Methoxychlor As a Model for Environmental Estrogens

Critical Reviews in Toxicology

ISSN: 1040-8444 (Print) 1547-6898 (Online) Journal homepage: http://www.tandfonline.com/loi/itxc20

Methoxychlor as a Model for Environmental Estrogens

Audrey M. Cummings

To cite this article: Audrey M. Cummings (1997) Methoxychlor as a Model for Environmental Estrogens, Critical Reviews in Toxicology, 27:4, 367-379

To link to this article: http://dx.doi.org/10.3109/10408449709089899

Published online: 25 Sep 2008.

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Download by: [University of Lethbridge] Date: 26 September 2015, At: 00:46 Critical Reviews in Toxicology, 27(4):367-379 ( 1997)

Methoxychlor as a Model for Environmental Estrogens

Audrey M. Cummings * Endocrinology Branch, Reproductive Toxicology Division, NHEERL, USEPA, Research Triangle Park, NC

* Address all correspondence to: Audrey M. Cummings MD-72, NHEERL, USEPA, Research Triangle Park, NC 277 1 1

ABSTRACT: Estrogens can have a variety of physiological effects, especially on the reproductive system. Chemicals with estrogenic activity that are present in the environment may thus be considered potentially hazardous to development and/or reproduction. Methoxychlor is one such chemical, a chlorinated hydrocarbon pesticide with proestrogenic activity. Metabolism of the chemical either in vivo or using liver microsomes produces 2,2-bis(p-hydroxyphenyl)- 1,l,l -trichloroethane (HPTE), the active estrogenic form, and the delineation of this mechanism is reviewed herein. When administered in vivo, methoxychlor has adverse effects on fertility, early pregnancy, and in utero development in females as well as adverse effects on adult males such as altered social behavior following prenatal exposure to methoxychlor. Effects of methoxychlor on the female have been studied extensively, whereas reports on the chemical's effects on males are less common. From the studies reviewed here, the reproductive toxicity of methoxychlor is evident, but the significance of this toxicity with respect to human health remains to be determined.

KEY WORDS: methoxychlor, proestrogen, HPTE, estrogenic pesticide.

1. INTRODUCTION mone binds to its receptor, a conformational change results in active or transformed receptors. The ac- Any exogenously administered estrogen can tive, dimer form binds to the genome at an accep- have toxic effects, depending on the dose applied. tor site or steroid response element (SRE), thus For example, estrogen administered during early activating or inhibiting the transcription of spe- pregnancy can block implantation or terminate cific messenger WAS.Estrogen-induced mRNAs pregnancy.lg2This hormone can also accelerate em- are subsequently processed and then translated by bryo transport through the reproductive tract that, in ribosomes in the cytoplasm to produce a new itself, can impair early pregnan~y.~Effects of ex- protein, such as an enzyme. This new protein thus cess estrogen on the decidualization of the uterus, alters the function of the cell and, ultimately, the a growth and differentiation of the endometrium hormone target tissue.6,

Downloaded by [University of Lethbridge] at 00:46 26 September 2015 that is necessary in the rat and human for implanta- The uterus is a primary target organ for estro- tion, can include a reduction or obliteration of decid- gens. Estrogen is uterotrophic. The administration of ual tissue formati~n.~?~However, the same hormone estrogen to immature or ovariectomized rats will that has all of these effects is necessary for preg- result in the rapid growth of uterine tissue, imbibi- nancy and decidualization when present at the tion of fluid, and increase in uterine weight by 6 h.7 proper levels and in proportion with progester- Estradiol can promote the activity of a number of one. uterine enzymes, including omithine decarboxy- The mechanism by which estrogen exerts many lase,8 creatine kinase?JO uterine peroxidase,I1J2 of its effects is well known (Figure 1). The circulat- glucose-6-phosphate dehydr~genase,'~and others. ing hormone binds to a receptor molecule in the The uterotrophic response and these specific en- cytoplasm or nucleus of target cells (those cells of zyme activities have been used as markers for the organs containing receptors). When the steroid hor- action of environmental estrogens in animals.

1040-8444/97/$.50 0 1997 by CRC Press LLC 367 Downloaded by [University of Lethbridge] at 00:46 26 September 2015

