SHORT COMMUNICATION EFFECT OF SINGLE AND REPEATED IN VITRO EXPOSURE OF OVARIAN FOLLICLES TO o,p’-DDT AND p,p’-DDT AND THEIR METABOLITES

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SHORT COMMUNICATION

EFFECT OF SINGLE AND REPEATED IN VITRO EXPOSURE OF OVARIAN FOLLICLES TO o,p’-DDT AND p,p’-DDT AND THEIR METABOLITES

Anna K. Wójtowicz

^1,#

, Ewa L. Gregoraszczuk

¹

, Anna Ptak

¹

, Jerzy Falandysz

²

Laboratory of Physiology and Toxicology of Reproduction, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, Ingardena 6, PL 30-060 Kraków; University of Gdañsk, Faculty of Chemistry, Chair of Analytical Chemistry, Sobieskiego 18, PL 80-952 Gdañsk, Poland

Effect of single and repeated in vitro exposure of ovarian follicles to o,p’-DDT and p,p’-DDT and their metabolites. A.K. WÓJTOWICZ, E.L. GREGORASZCZUK, A. PTAK, J. FALANDYSZ. Pol. J. Pharmacol., 2004, 56, 465–472.

The aim of the presented study was to compare the effect of o,p’-DDT [1,1-dichloro-2,2-bis-(p,p’-chlorophenyl)-ethylene] and p,p’-DDT [1,1,1- trichloro-2,2-bis-(p-chlorophenyl)-ethane] and their metabolites DDE and DDD on estradiol secretion by ovarian follicles, the target organs of environmental estrogens. Theca interna (Tc) and granulosa cells (Gc) were collected from medium size porcine follicles and cultured as a monolayer. The cells were initially cultured for 24 h to allow attachment to the plates and then media were changed for the new ones and o,p’-DDT and p,p’-DDT and their metabolites: o,p’-DDE, p,p’-DDE and o,p’-DDD were added at doses of 4, 40, 400 ng and 4mg/ml medium to investigate dose-dependent effects. Me- dia were collected after 24 h and frozen for estradiol content determination.

When the effect of single and repeated exposure was investigated, the lowest dose of 4 ng/ml and the highest one of 4mg/ml were chosen on the basis of the results of Experiment 1. o,p’-DDT exerted antiestrogenic action at all doses used while its metabolites and p,p’-DDT and its metabolites decreased estradiol secretion only when present in the medium at a dose of 4 ng/ml.

The highest doses caused the increase in estradiol secretion. Parent o,p’- DDT and its metabolites showed antiestrogenic action after single exposure to 4 ng/ml while parent p,p’-DDT and its metabolites caused estrogenic action. All investigated compounds, except o,p’-DDT, increased estradiol secretion after single exposure to the dose of 4mg/ml. Repeated exposure resulted in a massive antiestrogenic action of all investigated chemicals.

In conclusion, our study points to time-dependent effect of DDT and its metabolites on ovarian follicles with the strongest estrogenic properties observed after single exposure and antiestrogenic action caused by repeated exposure. Given the duration of folliculogenesis, one can imagine many different potential mechanisms by which DDT could influence steroidogenesis.

Key words: granulosa and theca cells, o,p’-DDT, p,p’-DDT, steroid se- cretion

ISSN 1230-6002

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INTRODUCTION

It was found that the insecticide p,p’-DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane] and its most stable metabolite p,p’-DDE [1,1-dichloro-2,2-bis-(p,p’-chlorophenyl)-ethylene] have adverse effects on reproduction [3]. In mammals p,p’-DDT is converted by reductive dechlorination to p,p’-DDD and then by dehydrochlorination to p,p’- DDE [9]. Dietary exposure to DDTs results in their accumulation in the lipid phase of the tissues and cells including human blood, adipose tissue, and ovarian follicular fluid [29]. Hirshfield [19]

showed that those xenobiotics could impair fertility by altering follicular growth and hormone biosynthesis.

In mammalian species, dose-response studies of toxic effects of these substances, such as distur- bance of sexual development and reproductive function across species, tissues, and ages are very complicated, expensive and time consuming and, therefore, very limited. In vitro systems are uniquely suited to investigate specific cellular and molecular mechanisms of toxic effects in potential target organs such as the ovary and, thus, can improve risk assessment. Original toxicants, their precursors or selective inhibitors can be individually added to isolated cell types to evaluate specific toxicity mechanisms. In our laboratory, we collected follicular cells from the porcine ovary excised from animals showing natural estrous cycle to study the direct effect of DDT on ovarian steroidogenesis.

The aim of the present study is to compare dose- and time-dependence of effects of insecticides p,p’-DDT, o,p’-DDT as well as their metabolites (DDD and DDE), on estradiol secretion by follicular cells in primary co-culture of granulosa cells (Gc) and theca cells (Tc) isolated from antral porcine follicles. The rationale of performing the co- culture experiment was based on an idea that theca-derived androgens will serve as a substrate for estradiol production by co-cultured Gc [7, 34].

