Estrogenic Activity of p,p'-DDT

Sergio Bustos, Jorge Soto, and Andrei N. Tchernitchin*

Laboratory of Experimental Endocrinology and Environmental Pathology, the Departments of Experi- mental Morphology and Experimental Medicine (Campus Norte), University of Chile Medical School, Casilla 21 104, Correo 21, Santiago, Chile

DDT is a pesticide still indiscriminately used in several Third World countries. It contaminates the environment, including food, thus affecting human health and the survival of wild animals. It has been reported that its o,p' isomer exerts estrogen-like activity that has not been detected for the p,p' isomer. This study investigates the possible estrogenic activity of p,p'-DDT on genomic and nongenomic parameters of estrogen action. Additionally, its effects on the degranulation of eosino- phi1 leukocytes in the blood were investigated. Female nonovariectomized immature rats and female adult ovariectomized rats were intravenously injected with p,p'-DDT dissolved in dimethyl sulfoxide or with estradiol-l7p. The animals were killed at 6 or 24 h after treatment, the uterine horns excised, fixed in neutral formalin, and submitted to further histological procedure. Samples of blood were collected into glass vials containing EDTA and processed for evaluation of eosinophil degranulation. The results revealed myometrial hypertrophy and the development of edema in superficial and deep endometrial stroma under the effect of DDT, at 24 h after treatment, similarly to the effect of estradiol- 17p. Further, they revealed an increase in degranulation of the blood eosinophils at 6 and 24 h after treatment with estradiol or DDT. The results confirm the estrogenic activity of the least toxic DDT isomer, for both genomic responses and eosinophil-mediated nongenomic responses and explain in part the reproductive effects of the pesticide in humans and in wild animals. Further, considering its effects on blood eosinophils, suggests a possible interaction with immune regulation and other processes where eosinophils play a role. 0 7996 by John Wiley & Sons, Inc.

INTRODUCTION

DDT (1, 1 , l ,-trichloro-2,2-bis[chlorophenyl]ethane) is a pesticide still indiscriminately used in several Third World countries (Metcalf, 1973) that contaminates the environment, including soil, water, and food. This con- tamination can affect human health as well as the sur- vival of wild animals, suggesting the importance of the investigation of its toxic effects in mammals.

It is well known that o,p'-DDT exerts estrogen-like activity (Foster et al., 1975; Kupfer, 1975; McBlain,

* To whom correspondence should be addressed at Laboratory of Experimental Endocrinology and Environmental Medicine, P.O. Box Casilla 21104, Correo 21, Santiago, Chile.

1987; Galand et al., 1987), through interaction with cytosol-nuclear estrogen receptors (Kupfer and Bulger, 1977). Preliminary studies performed in our laboratory have demonstrated that the least toxic and most widely used of the DDT isomers, p,p'-DDT, also displays estrogenic action in the rat uterus (Bustos et al., 1988).

To investigate the estrogenic actions of DDT, it is necessary to consider that estrogens induce separate groups of responses through independent mechanisms of hormone action in which different kinds of estrogen receptors are involved. Accordingly, the increase in uterine RNA and protein synthesis are genomic re- sponses to hormone stimulation induced through hor- mone interaction with cytosol-nuclear receptors in the various uterine cell types (Jensen and DeSombre,

Environmental Toxicology and Water Quality: An International Journal, Vol. 11 (1996) 265-271 0 1996 by John Wiley & Sons, Inc. CCC 10Yd-4725/96/030265-07

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266 BUSTOS ET AL.

