1



### The Influence of 1, 2 , and 4 Days of Atrazine Treatments on the LH Surge in Ovariectomized / Estr adiol

- primed Rats: Preliminary Report to the Office of Chemical Safety and Pollution Prevention

Jerome Goldman, Lori Davis, Ashley Murr and Ralph Cooper

Endocrine Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711

This internal report was prepared for the US EPA O CSPP and submitted on 28 April 2011

Disclaimer: This report has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency and approved for dissemination. Approval does not signif y that the contents necessarily reflect the views or policy of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.2

Introduction
Previous studies from our laboratory had shown that the chlorotriazine herbicide atrazine administered to cycling female rats for 4 consecutive days diminished the surge of luteinizing hormone (LH) from the pituitary that serves as the proximal trigger for ovulation (Fig. 1 ) (Cooper et al., 2000; 2007)

. Perhaps the most compelling evi dence supporting this finding is the fact that atrazine exposure alters the pulsatile release of gonadotropin releasing hormone (GnRH) , the final common signal emanating from the brain to regulate LH release (Cooper et al.,
2 007; Foradori et al., 2009). Recent studies from our laboratory on both the hypothalamic

pituitary

- gonadal (HPG) and hypothalamic

- pituitary

- adrenal (HPA) axes have continued to provide some insight into the sequence of neuroendocrine changes that take pla ce following the initial exposure to this chemical and how these changes ultimately lead to a disruption of gonadal function. This includes the observation that atrazine (and atrazine metabolites) activates the HPA axis within minutes in both males (Laws et al., 2009) and females (Fraites et al., 2009). Below we discuss the effects of atrazine on these two broad ne uro endocrine axes and those events that lead to impaired LH secretion

. As indicated, the accepted mode of action for the atrazine

- induced i mpairment of LH secretion is a disruption in the central (CNS) mec hanisms controlling LH release (i.e., impaired GnRH release) (Cooper et al., 2007; Foradori et al., 2009). Direct exposure of pituitary tissue in vitro did not affect LH secretion (Fig. 2). This central mode of action was reviewed by the 2003 atrazine Science Advisory Panel and supported by research from several laboratories (McMullin et al., 2004; Foradori et al., 2009). However, there is little known about the exact molecular or

neuro nal events responsible for an a trazine

- induced change in GnRH /LH release. It is assumed that the changes in GnRH occur as a result of either (1) a direct effect of the chlorotriazines on the GnRH neurons themselves (2) an effect of the herbicide on the n euronal mechanisms known to regulate GnRH (and thus affect GnRH cellular activity indirectly ) or (3) an effect on both the GnRH neurons and those neurons regulating GnRH activity.3

R ecent studies revealed that atrazine (and exposure to atrazine metaboli tes) leads to a rapid (within minutes) increase in both adrenocorticotropin (ACTH) secretion and adrenal steroid hormone pro d uction ( corticosterone and progesterone) in both male ( Laws et al., 2009) an d female (Fraites et al., 2009) rats . The rapidity of this dose

- dependent, atrazine

- induced HPA
response , and the finding that this response can be initiated following intravenous injection with the atrazine metabolite desisopropylatrazine (DIA) (Fraites et al., 2009) suggested that atrazine activates the a drenal axis by targeting the CNS ( putatively the hypothalam us ) directly to alter the control of ACTH. In addition, other data (Fraites et al., 2009) indicate d that it is unlikely that atrazine causes a non - specific stress respon s e in the animal. The significance of these observations for interpreting the events leading to impaired LH secretion becomes clear when one considers the role of progesterone (both adrenal and ovarian) i n the regulation of LH , as well at the impact that an inappropriate ex posure to corticosterone has on the HPG axis in general.
Estradiol and progesterone have long been known to play coordinate roles in the generation of the surge (Mann & Barraclough, 1973). In a f emale rat exhibiting a normal 4

- day estrous cycle, estradi ol begins to rise on diestrus day 2, reaching a midday apex on the following day of proestrus. This increase serves to increase the responsiveness of the hypothalamic mechanisms that promote the secretion of GnRH into the hypothalamic

- pituitary portal ves sels and the subsequent surge of LH on the afternoon of proestrus. Progesterone, on the other hand, shows a rise beginning around noontime on proestrus and acts synergistically with estradiol to generate a surge (Brown

- G rant and Naftolin, 1972). This effect of progesterone in combination with estradiol is clearly demonstrated in the ovariectomized rat ( Fig. 3 )