Cell Membrane Nuciear Membrane

RWA

UAA

FIGURE 1. Molecular pathway for steroid hormone action. S, steroid hormone, NHP, non-histone protein. (Reproduced with permission.6) Estrogens have significant effects following of 10 to 100 mgkg; the chemical also produced perinatal exposure, specifically in brain and precocious vaginal opening, persistent vaginal reproductive tract development. Perinatal admin- estrus, and anovulation.22The polychlorinated istration of estrogen can induce a male pattern of biphenyl (PCB) Aroclor 1221 produced uterine brain differentiati~n.’~The synthetic estrogen di- growth in weanling rats, and when administered ethylstilbestrol (DES), administered to rodents to pups on days 2 and 3 of life it induced preco- perinatally, can reduce fertility and produce struc- cious puberty and persistent vaginal estrus, which tural abnormalities of the oviduct, uterus, cervix, are hallmarks of an estrogen.23Using the 18-h and ~agina.’~J~DES also alters sex differentia- glycogen response of the immature rat uterus, tion of the rodent central nervous system, where, Bitman and evaluated a number of PCB for example, the sexually dimorphic nucleus in compounds. PCB’s numbers 43, 44, 45, and 46 the preoptic area (SDN-POA) of the rat hypo- (corresponding to Aroclors 1221,1232,1242,and thalamus is considerably larger in male rats than 1248) were shown to be estrogenically active. 4- in females.I4 Octylphenol is a wastewater treatment plant ef- DDT was one of the first estrogenic pesticides fluent chemical that is a breakdown product of found in the environment to be banned. Original- detergents. It blocks early pregnancy in rats (un- ly, Burlington and LindemanI7demonstrated that published data) and has been shown to be estro- DDT inhibited testes growth and secondary genic.25 sexual characteristics in cockerels, suggesting Methoxychlor (MXC) is a pesticide that was that it was estrogenic. Later it was reported that developed to replace DDT. Considerable inter- 2,2’-bis-(pp-hydroxyphenyl)-1, 1 , 1 -trichloroethane est has been paid to MXC due to the fact that it appeared to have estrogenic activity, while DDT continues to be used, is found in the environ- did not.’* When o,p’ DDT (l,l,l-trichloro-2(p- ment, and is a proestrogen. chlorophenyl)-2-(o- chloropheny1)ethane) was compared with p,p’-DDT by the measure of increased uterine and oviductal weight, water con- II. GENERAL CHARACTERISTICS tent, glycogen and RNA in rats, chickens, and OF METHOXYCHLOR quail, o,p‘ but not p,p’-DDT was found to be estrogenically active. l9 Methoxychlor has a chlorinated, double ring Further work with DDT analogues have structure (Figure 2). Technical-grade MXC con- shown that 500 pg of o,p’-DDT injected daily tains 50 to 88% of the pure chemical, and the into young rats advances vaginal opening and remainder is isomers and reaction products. It increases ovarian and uterine weights.20In ma- was first synthesized in 1893, and commercial ture ovariectomized rats, o,p’-DDT treatment production in the U.S. began in 1946. MXC is with 10 mg/d resulted in an increase in uterine approved for use as an insecticide on 87 agricul- weight, a thickened vacuolated uterine epithe- tural crops and on beef cattle, dairy cattle, goats, lium, and the induction of cornified vaginal sheep, and swine.26Tradenames include Maralate, Downloaded by [University of Lethbridge] at 00:46 26 September 2015 smears with reduced serum luteinizing hormone Marlate, and Metox, and the chemical name is (LH).20 Two homologues of DDT, p,p‘-DDD 2,2-di(p-methoxy phenyl) 1, 1,l-trichloroethane. (2,2-bis(p-chlorophenyl)-1,l -dichloroethane) and A report published in 198327stated that MXC p,p’-DDE (2,2-bis(p-chlorophenyl)-1,l -dichloro- was found in 1.2% of composite samples of food ethylene) were not estrogenic in these studies, in the U.S., representing an intake of 0.1 to 0.8 pg while one of the major metabolites of o,p’-DDT, MXC/d for 16- to 19-year old males. When admin- p,p’-DDA (2,2-bis(p-chlorophenyl)acetic acid) istered orally to mice, 98.3% of the MXC is elimi- was found to exhibit estrogenic activity. nated by 24 h.28Both mice and insects metabolize Chlordecone (Kepone) is another pesticide methoxychlor to 2-(p-hydroxyphenyl)-2-(p- with estrogenic characteristics that is no longer methoxypheny1)-l,l,l-trichloroethane and 2,2-bis- used due to its toxicity.21Kepone has been shown (p-hydroxypheny1)- l,l,1-trichloroethane via to produce uterine growth in weanling rats at doses O-demethylation.28When rats were fed methoxy-

369 CI-c -CI I CI

CHEMICAL FORMULA:

FIGURE 2. Structure of methoxychlor: 1,1,1-trichloro-2,2-bis(prnethoxyphenyl)ethane. (By permission, Ref. 6.)

chlor at 25 mgkg in the diet, the chemical was not positive and negative, the National Cancer Insti- detected in body fat at 18 weekszyRats receiving tute concluded that methoxychlor is not an animal MXC in the diet for 2 years at 25, 200, or 1600 ~arcinogen.~~ mgkg did store the chemical in the fat, and MXC was also found in the kidney, liver, and brain.’, The first publication of a chronic oral study 111. CHEMISTRY OF METHOXYCHLOR was published in 1950.’’ The LD,, in rats at 72 h AND ITS MECHANISMS was found to be 5 g/kg for the technical-grade MXC. The purified (recrystallized) material was In one of the early studies investigating the less toxic; at 72 h a mortality of 10% was ob- chemical mechanism of the effects of methoxy- served at 5.8 g/kg. No specific abnormalities were chlor, a report by Welch, Levin, and C~nney~~ recorded after necropsy and histopathological showed a parallel increase in rat uterine weight 6 h ecaluation except for a drastic reduction in testes after methoxychlor administration, and a decrease wight and atrophic changes indicated in sections in the uptake of 17~-estradiolby the rat uterus if ot the treated testes. This level of toxicity is consid- 50 mgkg MXC was administered 2 h prior to erably less than that of DDT (LD,, = 0.25 g/kg), labeled estradiol in vivo. In a study where various the pesticide that MXC was expected to replace. chlorinated hydrocarbons, including MXC, were Downloaded by [University of Lethbridge] at 00:46 26 September 2015 More recently, the toxicity of MXC was exam- added to a rat uterine supernatant preparation, both ined in the freshwater teleost Puntius conchonius.’2 technical and purified MXC inhibited the binding It was found that there was selective damage to of 3H-estradiol in vitro; a parallel increase in uter- the oocytes in the absence of general toxicity. ine weight was found following in vivo adminis- The majority of investigators reporting on the trati~n.~~Evidence that the induction of hepatic carcinogenicity of MXC concluded that the re- monooxygenase by pretreatment of animals with sults were negative,''^^^ although a few reports CCI, interferes with the uterotrophic action of the found positive results.35 A similar apparent con- chemicals tested3*suggested that metabolite(s) of tradiction was raised in a recent report where MXC were the active species binding to the estro- MXC was shown to be positive for mutagenicity gen receptor.40Methoxychlor and technical meth- in the mouse lymphoma assay (MLA) but nega- oxychlor were shown to be more effective inhibi- tive in the Chinese hamster ovary cell (CHO) tors of 3H- estradiol binding if they had been a\say.i6 After taking into account all of the data, pretreated with liver microsomes, suggesting that