1972). Estrogen-induced uterine edema, increase in vascular permeability, and release of histamine are nongenomic responses (Tchernitchin and Galand, 1982; Tchernitchin et al., 1985b) induced through hor- mone interaction with eosinophil leukocyte estrogen receptors (Tchernitchin et al., 1985b, 1989), which me- diate the migration of these cells from the blood to the uterus (Tchernitchin et al., l974,1985b), their degranu- lation (Tchernitchin et al., 1985a, 1989), and the release of enzymes and agents involved in the development of the eosinophil-mediated responses (Tchernitchin et al. 1985b, 1989). Additional mechanisms of estrogen ac- tion, involving lower affinity kind I1 cytosolic and nu- clear estrogen receptors (Clark and Peck, 1979), uterine membrane estrogen receptors (Pietras and Szego, 1977; Miiller et al., 1979; Nenci et al., 1981), cyclic AMP (Hechter et al., 1965; Kvinnsland, 1976), and prosta- glandins (Resnik et al., 1975; Penney et al., 1981; Soto-Feine et al., 1981) have been proposed as well. The existence of multiple and independent mecha- nisms of estrogen action for the different responses to hormone stimulation results in a dissociation of these responses under a number of conditions (Tcher- nitchin and Galand, 1982; Galand et al., 1985; Tcher- nitchin et al., 1985b). In addition, estrogenic com- pounds may interact selectively with some receptor systems, perhaps in some cell types but not others, affecting some parameters of hormone stimulation only (Tchernitchin et al., 1985b). Therefore, the study of any agent displaying estrogen action must consider the different mechanisms and the wide spectrum of responses to hormone stimulation in the target organ under study. The present study investigated various estrogenic responses of p,p’-DDT action separately in the different uterine cell types, taking into consider- ation the two best known mechanisms of estrogen action.

MATERIALS AND METHODS

Female rats from a Sprague-Dawley derived colony bred at the vivarium of the Faculty of Medicine, University of Chile, were used in the present study. In the first experiment, immature rats were used at the age of 21 days, weighing between 40 and 49 g. In the second experiment, adult females were ovariectomized at the age of 3 months and used 15 days after ovariectomy. For each kind of animal, the individuals were intravenously injected (into the jugular vein, under ether anesthesia) with p,p’-DDT, its vehicle (dimethyl sulfoxide, DMSO), estradiol-176 or its vehicle.

The different parameters of estrogen stimulation in

the uterus were investigated in the immature rats. Es- tradiol-17P was administered at the dose of 30 pg/IOO g body wt, and DDT was administered at the doses of 1 or 2 mg/100 g body wt. Both substances or vehicle was administered intravenously under ether anesthe- sia. The uteri were excised under ether anesthesia, 6 or 24 h after treatment, for wet weight determina- tion, fixed in 4% netural formalin, and subjected to further histological procedure for eosinophil quantifi- cation and morphometry (Tchernitchin and Galand, 1983). The following estrogenic responses were inves- tigated: myometrial hypertrophy was measured as increase in the reciprocal value of cell density (RVCD) in circular myometrium, edema in deep and superficial endometrial stroma was evaluated as increases in RVCD in these histological locations (Grunert et al., 1984, 1986), and uterine eosinophilia was measured as total number of eosinophils located in both uterine horns (Tchernitchin et al., 1974).

The degranulation of the blood eosinophils was in- vestigated in the adult ovariectomized rats. Estradiol was administered at the dose of 30 pg/lOO g body wt, and DDT was used at the dose of 25 pg/IOO g body wt. Six and twenty-four hours after treatment, 0.1 mL of blood was collected from the tail under ether anes- thesia into glass vials containing a 5% (w/v) solution of EDTA in distilled water, obtaining a 9 : 1 dilution of blood in EDTA solution. Eosinophil quantification and evaluation of eosinophil degranulation was performed according to the method of Tchernitchin et al. (1985a), as follows: the blood samples were incubated in the glass vials at 18°C. Aliquots were taken immediately after blood sample collection and at 15 min of subse- quent in vitro incubation and diluted 1 : 10 with freshly prepared eosin stain solution (1 mL of 1% eosin Y stock solution in 100% ethanol diluted in 10 rnL of distilled water and 1 mL of acetone). Subsequently, an aliquot of blood-stain solution was transferred to a Neubauer chamber for eosinophil quantification and evaluation of their degranulation. For this purpose, each eosinophil was classified as degranulated or non- degranulated according to its histological characteris- tics (Tchernitchin et al., 1985a). Eosinophils that ap- peared with a marked decrease in the number of eosin- stained cytoplasmic granules-i.e., that contained less than half of the normal granule content, assessed visu- ally, or that exhibited cytoplasmic areas free of gran- ules, or that had lost almost all granules-were con- sidered to be degranulated. Almost completely degran- ulated eosinophils were recognized because there was always a few eosin-stained granules in the vicinity of the nucleus that conferred a slight shade of pink color to the nuclei of the eosinophils but not to the nuclei of other white blood cells.