. Progesterone treatment alone in these female s is with out effect on the induction of a surge (e.g., Caligaris et al., 1971) . In contrast to t he synergistic effect of progesterone, longer
exposures are ess entially inhibitory (e.g., Caligaris et al., 1971; Banks & Freeman, 1978; 1980; Prilusky et al., 1984 ). In fact, LH in general and the ovulatory s urge of LH in particular are suppressed by extended elevations in progesterone and other progestins. This is the basis for the actio n of many oral contraceptives. Furthermore, increased levels of progesterone during a woman’s pregnancy, secreted first from the ovaries and subsequently from the p lacenta,4

prevent the appearance of a surge and ovulation over that time. Finally, it is noteworthy that treatment with progestins also lowers serum gonadotropins and testosterone production in men (e.g., Amory, 2004) and in combination with a synthetic a ndrogen has promise as a male hormonal contraceptive (Roth and Amory, 2011)

. Similarly, extended exposure s to corticosterone also impair normal hypothalamic

- pituitary

- gonadal function. This biphasic effect of progesterone on the LH surge suggest s that t he effectiveness of atrazine in modifying LH secretion depends not only on the dosage administered, but also on the duration of exposure prior to the appearance of the surge. Thus, we hypothesized that a single dose of atrazine (which increases adrenal p rogesterone secretion in the ovariectomized rat) administered on t he expected day of the estrogen

- induced LH surge would incr ease the amount of LH released ( peak amplitude and area under the curve). In contrast, we also hypothesized that multiple days of exposure will suppress both the peak height of the surg e and the amount of LH secreted as a result of the atrazine

- induced daily increase in both progesterone and corticosterone.

Materials and methods

a. Animals
Upon arrival, 60

- day

- old Sprague

– Dawl ey female rats (Charles River Labs, Raleigh, NC) were provided with food/water ad libitum and acclimated to their new surroundings. They were housed under a 14

- h light:10

- h dark photoperiod (lights on 05 00 h) with controlled temperature (22 ± 2 0 C) and hum idity (40

– 50%). Vaginal lavages were taken daily for 2 weeks to determine cycling status, and any non

- cycling animals were eliminated from the study. All experimental procedures conformed to NIH standards for laboratory animal research and were approved by the Animal Care and Use Committee at the National Health and Environmental Effects Research Laboratory.
b. Dosing Materials
Atrazine (97.1% purity) was a gift from Syngenta Crop Protection, Inc. (Greensboro, NC). It was administered by oral gavage in a suspension of 1% methyl cellulose (M

- 7140, Sigma Chemical Co., St. Louis, MO) in reverse osmosis

- purified deionized water. The dosing volume was 5.0 ml /5

kg body weight. The dose selecte d (100 mg/kg) was based upon previous results (F ig. 4 ) using an ovariectomized (OVX) / estradiol

- primed animal model that showed a complete suppre ssion of the LH surge in response to three daily treatments at this concentration

. Control animals received 1% methyl cellulose vehicle (5.0 ml / kg body we ight)

.

c . Experimental Procedures
Previously cycling rats were bilaterally OVX under ketamine/xylazine anesthesia and subcutaneously implanted with a 6 mm silastic capsule containing estradiol benzoate (4 mg/ml in sesame oil) to induce a daily surge of LH (L egan et al., 1975). Capsules were soaked in saline prior to implantation to preclude an initial uneven rise in circulating concentrations before they would fall to a relatively constant level. Th ree days after surgery , ~250 l aliquots of blood were gent ly expressed from a nick in a lateral tail vein into small serum separation tubes at 1400, 1600 and 18 00 h. The animals were then killed at 20 00 h and trunk blood taken. O n the day blood was sampled , all animals were dosed at 1300h. One group received treatment only on that day. A second group was treated daily for 2 consecutive days, while a third was dosed for 4 days beginning just prior to ovariectomy. This experiment al paradigm is shown in Figure
5

. d. Hormonal Assays
Sera from all time points w ere analyzed for LH by radioimmunoassay using materials obtained from the National Hormone and Peptide Program through Dr. A.F. Parlow ( University of California

- Los Angeles Medical Center, Torrance, CA). Tracer iodination with 125 I was performed in