370 some metabolite of MXC has a higher affinity for of 3H-E2binding to receptor preparations, HPTE the receptor than does MXC.41 and the parallel metabolite were most effective in Investigations by Bulger, Muccitelli, and inhibiting the binding of 3H-E, to uterine cytosol. Kupfef', revealed that laboratory-grade MXC was In a landmark paper by Kupfer and Bulger,44the contaminated with a material that specifically bound authors defined a proestrogen as a compound that the estrogen receptor from rat uterus. The contam- requires metabolic activation to achieve the trans- inant was eliminated by base washing, suggesting location of the cytosolic estrogen receptor (&)into the presence of an acidic, phenolic compound. The the nucleus, yielding an increase in the nuclear base-washed, recrystallized MXC did not translo- form (R,,).Initially, it was shown that the incuba- cate the estrogen receptor, whereas the phenolic tion of purified methoxychlor with rat uteri did contaminant was shown to compete with 3H-estra- not cause the translocation of the estrogen recep- diol for binding to the receptor. When the DDT tor. Both l7p-estradiol and HPTE decreased cy- analog 2,2-bis(p-hydroxyphenyl)-l,1,l -trichloro- tosolic estrogen receptors (&) and elevated nuclear ethane (HPTE), known to be a major metabolite estrogen receptors (R,,). Methoxychlor and of methoxychlor, was investigated, HPTE was o,p'DDT were independently incubated with rat shown to markedly inhibit the binding of 3H-E, liver microsomes in the presence of immature rat with high affinity. It was concluded, albeit erro- uteri. The effect of metabolism on the distribution neously, that the estrogenic activity of MXC re- of cytosolic (R,) and nuclear (RJ estrogen recep- sults from the activity of a contaminant and that tors was determined. The incubation of methoxy- the contaminant may be HPTE. In a subsequent chlor resulted in diminished R, and elevated R,. evaluations using ornithine decarboxy- These data, with the range of appropriate controls, lase (ODC) activity (an indication of estrogenic showed that the transformation of MXC by rat activity) and uterotrophic activity showed that liver microsomes into chemicals with high estro- both laboratory-grade and purified methoxychlor gen-like activity is catalyzed by the microsomal (5 mg/rat) stimulated both parameters after 7 h, monooxygenase system. The chemical o,p'DDT indicating that methoxychlor was active in vivo. had equivalent estrogenic activity with or without However, 100 pg/rat of HPTE markedly elevated metabolic activation, signifying that it is estro- ODC activity and uterotrophic activity after 6 h. genic, not a proestrogen. The higher potency of the MXC metabolite, HPTE, Based on the prior knowledge28that the major and the shorter time required for its activity sug- metabolites of MXC result from 0-demethylation gested that, in vivo, methoxychlor is bioactivated and consist of a mono- and a bis-phenol, Ousterhout to an estrogen metabolite that may be HFTE. Sub- et al.45synthesized the two compounds and evaluat- sequently, methoxychlor was incubated with he- ed the ability of each chemical to inhibit the binding patic microsomes, and the resulting metabolites of E, to rat uterine cytosol in vitro. The bis-phenol were isolated and recovered via thin layer chro- had a higher affinity for the receptor as indicated by matography (TLC). Results indicated that meth- the fact that a lower concentration was necessary to oxychlor is 0-demethylated in vitro by rat liver inhibit l!$ binding by 50%. Sucrose density gradient Downloaded by [University of Lethbridge] at 00:46 26 September 2015 microsomes and that methoxychlor demethylase centrifugation showed that 5 was displaced from activity is enhanced by pretreatment of animals the 8s receptor by the bis-phenol. Liver microsomal with an inducer of hepatic monooxygenase activ- incubation of methoxychlor resulted in predomi- ity. When extracts from the hepatic incubations nantly the mono-phenol. From these data, the au- were evaluated for receptor binding, it was found thors erroneously concluded that the estrogenic ef- that the demethylation products of methoxychlor fects of methoxychlor in vivo are primarily due to inhibit 3H- E, binding to the 8s estrogen receptor metabolism to the mono-phenol. of rat uterine cytosol under both saturating and A report by Bulger, Feil, and KupfeF re- non- saturating conditions with respect to 3H-E2. vealed the error of the conclusions of Ousterhout Identification of the metabolites separated by TLC et al.45 by using the liver microsomehat uteri revealed the presence of a compound chromato- incubation technique to examine the identity and graphically identical to HPTE. Three metabolites activity of the contaminants and metabolites of were tested, and while all produced an inhibition MXC. MXC was purified by base washing, recrys-

