the SD rat (Figure 1) appears to result from a breakdown in the capacity of the hypothalmus to convert the estrogen signal from the ovary into an L H surge sufficient to induce ovulation (16-19). The breakdown is evidenced by a gradual transition from normal 4 to 5 day estrous cycles to extended periods of estrus with continuous endogenous estrogen secretion, and finally to a state of persistent estrus (20,21). This persistent estrus state, which can last the remainder of the animal's life (20,21), has a profile of moderate, continuous elevation and secretion of serum estradiol and low levels of serum progesterone (21). Serum prolactin levels are elevated as a result of the increase in estradiol, which acts on the anterior pituitary to stimulate prolactin synthesis and secretion (22). Given this markedly different endocrine environment during reproductive decline in SD rats and humans, the SD rat seems to be a poor surrogate model for reproductive senescence in the human female.

In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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Normal Young


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Figure 1. Schematic Representation of Preovulatory L H Surges in the Young Adult (Upper Panel) and Mid-Aged (Lower Panel) Female Sprague-Dawley (SD) Rat. In young SD rats, a rising estrogen secretion from developing ovarian follicles primes a surge of L H after noon of the day preceding ovulation. Estrogen secretion then declines as the follicular source disintegrates with ovulation. In middle age, declining hypothalamic function prohibits rising follicular estrogen from inducing a sufficient L H signal, so ovulation fails to occur and the ovarian follicles persist. Instead of normally cyclic elevations of estrogen, mid-aged SD rats maintain estrogen secretion at continuously elevated levels. In this case, vaginal cytology displays repeated days of heavy cornification.



402 The F-344 rat exhibits a late life reproductive senescence that runs a very different course than that observed in the SD rat. Through about 1.5 years of age, the majority of F-344 rats maintain normal 4 to 5 day estrous cycles (23). By 2 years of age, the rats have entered a senescent reproductive pattern of normal estrous cycles interspersed with periods of extended maintenance of the corpus luteum and the resultant hypersecretion of ovarian progesterone (23,21). This condition is appropriately called repeated pseudopregnancy. Anterior pituitary weights remain normal through 2 years of age, serum prolactin is slightly elevated and serum L H concentrations are slightly reduced (23). More importantly, the hypothalamic-pituitary axis maintains the capacity to mediate the estrogen-induced hypersecretion of L H and normal ovulation that is common in aged F-344 rats (2325). The only known neuroendocrine defect in the F-344 rat is the inability to reduce episodic prolactin surges (26,23), which maintains the corpus luteum (27). While not completely similar to the human in its pattern of reproductive senescence, the F-344 does share with the human female the following features; both have a late life reproductive senescence; both experience low estrogen levels during late life, and both maintain the ability to control L H secretion during reproductive senescence. As such, the F-344 rat more closely models the human female than does the SD rat. Evaluation of the Mode of Action of Atrazine in the Strain-Specificity of the Mammary Tumorigenic Effects in the SD Rat It has been proposed that the strain- and sex specificity of the earlier appearance of tumors with 2 years of high-dose atrazine feeding in the SD rat was a result of the superimposition of an atrazine effect on the early appearance of persistent estrus in the SD rat (28,29,2,3). It is important to recognize that atrazine feeding results in an earlier appearance of a spontaneous reproductive senescence event in the SD rat, i.e., mammary tumors. Exposure to atrazine does not result in the development of a new mammary pathology. If this hypothesis is correct, then two predictions should follow. First, atrazine feeding at the MTD, a dose associated with an earlier appearance of mammary tumors in the SD female rat, should cause an earlier appearance of persistent estrus in the SD, but not in the F-344 rat. The chapter by Eldridge et al in this same volume documents that atrazine-induced early persistent estrus does occur. Second, atrazine treatment should induce an earlier appearance of a neuroendocrine deficit that would lead to the appearance of persistent estrus in SD female rats, i.e., a decrease or attenuation, of the proestrous L H surge. Indeed, a preliminary study by Cooper and colleagues (1996) (30) had suggested just this possibility. The evidence for the latter effect of atrazine treatment is the subject of the remainder of this chapter. Effects of Atrazine Treatment on the Estrogen-Induced L H and Prolactin Surges: Acute Study As an initial evaluation of the effects of atrazine treatment on the L H surge, female rats were ovariectomized and simultaneously implanted with an estradiolcontaining sustained-release capsule (4mg/ml sesame seed oil). This mode of estradiol administration produced levels seen during normal preovulatory surges of L H and has also been shown to produce daily surges of L H in young rats (18,19). In Triazine Herbicides: Risk Assessment; Ballantine, L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