- house using chloramine

- T, and e ach labeled hormone was separated from unreacted iodide by gel filtration chromatography. The assays were carried out according to

recommen dations accompanying each kit, with the sensitivity optimized by a 24 h co

in cubation of sam ple and first antibody prior to the addition of 125 I

- labeled tracer. G oat anti

rabbit gamma - globulin (Calbiochem , San Diego, CA ) was used as second antibody. All samples

were run in duplicate and included in a single as say, with a sensitivity of 0.11 ng/ml, and an 5.8% intra

- assay coefficient of variation.6

Serum progesterone (all sampling times) and estradiol (2000h) were analyzed in duplicate using antibody

- coated tube kits purchased from Siemens Healthcare Diagnostic (Deerfield, IL). The sensitivi ties were 0.08 ng/ml (progesterone) and 10 pg/ml (estradiol). Intra

- assay coefficients of variation were less than 10% for both steroid hormones.
e. Statistical Analyses

Serial LH samples were analyzed by a repeated measures analysis of variance ( SAS 9.1 , Cary, NC). Because the time of appearance of a surge peak can vary among animals ( in our studies at the 1600 or 1800h sampling tim es ) , the four LH values fo r each animal were shifted along the horizontal axis so that analyses could be performed on the peaks (or highest LH concentration s ), along with times designated as

- 6,

- 4,

- 2, + 2, + 4, and + 6 h ours from the peak

. S ampled progesterone concentrations were also analyzed by a repeated measures analysis of varianc e ( SAS 9.1 , Cary, NC). Areas under th e curve (AUC) for the sampling times were determined using MedCalc software (Mariakerke, Belgium) , and the data for the different dosing regimens analyzed by t

- test. Estradiol concentrations were analyzed by analysis of variance. Statistical significance was set at p < 0.05 for all statistical tests.

Results Figure 6 shows the progeste rone response to dosing at the four sampling times. For animals receiving only a single dose, a statistically significant elevation is present at 1400h, 1 hour after atrazine treatment, but thereafter concentrations are comparable to the control group. However, after 4 days of daily exposures no differences were present at any of th e sampling times , results that were comparable to those seen after 2 days of dosing (data not shown). No difference s in estradiol from the implanted capsules were seen in trunk blood taken at 2000h (Fig. 7)

. For LH, 4 days of dosing caused a significant suppression in peak height and AUC throug h the serial samplings (Fig. 8 ). In response to two daily tre atments, peak height was not significantly diminished, although AUC remained decreased. The single administration of7

atrazine, on the other hand, caused a marked and statistically significant elevation in both the peak height of the surge and AUC, the latter value was 184% over the control AUC

.

Discussion

The disparity in the influence of a single vs. multiple daily doses of atrazine on the LH surge is like ly attributable to an influence of this herbicide on adrenal progesterone and perhaps corticosterone secretion . This conclusion is based on what is known about the nature of the
te mporal relationships between these two adrenal steroids and the sur ge. From the data reported by Fraites et al. (2009), atrazine was able to induce a rapid elevation in progesterone
to approximately 19 ng/ml just 15 minutes after a single 75 mg/kg exposure ( greater than 4

- fold over controls ) (Fig. 9) . Although in the p resent work, we did not observe a significant difference from controls a t 1 hour following the 2 nd or 4 th daily treatments , it is likely that progesterone was elevated prior to this time. At 15 minutes after the last of 4 daily atrazine doses, Fraites found that progesterone , while statistically significant at 7 ng/ml , w as down to 3

- fold over controls, providing evidence of a decline in the stimulation of the adrenal progesterone response with multiple exposures. It would appear then that in th e present experiment, the lack of a statistically significant atrazine

- induce d increase m a y well be due to a reduction in the magnitude of the rapid increase in prog esterone, so that by 1 hour circulating levels have bee n sufficiently cleared to approach c ontrol concentrations

. Because progesterone and corticosterone are co

- secreted from the adrenals, both steroids may have contribute d to a combined effec t on the LH surge. However, it appears unlikely that an increase in blood concentrations of corticosterone was involved in the 1 day effect o n the surge, since previous data obtained employing this OVX / estradiol

- primed animal model showed no difference in the surge when a single injection of corticosterone was given 5 hours prior to the anticipated LH peak (Fig. 10). In contrast, corticosterone administration on the previous day produced a statistically significant attenuation in LH, indicating that in the present experiments a combination of both adrenal steroids , along with a possible no n - steroid

- linked mode of action , could have induced the observed response to multiple atrazine dosages.8