371 tallization, and preparative HPLC. The in vitro eterious to reproduction. Six weeks of treatment determination of proestrogenic and estrogenic ac- at 2500 or 5000 ppm in the feed resulted in fewer tivity involved mixtures of rat hepatic microsomes matings, litters, and implantation sites.47Doses of (capable in the presence of NADPH of transform- 1000 ppm did not impair fertility, but offspring ing the proestrogen into an active estrogen) and were affected: deleterious effects included reduced immature rat uteri (target monitor for E2 activity, mating and fewer litters. The time from birth to where ER, and ER, are measurably changed) with vaginal opening was shortened in pups from dams or without NADPH for the proestrogenic vs. estro- fed 1000 or 2500 ppm MXC prior to breeding. genic activity, respectively. Compounds testing Pups from control dams that were exposed to MXC positive for estrogenic activity were subjected to from birth and postweaning exhibited reduced mat- a second in vitro assay, similar to the first, except ing and fewer litters. MXC also produced persis- that no microsomes were present in either vial. tent estrus, identified by vaginal smears, if admin- Compounds were also tested directly in vim for istered long enough (5 weeks) and at high enough ( 1 ) their relative binding affinity with 3H-estra- doses (5000 ppm) to adult females.47(The authors diol in rat uterine cytosol and (2) saturation analy- point out the relative potency of technical MXC: sis of the inhibition of ?H-E2binding to rat uterine 0.10 pg diethylstilbestrol is equivalent to 1 mg cytosol. In metabolism studies. demethylation was technical MXC, as derived from their previous determined for compounds by analyzing formalde- In a similar study, groups of female rats hyde evolution. Finally, estrogenic activity in vivo received MXC at 100 or 200 mg/kg/day for 14 d uas determined by the administration of compounds prior to mating and throughout pregnancy. On to immature female rats and, 6 h later, the evalu- gestation day 13, no implantations were found; ation of uterine weight and ODC activity. Of the evaluation of the ovaries revealed a lack of cor- 16 compounds (including methoxychlor) that make pora lutea and follicular atre~ia.~~In another study, up 99.5% of technical-grade methoxychlor, MDDE rats were dosed with 90% pure MXC from wean- (compound 34; 1,l-dichloro-2,2-bis(4-methoxy- ing through puberty and gestation to day 15 of pheny1)ethane) and methoxychlor required meta- lactation; doses ranged from 25 to 200 mg/kg/d bolic transformation for estrogenic activity to be MXC.'@The administration of 100 or 200 mg/kg/d manifested. Two base soluble compounds, the mono- MXC produced a significant reduction in fertility; phenolic denvatives of methoxychlor and of MDDE fertility was 0% at 200 mg/kg/d.4y (mono-OH-methoxychlor and mono-OH-MDDE) In Tullner's original report in 1961, technical- were active per se. When the two sets of demethy- grade methoxychlor was first shown to produce an lated metabolites of MDDE and of MXC were increase in uterine weight in rodents.50 A direct evaluated directly in the cytosolic estrogen recep- effect on the uterus was identified, and the author tor binding assay, the relative binding affinity recognized that the active moiety was an isomer or (RBA) was found to be his-OH-MDDE >> bis- contaminant of the technical-grade chemical. In OH-MXC (HPTE) > mono-OH-MDDE > mono- work reported by Welch et al.,38purified MXC was OH-MXC. Thus, the most active metabolite of used in the 6-h uterotrophic test and increased the Downloaded by [University of Lethbridge] at 00:46 26 September 2015 MXC was shown to be HPTE, and components of weight of immature female rat uteri by 37% at 50 technical-grade methoxychlor were shown to be mgjkg. When purified MXC (50 mag)was admin- either estrogenic per se, proestrogenic, or inactive. istered to immature rats 2 h prior to 1.5 clg/kg 3H-17- P-estradiol, the chemical decreased the uptake of the tritiated estradiol by the uterus by 28% com- IV. REPRODUCTIVE EFFECTS pared with a value of 61% for 30 pgkg diethyl- OF METHOXYCHLOR stilbestrol.'s Relative binding to the estrogen receptor also appeared to be the critical factor in the A. Fertility and Related Mechanisms initiation of implantation and maintenance ofpreg- nancy by a variety of chemicals, including MXC in Treatment of rats with technical-grade MXC a hypophysectomized,progesterone-treated model of in the feed prior to breeding was extremely del- early pregnan~y.~'MXC was more successful than