ESTROGENIC ACTIVITY 267

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Fig. 1. Effect of p,p’-DDT or estradiol-17p on the number of uterine eosinophils. The immature rats were intrave- nously injected with estradiol-17p at the dose of 30 pg/ 100 g body wt [E (6)] or its vehicle [C (E)], or with DDT at the dose of 1 [D 11 or 2 [D 21 mg/100 g body wt or its vehicle [C (D)]. The uteri were excised 6 h [(S)] or 24 h [(24)] after treatment. Results are expressed as % of response to estrogen, measured at 6 h after treatment, LSEM. For analysis of significance, the LSD test was used. (*) p < 0.05, (**) p < 0.01, and (***) p < 0.001, compared to vehicle-injected animals.

Statistics

For comparisons between the controls and experi- mentals and between estrogen-treated and DDT- treated animals, the least significant difference (LSD) test was used. The common variance used in this test was obtained from the one way analysis of variance (ANOVA), and no significant differences were declared unless significant (Snedecor and Coch- ran, 1967). In the study of in vivo induced in vitro eosinophil degranulation, the possible loss of eosino- phils throughout the incubation period in the glass vials, which could alter the results on degranulation percentage, was monitored by comparing the eosino- phi1 numbers before and after the 15 min in vitro incubation by ANOVA. In none of these comparisons

a significant difference in the number of eosinophils was detected.

RESULTS

Parameters of Estrogen Stimulation in the Uterus of the Immature Rats

Uterine Eosi noph i lia

Figure 1 shows the comparison between p,p‘-DDT in- duced uterine eosinophilia 6 or 24 h after treatment and the response induced by estradiol-17P 6 h after treatment. The response induced by estradiol at 24 h of treatment is not shown because of its high variability between animals. It can be observed that the dose of 2 mg/100 g body wt DDT only induces a significant increase in uterine eosinophilia at 6 h after treatment, and that both 1 and 2 mg DDT cause an increment in uterine eosinophilia at 24 h of treatment, that is similar to that induced by estradiol at 6 h.

Edema in Superficial Endometrium

Figure 2 shows the comparison between p,p’-DDT- induced edema in superficial endometrium, evaluated as increase in the RVCD in this location, 6 or 24 h after treatment, and the response induced by estradiol-17p 6 and 24 h after treatment. It can be observed that DDT, at the dose of 1 mg/100 g body wt, induces edema in superficial endometrium at 24 h after treatment.

Edema in Deep Endometrium

Figure 3 shows the comparison between p,p’-DDT- induced edema in deep endometrium, evaluated as in- crease in the RVCD in this location, 6 or 24 h after treatment, and the response induced by estradiol-17P 6 and 24 h after treatment. It can be observed that DDT, at the dose of 1 mg/100 g body wt, induce edema in deep endometrium at 24 h after treatment; this re- sponse is significantly weaker than that induced by estradiol at the same time after treatment.

Hypertrophy in Circular Myomet ri u m

Figure 4 shows the comparison between p,p’-DDT- induced hypertrophy in the circular layer of the myo- metrium, evaluated as increase in the RVCD in this location, 6 or 24 h after treatment, and the response induced by estradiol-17P 6 and 24 h after treatment. It can be observed that DDT, at the dose of 1 mg/100 g body wt, induce a weak hypertrophy in circular myo- metrium at 24 h after treatment; this response is sig- nificantly weaker than that induced by estradiol at the same time after treatment.

268 BUSTOS ET AL.

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Fig. 2. Effect of p,p’-DDT or estradiol-17P on the RVCD in superficial endometrium. The immature rats were intra- venously injected with estradiol-17p at the dose of 30 fig/ 100 g body wt [El or its vehicle [C (E)], or with DDT at the dose of 1 mg/100 g body wt [D] or its vehicle [C (D)]. The uteri were excised 6 h [E6, D6] or 24 h [(E24, D24)) after treatment. Results are expressed as % of the maximal response to estrogen, which was at 24 h after treatment, -r-SEM. For analysis of significance, the LSD test was used. (****) p < 0.0001, compared to vehicle-injected animals; (++++) p < 0.0001, compared to the response to estradiol. within the same time of treatment.