From what is known about the relationships between estradiol and progesterone on the temporal course of facilitation and inhibition of the LH surge, it doesn’t seem that the present effects of atrazine are attributable to changes in circulating estradiol. Although Sanderson et al. (2000) found that estradiol concentrations were elevated following in vitro exposures of the H295R adrenocortical tumor cells to atrazine, this effect was due to an augmentation in synthesis through an increase in aromatase activity. In the present experiment, estradiol is exogenously provided via implant, and the concentration should remain c onsistent within a defined range.
In summary, t he principal findings of this preliminary report demonstrate two important factors concerning the HPA/HPG interactions. First, a single dose of atrazine does affect the adrenal axis by increasing the amount o f LH secreted during the afternoon LH surge. The fact that this same single dose of atrazine increases adrenal hormone secretion (primarily progester one) would explain the enhancement of the surge. Second, multiple (four daily) doses of atrazine resulted in a suppression of the LH surge. Again, we attribute this ultimate suppression of the surge to the activation of adrenal hormone secretion following each dose of atrazine. As discussed above, the effect of continued exposure to both progesterone and co rticosterone on LH secretion in general and on the LH surge in particular is well known. Although this preliminary study employe d ovariectomized animals and a single dose of atrazine, it demonstrates that the changes in LH secretion can be predicted by a recognition of the capacity of this herbicide to affect adrenal steroid hormone production and the temporal impact of these steroids on LH. These data, when considered in combination with those results describing the hypothalamic response to atrazine expo sure, depict a complex relationship between the HPG and HPA axes in the initiation of a cascade of neuroendocrine changes that ultimately lead to altered ovarian function.9

References

. Amory JK

. Testosterone/progestin regimens: a realistic option for male contraception? (2005)

. Curr Opin Investig Drugs. 5 :1025

- 10 30

. Banks, JA, Freeman, ME. (1978). The temporal requirement of p rogesterone on proestrus for extinction of the estrogen

- induced daily signal controlling luteinizing hormone release in the rat. Endocrinology 102:426

- 432. Banks, JA, Freeman, ME. (1980). Inhibition of the daily LH release mechanism by progesterone acting at the hypothalamus. Biol. Reprod. 22:217

- 222.

B rown - Grant, K, Naftolin, F. (1972). Facilitation of LH secretion in the female rat by progesterone. J. Endocrinol. 53:37

- 46. Cooper, RL, Stoker, TE, Tyrey, L, Goldman, JM, McElroy, WK . (2000). Atrazine disrup ts the hypothalamic control of pituitary

- ovarian function. Toxicol. Sci. 53:297

- 307. Cooper, RL, Laws, SC, Das, PC, Narotsky, MG, Goldman, JM, Tyrey, EL, Stoker, TE. (2007). Atrazine and reproductive function: Mode and mechanism of action studies. Birth De f. Res., pt. B. 80: 98

- 112. Fraites MJ, Cooper RL, Buckalew A, Jayaraman S, Mills L, Laws SC. (2009). Characterization of the hypothalamic

- pituitary

- adrenal axis response to atrazine and metabolites in the female rat. Toxicol. Sci . 112: 88

- 99. Foradori CD, Hinds LR, Hanneman WH, Legare ME, Clay CM, Handa RJ. (2009). Atrazine inhibits pulsatile luteinizing hormone release without altering pituitary sensitivity to a gonadotropin

releasing hormone receptor agonist in female Wistar rats. Biol Reprod

. 81:40

- 45. Goldman, JM, Cooper, RL. (2010). The impact of centrally acting p esticidal/ environmental t oxicants on the neuroendocrine regulation of r eproductive function in the f ema le rodent:
r elevance to human reproductive risk a ssessment . In: JC Eldridg e, JT Stevens (Eds.), Endocrine Toxicology (3rd edition). In forma Healthcare: London. pp. 210

- 239. Laws ̧ SC, Hotchkiss, M, Ferrell, J, Jayaraman, S, Mills, L, Modic, W, Tinfo, N, Fraites, M, Stoker, T, Cooper, R. (2009) Chlorotriazine herbicides and meta bolites activate an ACTH

- dependent release of corticosterone in male Wistar rats. Toxicol. Sci. 112:78

- 87.10

Legan , SJ, Coon, GA, Karsch, FJ (1975). Role of estrogen as initiator of daily LH surges in the ovariectomized rat. Endocrinology 96:50