3 72 other polychlorinated hydrocarbons in initiating to the effects of MXC than the postimplantation and maintaining pregnancy according to the de- period. gree to which the chemicals bind the estrogen The decidual cell response (DCR) is a tech- receptor, thus supporting the role of the estrogen nique that utilizes the phenomenon of induced receptor in the biological effects of MXC.51 pseudopregnancy (a state endocrinologically corre- In an examination of estrogen-induced behav- sponding to pregnancy but lacking fertilized eggs) ior and its modification by MXC, Gray et d.52used and a surgical technique that induces a response running wheel activity (RWA; an estrogenically con- from the uterus that mimics the growth and devel- trolled, normally cyclic phenomenon) and induced opment of the endometrium that occurs during sex behavior. Exposure to MXC at 400 mg/kg/d early pregnan~y.~~?~~The result is a uterus filled produced high levels of non-cyclic RWA and persis- with decidual tissue, a deciduoma, that can weigh tent vaginal estrus. When 200 mgkg/d MXC was up to 2 g per horn. Estrogen and progesterone, in administered to ovariectomized female rats and the proper amounts and ratio, are critical for the the animals then received progesterone, the proges- development of these deciduomata. Excess estro- terone blocked the MXC-stimulated increase in gen will impair both the development of the sen- RWA, indicating a mechanism mediated by estro- sitivity of the uterus to stimulation and the growth gen receptors in the CNS.52Finally, MXC-treated, of the tissue following ind~ction.~.~The adminis- ovariectomized rats that were injected with proges- tration of 98% pure MXC for 8 d to female rats terone displayed reproductive behavior, in a man- prepared for the DCR inhibited the decidualiza- ner similar to estrogen plus progesterone-treated tion of the uterus in a dose-dependent manner: animals, where the estrogen and the MXC is act- 100 mg/kg/d MXC had no effect, but doses of ing on areas of the CNS other than those involved 200, 300, 400, and 500 mg/kg/d produced incre- in RWA.52However, unlike estrogen, MXC did mental inhibition of this phen~menon.~~These not block the ovariecromy induced increase in data suggest that MXC exerts an effect directly on FSH or LH.52 the uterus (no effect on serum progesterone was found in this study), and such an effect may contribute to the antifertility effect of MXC. Using this system as a comparison between MXC and a B. Early Pregnancy positive control, estrone, the estrogenic activity of 98% pure MXC was estimated to be 1/20,000 When a range of dosages of MXC (98% pure) times the activity of estrone or 1/200,000 times were administered to rats to evaluate the chemi- the activity of estradiol or DES. In a related study, cal’s effect on early pregnancy, exposure was lim- ovariectomized rats hormonally prepared to re- ited to days 1 to 8 of pregnancy (day 0 = sperm +). produce pseudopregnancy and surgically induced Doses of 300 mg/kg/d and above reduced implan- on the proper day were treated with 500 mg/kg/d tation sites to zero, 200 mg/kg/d resulted in a MXC or oil. MXC blocked the DCR in the ab- significant reduction in the number of sites, where- sence of ovaries, confirming a direct effect of the Downloaded by [University of Lethbridge] at 00:46 26 September 2015 as 100 mg/kg/d had no effect when evaluations chemical on the uterus.53 were made on day 9.53Serum progesterone levels Although direct effects on the uterus were evi- were reduced at all doses of MXC. A protocol in dent, the sensitivity of the preimplantation interval which the sensitivity of the preimplantation inter- of pregnancy to the effects of MXC indicated the val (days 1 to 3) is compared with the postimplan- presence of other potential mechanisms such as tation interval (days 4 to 8) was applied to MXC. embryo transport rate, which is also susceptible to Whereas dosing with MXC during days 4 to 8 had estrogenic effect^.^ MXC was administered on days no effect, exposure during days 1 to 3 resulted in 1 to 3 of pregnancy (day 0 = sperm +) and the dis- partial (at 200 mg/kg/d) or total (at 500 mg/kg/d) tribution of the embryos between the oviduct and loss of embryos as judged by the number of im- the uterus was assessed on the afternoon of day 1 plantation sites seen on day 9.53Thus, the preim- and at morning and afternoon times on days 2 and plantation interval was found to be more sensitive 3. While no effect of MXC was seen on day 1, on