Degranulation of Eosinophils from the Blood of Adult Ovariectomized Rats In Vivo Induced In Vitro Eosinophil Deg ran u lat ion

Figure 5 shows the comparison between p,p’-DDT in vivo induced in vitro eosinophil degranulation 6 or 24 h after treatment and the response induced by estradiol- 170 6 or 24 h after treatment. It can be observed that DDT, at the dose of 25 ,ug/IOO g body wt, induces degranulation of blood eosinophil leukocytes, at 6 and 24 h after treatment. At 6 h after treatment, the re- sponse is similar to that induced by estradiol; at 24 h,

however, it is significantly lower than that induced by the hormone.

DISCUSSION

The present study confirms and extends previous re- sults (Bustos et al., 1988,1995) showing that p,~’-DDT, considered as the least toxic DDT isomer and therefore the most appropriate for use, displays estrogenic activ- ity in the uterus and additionally degranulates blood eosinophil leukocytes.

The responses to estrogen induced by p,p’-DDT in the uterus are myometrial hypertrophy, uterine eosino- philia, and edema in deep and superficial endometrium.

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Fig. 3. Effect of p,p’-DDT or estradiol-17p on the RVCD in deep endometrium. The immature rats were intravenously injected with estradiol-17p at the dose of 30 pg/lOO g body wt [El or its vehicle [C (E)], or with DDT at the dose of 1 mg/100 g body wt [D] or its vehicle [C (D)]. The uteri were excised 6 h [E6, D6] or 24 h [(E24, D24)] after treatment. Results are expressed as % of the maximal response to estrogen, which was at 24 h after treatment, kSEM. For analysis of significance, the LSD test was used. (****) p < 0.0001, compared to vehicle-injected animals; (+ + + +) p < 0.0001, compared to the response to estradiol, within the same time of treatment.

ESTROGENIC ACTIVITY 269

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Fig. 4. Effect of p,p'-DDT or estradiol-17p on the RVCD in the circular layer of the myometrium. The immature rats were intravenously injected with estradiol-17p at the dose of 30 pg/lOO g body wt [El or its vehicle [C (E)], or with DDT at the dose of 1 mg/lOO g body wt [D] or its vehicle [C (D)]. The uteri were excised 6 h [E6, D6] or 24 h [(E24, D24)] after treatment. Results are expressed as % of the maximal response to estrogen, which was at 24 h after treatment, ASEM. For analysis of significance, the LSD test was used. (****)p < 0.0001, compared tovehicle- injected animals; (++++) p < 0.0001, compared to the response to estradiol, within the same time of treatment.

Myometrial hypertrophy was shown to correspond to a genomic response mediated by cytosol-nuclear recep- tors (Grunert et al., 1984), while uterine eosinophilia and endometrial edema correspond to agroup of nonge- nomic responses to estrogen that are mediated by the high affinity estrogen receptors located in the surface of the eosinophils (Tchernitchin and Galand, 1982; Tchernitchin et al., 1989). The higher potency of DDT to induce the eosinophil-mediated responses than myo- metrial hypertrophy is in agreement with the existence of different kinds of receptors and separate mecha- nisms involved in these responses and suggests a differ- ence in affinity of DDT for receptors involved in these parameters of estrogen action.