403 Atrazine was then administered by gavage daily at a dose of 300 mg/kg body weight for 3 days. On the third day of treatment, animals were sacrificed by decapitation at 11:00, 13:00, 15:00, 18:00 and 22:00 hours, a time during which the L H surge was expected. The vehicle-treated animals showed the expected estrogen-induced L H surge, with L H levels increasing at 15:00 h, peaking at 18:00 h and diminishing at 22:00 h (Figure 2). By contrast, the atrazine-treated rats failed to show an increase in serum L H at any interval through 18:00 h and exhibited only a slight increase in L H at 22:00 h. Assessment of prolactin concentration in these same animals revealed that the estrogen-induced surge of this hormone was also blunted or perhaps delayed, as reflected by diminished prolactin levels at the times of the peak prolactin concentrations in control animals at 15:00 h and 18:00 h (Figure 3). These results indicate that atrazine treatment at a level > the MTD was able to disrupt a neuroendocrine mechanism that transduces the estrogen signal to an L H response. As such, the hypothesis that atrazine blunts the preovulatory L H surge was supported. Additionally, in as much as the prolactin surge was also affected, it appears that treatment altered a very fundamental mechanism that converts the estrogen signal into neuroendocrine responses. Dose-Dependent Effects of Atrazine on the Estrogen-Induced L H Surge: 4Week Study To determine the dose-dependent effects of atrazine on the estrogen-induced L H surge, an experiment similar in design to the acute study was conducted. SpragueDawley rats were treated by gavage, to 0,2.5,5.0,40, or 200 mg/kg/day doses of atrazine for 30 days. Three days prior to sampling, animals were ovariectomized and immediately implanted with a capsule containing estradiol as described above previously. On the 30th day of atrazine treatment, animals were sacrificed at 6 intervals, from 13:00 to 23:00. As expected, the vehicle-treated control showed a "preovulatory-like" L H surge that peaked at 16:00 to 18:00 and declined thereafter (Figure 4). Responses to 2.5, 5 and 40 mg/kg were quite similar to vehicle-dosed animals. In contrast to these low doses of atrazine, the 200 mg/kg dose of atrazine caused a reduction of L H at the peak time of 16:00 (Figure 4). Because the time of peak L H secretion in response to estrogen treatment is variable, another set of animals, dosed (0, 2.5, 5.0, 40 or 200 mg/kg/day) for 30 days, was sampled sequentially on the third day after ovariectomy and estrogen implantation. L H data were normalized to the time of the peak response and, when expressed on this basis, the effect of high dose atrazine treatment becomes even more clear. The control, 2.5 and 5 mg/kg doses of atrazine showed similar increases in L H secretion to peak levels and a decline over the next 4 h (Figure 5).


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Figure 2. Suppression of the Estrogen-Induced L H Surge After 3 Days of Atrazine Treatment. Ovariectomized young adult SD rats were implanted with an estradiol-containing silastic capsule and were administered atrazine (ATR) by gavage, 300 mg/kg/day for 3 days, or gavage vehicle (VEH). Blood samples were collected after decapitation at the indicated times and plasmas were analyzed for L H . Points represent means + S.E.M. of 10 animals per interval. Group mean values in the ATR-treated animals were significandy different from V E H means at each time interval except 13.00 (p

Role of Strain-Specific Reproductive Patterns in the Appearance of

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