373 days 2 and 3 both 200 and 500 mg/kg/d MXC cytosol from MXC or estradiol treated rat uteri accelerated the transport of embryos from the ovi- also yielded a peak at 49,000. Such estimates are duct to the uterus, and in the case of 500 mg/kg/d, near the established mol wt of creatine kinase of moved the embryos out of the tract alt~gether.~~By 46,000. These data, again, demonstrate parallel day 3, even 100 mg/kg/day MXC was effective in mechanisms, at a biological level, between MXC accelerating embryo transport and 200 mgkg/d (HPTE) and e~tradiol.~~ MXC reduced total embryo recovery. Comparing Epidermal growth factor receptor (EGF-R) bind- these data with previous results where 200 mg/kg/d ing has been shown to be stimulated by estradiol in MXC was the minimal effective dose for blocking immature female rat uteri.6oFollowing the admin- the DCRs6and 200 mg/kg/d was the lowest dose at istration of either 20 pg/rat estradiol or 500 mgkg which a significant reduction in the number of MXC (>99% pure), the binding of lZI-labeled EGF implantation sites was found,s3it would appear that to uterine membranes was increased.61Actinomy- both the acceleration of embryo transport and the cin D blocked the stimulation of EGF-R binding inhibition of decidualization contribute in a more by MXC and estradiol, but cycloheximide did not. or less equivalent manner to the early pregnancy This may be due to the high doses of MXC and failure produced by MXC. estradiol required to gain stimulation and the in- adequacy of the near lethally toxic dose of cycloheximide (100 pg/rat) used to attempt blockade. Scatchard analysis revealed an increase in maxi- C. Markers of MXC/Estrogen Action mal binding (BmJ by both MXC and estradiol in the Uterus and no change in the dissociation constant (KJ as a result of treatment with either chemical.6' These With the knowledge that the active metabo- data also show a parallel biological mechanism lite of MXC, HPTE,can bind to the estrogen recep- between estradiol and MXC (HPTE). tor, studies on resultant protein synthesis specific to estrogen action were performed. When immature female rats were treated with >99% pure MXC, a dose-dependent stimulation of the activ- D. Teratogenic and Developmental ity of uterine peroxidase was seen in the uterus.s8 Effects Concurrent treatment with actinomycin D, cycloheximide, progesterone, or tamoxifen blocked the To evaluate the chemical's potential teratoge- MXC-stimulated increase in uterine peroxidase nicity, two samples of methoxychlor were tested, activity, indicating a mechanism of MXC (HPTE) technical-grade (MXT) and a formulation (MXF) action that is parallel to that of estradiol.'* containing 50% technical methoxychlor and the rest In a similar manner, parallels between the were unknown ingredients. Treatment of rats with mechanisms of MXC at 500 mgkg and estradiol either sample during days 6 to 15 of gestation re- at 10 pg/rat were demonstrated using the enzyme sulted in reduced body weight gain at doses from 50 Downloaded by [University of Lethbridge] at 00:46 26 September 2015 creatine kinase, a protein originally known as estro- to 400 In addition, both types of methoxy- gen-induced protein or IP. Immature female rats chlor were fetotoxic at 200 and 400 mg/kg/d. When were treated with MXC or estradiol, and IP was fetuses were evaluated on gestation day 22, both measured in uteri using an in vim radiolabeling formulations were found to have produced an in- technique and the separation of labeled proteins crease in wavy ribs at 100,200, and 400 mg/kg/d.62 on nondenaturing gels. Both MXC and estradiol These data provided the initial evidence that meth- stimulated the synthesis of IP, and concurrent treat- oxychlor might produce developmental effects if ment with actinomycin D or cycloheximide blocked exposure occurred during pregnancy. the MXC-stimulated synthesis of IP.s9 Immuno- Developmental landmarks were also evalu- precipitation of creatine kinase by a monoclonal ated using a different paradigm. Rats were dosed antibody to the enzyme yielded a peak on SDS with 90% pure MXC from weaning through pu- gels with a mol wt of 49,000, and SDS gels of berty and gestation to day 15 of lactation; doses

374 ranged from 25 to 200 mgkgld MXC.4y MXC sion of production of gonadotropin by the estro- was shown to accelerate the age at vaginal open- genic action of technical metho~ychlor.”~~ ing and first estrus, and vaginal smears were corni- In the same study by Ba148 described earlier, fied, indicating estrogenicity of the chemical. Growth evaluations of the effects of MXC on the male retardation was noted at 100 and 200 mg/kg/d reproductive system were also made. Technical and fertility of the females was compromised. At methoxychlor was administered to adult male rats 200 mg/kg/d, mated females became pseudopreg- at 100 and 200 mg/kg/d for 70 d (which encom- nant following mating but had no implantation passes the spermatogenic cycle). Results showed sites. When the offspring of the 50 mg/kg/d dose a degeneration of Sertoli cells and morphological group were evaluated, vaginal opening was again abnormalities in the developmental stages of the accelerated, estrous cyclicity was abnormal when sperm in the seminiferous tubules. Abnormalities the rats reached middle age, and fecundity was of the ductus epididymis were also observed. Path- found to be reduced.4y ological alterations of the seminiferous tubules, and developing spermatozoa were also observed at the ultrastructural Goldman et a1.@explored the effects of MXC E. Effects in Males on the hypothalamus and pituitary. Male rats were administered 25 or 50 mg/kg/d MXC for 8 weeks In the same study by Gray et al. described beginning on day 21 of life. No changes in serum- above, developmental effects on male rats were luteinizing hormone (LH), follicle-stimulating hor- also observed. MXC was shown to reduce growth, mone (FSH), or prolactin or the pituitary concentra- seminal vesicle weight, cauda epididymal weight, tions of LH or FSH were seen. However, pituitary caudal sperm content, and pituitary weight; puberty prolactin was elevated and when pituitary seg- was also delayed.4yPituitary levels of prolactin, ments were perifused, the fragments released more thyroid-stimulatinghormone, and follicle-stimulat- prolactin than controls. Gonadotropin-releasing ing hormone were altered and serum thyroid-stim- hormone (GnRH) was elevated in the mediobasal ulating hormone was reduced by 50%. It should hypothalamus at 50 mgkg. The authors suggest be noted that serum testosterone, LH, and FSH an interaction between the GnRH and prolactin were not affected, there were no effects on male levels and suggest that prolactin is important ear- pituitary size or histology, and the testes were ly in the adverse action of MXC on the male only slightly affected with no histopathological reproductive system. lesions. The fertility of treated males was unaf- fected if bred with untreated fern ale^."^ In an early study, Tullner and Edg~omb~~examined the effect of MXC on male reproduction via F. Effects in Mice pair-feeding experiments where one of each pair received 1 % MXC in the diet. Rats received MXC Aside from the original experiments reported Downloaded by [University of Lethbridge] at 00:46 26 September 2015 for 33 to 55 d. MXC reduced body, seminal vesicle, by Tullner, investigations of the effects of MXC and ventral prostate gland weights, and testicular on reproduction in mice have been performed only atrophy was observed. Adrenal weights were in- fairly recently. In 1990, Cooke and Eroschenko creased and kidneys were enlarged. Histopatholog- examined the effects of technical MXC on the ic changes occurring following treatment included development of neonatal male and female mouse the finding of a lack of spermatozoa in the tubular reproductive tra~ts.~~,~~The technical MXC used lumina. Atypical spermatocytes were found in the was shown to contain quantities of several com- seminiferous tubules, and the number of sperma- pounds other than MXC that have estrogenic ef- tocytes was reduced. Leydig cells were also de- fects. Mice were treated for 10 d, starting on day 1 creased in number. The authors speculate that the of life, with 17P-estradiol or MXC at 0.05, 0.1, effects of MXC on the male reproductive system 0.5, or 1.0 mg/mouse/d. In male mice, serum may be regarded as a “consequence of the suppres- testosterone levels on day 10 were decreased;