- 56.
Mann, DR, Barraclough, CA. (1973). Role of estrogen and progesterone in facilitating LH release in 4

- day cyclic rats. Endocrinology 93:694

- 699. McMullin, TS, Andersen, ME, Nagahara, A, Lund, TD, Pak, T, Handa, RJ, Hanneman, W H. (2004). Evidence that atrazine and di aminochlorotriazine inhibit the estrogen/progesterone induced surge of luteinizing hormone in female Sprague

- Dawley rats without changing estrogen receptor action . Toxicol. Sci. 79: 278

- 286

. Prilusky, J, Vermouth, NT, Deis, RP. (1984). A dual modulatory effect of progesterone on the LHRH

- induced LH release. J. Steroid. Biochem. 21:107

- 110. Roth, MY, Amory, JK. (2011). Pharmacologic development of male hormonal contraceptive agents. Clin. Pharmacol. Ther. 89:133

- 136. Sanderson JT, Seinen W, Giesy JP, van den Berg M. (2000). 2

- Chloro

- s - triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estrogenicity? Toxicol. Sci. 54:121

- 127.11

Figure 1. Mean ( + SEM) concentrations of LH during t he afternoon of vaginal proestrus in confirmed regularly cycling rats dosed with atrazine (mg/kg) by gavage at 12:00 hr for 4 consecutive days. * p <0.05 versus control at same time point (from Cooper et al. Birth De fects Res. (Part B), 80:98

- 112, 2007).12

Fig. 2. 100 M a trazine has no effect on LH secretion from the pituitary when tested in vitro. Control and treated pituitary fragments were incubated for 2.5 hr and then exposed to two pulses of GnRH (85 nM) followed by a KCI challenge (60 mM). LH release was not different at any of the time points examined ( from Cooper et al. Atrazine disrupts the hypothalamic control of pituitary

- ovarian function. Toxicol Sci 53 :297

– 307, 2000)13

Figure 3. Influence of a subcutaneous (SQ ) injection of progesterone on an LH surge in ovariectomized rats implanted with an estradiol capsule at the time of surgery. Small aliquots of tail blood were removed over the afternoon of the third day after ovariectomy in rats with or without a SQ injection of progesterone (2.5 mg/0.2 ml sesame oil) administered just prior to serial sampling for LH (Goldman et al. , 2010)

. Concentrations are indicated in ng/ml + SEM. Progesterone given alone at tha t time would be unable to elicit a surge.14

Figure 4. Th e influence of three daily oral atrazine treatments on the LH surge in ovariectomized / estradiol

- primed rats. Trunk blood was taken from animals killed at 0, 1, 3 and 6 hours after the last 1300h dosing. *p<0.05 c ompared to the 0 mg/kg control group at the same time point

.15

Figu re 5. Study design using the ovariectomized / estradiol

- prime d model. Estradiol implants are inserted at the time of ovariectomy (OVX) , releasing the hormone over the ensuing 3 day s (horizontal arrows). Oral exposures to methylcellulose vehicle or 100 mg/k g atrazine are administered for 1, 2 , or 4 days at 1300h (vertical arrows) , and s erial blood samples are taken at 1400, 1600, 1800 , and 2000h (green triangles)

. The white and shaded boxes indicate the light:dark portions of the photoperiod.16

Figure 6. Serum concentrations of progesterone (ng/ml + SEM) over the sampling times for the 1

- and 4

- day atrazine (0 and 100 mg/kg) exposures. ** P <0.01 for the difference between the two doses at that time.17

Figure 7 . Serum estradiol concentratio ns (pg/ml + SEM) for the treatment groups determined at the 2000h sampling time.18

Figure 8 . The LH surge (ng/ml + SEM) and areas under the curve in response to 4, 2 and 1 day of atrazine treatment. The numbers in parentheses indicate the group sizes for each of the treatments. The number s within the 100 mg/kg columns at the right indi cate the percent change from the 0 mg/kg group. * p <0.05, ** p <0.0119

Figure 9

. Comparison of the increase in progesterone (ng/ml + SEM) at 15 minutes in response to 1 and 4 daily exposure s to 75 mg atrazine/kg . ** p <0.0120

Figure 10

. Influence on the LH surge of corticosterone administration (10 mg/rat) to ovariectomized / estradiol

- primed rats at two different times, 5 or 20 hours prior to the anticipa ted surge peak. * p <0.05 vs. control group at that time.