It was previously shown that a second class of estro-

gen receptors in the eosinophils, displaying a lower affinity for estradiol-17P but very high affinity for estra- diol-l7a, are involved in eosinophil-mediated re- sponses to estrogen (Tchernitchin et al., 1985b, 1989). The first class of eosinophil estrogen receptors displays a very high affinity for estradiol-l7p, is located in the surface of these cells, and is involved in the recognition of the endothelial vascular cells of the uterus by the eosinophils and their subsequent migration to the extra- vascular compartment of the organ. The second class of eosinophil estrogen receptors is locaed in the cyto- plasm of the cell and plays a role in eosinophil degranu-

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Fig. 5. Effect of p,p'-DDT or estradiol-17p on the in vivo induced in vitro degranulation of eosinophils from blood of adult ovariectomized rats. The animals were intrave- nously injected with estradiol-17p at the dose of 30 p g / 100 g body wt [El or its vehicle [C (E)], or with DDT at the dose of 25 pg/lOO g body wt [D] or its vehicle [C (D)]. The blood was collected from the tail of ether-anesthetized animals at 6 h [E6, D6] or 24 h [(E24, D24)] after treatment and incubated for 15 min. Results are expressed as % of the maximal response to estrogen, which was at 24 h after treatment kSEM. For analysis of significance, the LSD test was used (***) p < 0.001 and (****) p < 0.0001, compared to vehicle-injected animals; (+) p < 0.05, com- pared to the response to estradiol, within the same time of treatment.

270 BUSTOS ET AL.

lation. The dissociation between responses mediated by these receptors points to their different properties of the receptors. In this context, the higher potency of DDT to induce eosinophil degranulation than eosino- phi1 migration to the uterus and endometrial edema suggests that DDT displays higher affinity for the eosin- ophil cytoplasmic receptor. The previous finding that DDT inhibits estrogen-induced eosinophil degranula- tion has already suggested that DDT interacts with estrogen receptors involved in eosinophil degranula- tion (Bustos et al., 1995).

Eosinophil leukocytes that migrate to the uterus un- der estrogen stimulation must degranulate and release their content before eosinophil-mediated responses are elicited. It has been previously shown that agents or conditions degranulating the eosinophils interfere with parameters of hormone action where the eosinophils play a role (Tchernitchin et al., 1985a, 1989). The eosin- ophil-degranulating property of low doses of p,p‘-DDT suggest that the pesticide may interfere with eosino- Phil-mediated responses to estrogen. In fact, the com- parative higher potency of the pesticide to induce edema in superficial stroma than in deep stroma can be interpreted as caused by the increase in eosinophil degranulation, which occurs with very low doses of DDT, so that the enzymes released by these leukocytes can access sites such as superficial stroma, where nor- mally only scarce eosinophils can be found.

The most relevant finding of this study is the interac- tion of p,p’-DDT with the action of estrogens in the uterus, which occurs mainly with eosinophil-mediated nongenomic responses to estrogen, but also, to a lesser degree, with genomic responses. This interaction may explain the reproductive effects of the pesticide in hu- mans and in wild animals, and suggest a possible inter- action with other responses to estrogen, such as neuro- behavioral effects, that need to be investigated.

The decrease in fertility under the effect of exposure to p,p’-DDT (Lundberg, 1973) is well known. The pres- ent study supports the suggestions that it may occur through either of two mechanisms: a direct interaction with estrogen receptors of adult humans or experimen- tal animals (Bulger and Kupfer, 1983; Jordan et al, 1985), or as a delayed effect following prenatal expo- sure to estrogens (Herbst et al., 1971; McLachlan et al., 1982; Berger and Alper, 1986; Menczer et al., 1986; Tchernitchin and Tchernitchin, 1992). In addition, it was reported that p,p‘-DDE, an environmental metab- olite of DDT, displays potent androgen receptor antag- onist action (Kelce et al., 1995), and it was suggested that the decrease in sperm count in Denmark over re- cent years and the decrease in penile size in alligators in Lake Apopka, Florida, may be explained by expo- sure to this metabolite (Sharpe, 1995).

The interaction of p,p’-DDT with eosinophils is spe-

cially important, taking into consideration that these cells, in addition to their role in estrogen action and in reproduction, are involved in giucocorticoid-induced regulation of immune responses (Tchernitchin et al., 1989), in many hypersensitivity reactions including al- lergy and bronchial asthma, in defense against pluricel- Mar parasites, and in diseases such as hypereosinophi- lic syndrome. Therefore, DDT may affect all these processes where eosinophils play a role.

This research was financed by research grant DTI B-2684, University of Chile.

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