375 DNA contents of the seminal vesicles, bulboure- ment. The secretion of labeled proteins stimulated thral glands, and ventral prostate were also re- by MXC, identified after gel electrophoresis, ap- duced in both groups.6s These data indicated an peared “indistinguishable” from those stimulated inhibition of neonatal male reproductive tract by 17P-e~tradiol.~~Further effects of 50% technical development. Female mice exhibited precocious MXC on adult mice were evaluated by Swartz vaginal opening, complete vaginal cornification, et al.72in experiments where adult mice were ex- and increased tract weight.66In a related posed to MXC at 0.1,0.5,1.O, 2.5, or 5 .O mg/mouse/ technical MXC was administered for 14 d to neo- d for 4 weeks. Morphometric and transmission elec- natal female mice and ultrastructural effects of the tron microscopic (TEM) studies were performed. chemical were investigated in various tissues. Both MXC produced an increase in the height of uter- 0.5 and I .O mg/mouse/d doses of MXC increased ine epithelial cells and increased vacuolization the fluid weight of the tract and accelerated the and swelling of mitochondria, as indicated by TEM. time of vaginal opening. Ultrastructural changes Effects on microvilli of the uterine epithelial cells in vaginal epithelium and uterine epithelium elic- were also observed. The authors suggest that these ited by MXC differed from changes elicited by effects of MXC are not only estrogenic but 1 7P-estradiol, such as uterine “pitting”, uterine It is not clear whether the toxicity is due to the atypia, and distorted apical cell m~rphology.~~ estrogenic effect itself or if contaminants of the When administered to adult female mice at technical MXC have effects unrelated to their estro- 1.25. 2.5, or 5 mg/mouse for 2 or 4 weeks, 50% genicity. Finally, to examine the effect of purified technical-grade MXC produced persistent vaginal MXC (98% and base washed 98% MXC), neona- estrus and ovarian atrophy with atretic large folli- tal mice received 0.05,0.5, or 1.O mg/mouse/d for cles.68To assess effects of the chemical on preg- 14 d beginning on day 1 of life.73Base-washed nancy and subsequent litters, technical MXC was MXC had the same effect as 98% MXC and 170- administered at 2.5, 5.0, or 7.5 mg/mouse/d on estradiol. In a manner similar to that seen in pre- day 6 to 15 of pregnancyh9.Female offspring were vious reports for technical MXC, 98% pure MXC cross-fostered and sacrificed at 8 weeks of age. induced precocious vaginal opening, persistent vag- Dams exposed initially were rebred and the sec- inal estrus, increased reproductive tract weights, ond set of litters were evaluated. The highest dose and epithelial hypertrophy in the vagina and uter- of MXC prevented the mice from carrying to us.73When newborn mice received technical MXC term, and the length of gestation was increased by daily for 14 d and were evaluated as adults, an 5.0 mg/mouse MXC. In the second set of litters, increase in ovarian atrophy and a depletion of females exhibited accelerated vaginal opening; corpora lutea were found at 0.5 and 1.O mg MXC.74 the authors suggest that this is a residual effect of Exposure of pregnant mice to MXC during GDI I - MXC from the original pregnancy.69 The possi- GD17 led to an alteration in behavior in adult bility of the passage of MXC via nursing female males; the rate of urine marking in male adults mice wa\ investigated by Appel and Ero~chenko.~~was increased when doses of 1, 100, or 5000 pg ‘T’echnical-gradeMXC was administered to lactat- MXC were administered to dams.7s Downloaded by [University of Lethbridge] at 00:46 26 September 2015 ing mouse dams for 14 d. Suckling female pups were evaluated and alterations in their reproductive tract indicated that MXC was excreted in the V. SUMMARY milk in an active Rourke et aL7’compared the effects of technical It is clear that methoxychlor is considerably MXC ( 1 mg/mouse) and 17P-estradiol( 10 pg/mouse) less toxic than DDT, chlordecone, or other organo- administered for 14 d to ovariectomized adult mice. chlorine pesticides having estrogenic activity when Uterine luminal proteins were radiolabeled with considered from an LD,, , non- reproductive stand- “S-methionine and separated by two-dimensional point. However, reproductive toxicity is an impor- gel electrophoresis. Both 17P-estradiol and MXC tant aspect of an evaluation of an environmental altered reproductive tract weights, promoted cellu- estrogen. The most sensitive of all the assays or lar hypertrophy. and stimulated uterine develop- evaluations of MXC action in rats appears to be

376 vaginal opening. In the study by Gray et al.,49 accel- 3 Greenwald, G. S., Species differences in egg trans- erated vaginal opening was seen in even the off- port in response to exogenous estrogen, Anat. Rec., 157, 163, 1967. spring of the groups treated with mg/kg/d MXC; 50 4. Yochim, J. M. and DeFeo, V. J., Control of decidual 90% pure MXC was used. Also sensitive was the growth in the rat by steroid hormones of the ovary, embryo transport rate analysis;57 100 mg/kg/d Endocrinology, 7 1, 134, 1962. accelerated embryo transport when evaluated on 5. Yochim, J. M. and DeFeo, V. J., Hormonal control day 3 of pregnancy; 98% pure MXC was used. of the onset, magnitude and duration of uterine sensitiv- Thus, the potency of MXC in rats appears to be ity in the rat by steroid hormones of the ovary, Endo- crinology, 72, 317, 1963. greatest when the purified chemical and not tech- 6. O’Malley, B. W. and Tsai, M-J., Molecular path- nical MXC is used. Experiments comparing MXC ways of steroid receptor action, Biol. Reprod., 46, and estrone using the blockade of the decidual 163, 1992. cell response as the endpoint suggest that MXC 7. Astwood, E. B., A six-hour assay for the quantitative has 1/200,000 times the activity of estradi01.~~ determinationof estrogen, Endocrinology, 23,25,1938. 8. Cohen, S., O’Malley, B. W., and Stastny, M., Estro- Studies with mice used technical MXC almost ex- gen induction of ornithine decarboxylase in vivo and clusively. This makes comparisons with rat stud- in vitro, Science, 170, 336, 1970. ies and purified MXC results difficult. However, 9. Notides, A. and Gorski, J., Estrogen-inducedsynthe- vom Saal’s study75in which 100 pg of MXC ad- sis of a specific uterine protein, Proc. Natl. Acad. Sci. ministered prenatally altered the social behavior U.S.A., 56, 230, 1966. of male offspring appears to contain the most sen- 10. Reiss, N. A. and Kaye, A. M., Identification of the major component of the estrogen induced protein of stitive endpoint in mice (whether technical or puri- rat uterus as the BB isozyme of creatine kinase, J. fied MXC was used was not reported for the latter Biol. Chem., 256, 5741, 1981. study). 11. Lucas, F. V., Neufeld, H. A., Utterback, J. G., Overall, MXC can be considered a reproduc- Martin, A. P., and Stotz, E., The effect of estrogen tive toxicant and environmental estrogen that on the production of a peroxidase in the rat uterus, J. Biol. Chem., 214, 755, 1955. compares with DDT in its reproductive toxicity. 12. Lyttle, C. R. and DeSombre, E. R., Uterine peroxi- A metabolite of MXC binds the estrogen receptor dase as a marker for estrogen action, Proc. Natl. Acad. and the chemical has effects similar to those elic- Sci. U.S.A., 74(8), 3162, 1977. ited by 17P-estradio1, including the induction of 13. Barker, K. L. and Warren, J. C., Estrogen control specific uterine enzymes. It is estrogenic in the of carbohydrate metabolism in the rat uterus: path- CNS of the adult and neonatal rat. Pregnancy is ways of glucose metabolism, Endocrinology, 78,1205, 1966. compromised by MXC administration, and delete- 14. Gorski, R. A., Sexual differentiation of the brain: a rious effects on ovarian and uterine function are model for drug-induced alterations of the reproduc- clear. Whether the dose levels sufficient to elicit tive system, Environ. Health Persp., 70, 163, 1986. reproductive effects in animals are relevant to hu- 15. McLachlan, J. A., Newbold, R. R., Shah, H. C., man and environmental health is unknown at this Hogan, M. D., and Dixon, R. L., Reduced fertility in time. The relevance to human health of the data female mice exposed transplacentally to diethylstilbestrol (DES), Fertil. Steril., 38(3), 364, 1982. discussed here await specific studies that address 16. Downloaded by [University of Lethbridge] at 00:46 26 September 2015 Newbold, R. R. and McLachlan, J. A., Diethylstil- issues of exposure from experiential and epidemi- bestrol-associateddefects in murine genital tract devel- ological viewpoints. opment, in Estrogens in the Environment II: Influenc- es on Development, McLachlan, J. A,, Ed. Elsevier North-Holland, New York, 1985, 288. 17. Burlington, H. and Lindeman, V. F., Effect of DDT REFERENCES on testes and secondary sex characteristics of white leghorn cockerels, Proc. SOC.Exp. Biol. Med., 74,48, Haddad, V. and Ketchel, M. M., Termination of 1950. pregnancy and occurrence of abnormalities following 18. Fisher, A. L., Keasling, H. H., and Scheuler, F. W., estrone administration during early pregnancy, Int. J. Estrogenic action of some DDT analogues, Proc. SOC. Fertility, 14(1), 56, 1969. Exp. Biol. Med., 81, 439, 1952. Greenwald, G. S., The antifertility effects in preg- 19. Bitman, J., Cecil, H. C., Harris, S. J., and Fries, nant rats of a single injection of estradiol cyclopentyl- G. F., Estrogenic activity of o,p’-DDT in the mamma- propionate, Endocrinology, 69, 1068, 1961. lian uterus and avian opiduct, Science, 162,371,1968.

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