MATERIALS and METHODS

Chemicals and reagents

Parker medium M199, trypsin, and calf serum (CS) were purchased from the Laboratory of Sera and Vaccines, Lublin, Poland. Antibiotic, antimy- cotic solution (100x) was obtained from Sigma Chemical Co. St. Louis, MO, USA. DDT conge-

ners (p,p’-DDT, o,p’-DDT, p,p’-DDD, o,p’-DDD, p,p’-DDE and o,p’-DDE) were purchased from Reference Standards, EPA, Research Triangle Park, NC, USA. Stock solutions of these test compounds in DMSO were prepared and added to M199 supplemented with 5% CS at appropriate concentrations every 24 h starting from 24 h till 72 h of culture. The final concentration of DMSO in the medium was always 0.6%.

Cell cultures

Porcine ovaries obtained from a local abattoir were collected into a bottle filled with sterilized ice-cold saline and transported to the laboratory.

Approximately 1.5 h elapsed from slaughter to arri- val at the laboratory. Medium (6–8 mm in diame- ter) follicles were obtained from ovaries collected on day 16 of the estrus cycle as previously described by Gregoraszczuk and Ska³ka [14]. In each experiment, six ovaries from three animals were selected for cell preparation. Since each ovary yielded four to six follicles, the total number of follicles for each preparation varied between 24–36.

This procedure was chosen to minimize possible variations existing between follicles and animals.

Gc and Tc were subsequently prepared according to the technique described by Stok³osowa et al. [34].

Gc were scrubbed from the follicular wall with round-tipped ophthalmologic tweezers and rinsed several times with PBS. After isolation, Gc were washed three times in M199, collected and resus- pended in M199 supplemented with 10% CS (M199/CS). The Tc from the same follicles was prepared as previously described in detail by Stok³osowa et al. [33]. Briefly, the theca layers were placed in a drop of saline under a dissecting microscope. The theca interna was manually separated from the underlying theca externa. The isolated theca interna tissue was then washed with PBS, cleaned, cut with scissors and exposed to trypsinization with 6–7 ml of 0.25% trypsin in PBS for 10 min at 37°C. The cells were separated by de- cantation and the procedure was repeated three times. Finally, the cells were centrifuged and resus- pended in M199/CS. For co-culture experiments, the viability of granulosa and theca cells was determined by the Trypan blue exclusion test and subsequently they were inoculated at a concentration of 1.25 × 10⁵and 0.3 × 10⁵cells/well, respectively, in Nunc 48 well tissue culture plates. Thus, the ratio of the cells was comparable to that observed in vivo

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(Gc : Tc = 4:1) according to Stok³osowa et al. [34].

The cultures were maintained at 37°C in a humidi- fied atmosphere of 5% CO₂/95% O_2.

(Gc : Tc = 4:1) according to Stok³osowa et al. [34].

The cultures were maintained at 37°C in a humidi- fied atmosphere of 5% CO₂/95% O_2.

Experimental procedure

Experiment 1 was conducted to examine the dose-dependent effect of DDT and its metabolites on estradiol secretion by follicular cells. Cells were isolated and initially cultured without test compounds for 24 h to allow for cell attachment to the wells. After 24 h, the medium was discarded and 0.5 ml of fresh M199 medium supplemented with 5% CS was added at the control culture, while to the experimental culture o,p’-DDT and p,p’-DDT or their metabolites were added at concentrations of 4, 40, 400 ng and 4mg/ml medium. Media were collected after 24 h and frozen for estradiol determination. Every treatment was conducted in 4 wells and each experiment was repeated 3 times.

Experiment 2 was conducted to demonstrate the time-dependent effect of DDT and its metabolites on estradiol secretion by follicular cells. Cells were isolated and cultured without test compounds for 24 h to allow for cell attachment to the wells. After 24 h the medium was discarded and 0.5 ml of fresh

M199/CS alone was added to the control culture, while to the experimental cultures, o,p’-DDT or p,p’-DDT or their metabolites were added at a concentration of 4 ng/ml or 4mg/ml medium. This concentration of DDT and its metabolites was chosen based on the results from dose-response curves obtained in Experiment 1. Media were collected after single (24 h) or repeated (72 h) exposure, and frozen for estradiol determination. Thus, in the experiments with repeated exposure, the compounds were added 3 times during the experiment. Each treatment was conducted in 4 wells and the experiment was repeated 3 times.

Steroid analysis

17b-estradiol (E2) concentration in the media was determined by radioimmunological method using Spectra kits (Orion, Diagnostic, Finland) sup- plied by Polatom (Œwierk, Poland). The detection limit of the assay was 5 pg. The coefficients of variation between and within assays were 10.3%

and 2.9%, respectively. The mean recoveries were 85.6–108.9%. The cross-reaction with ethinylestra- diol was 1.4%. All other tested steroids (estrone, 0

1 2 3 4 5 6 7 8

C o’,p’-DDT o,p’-DDE o,p’-DDD p,p’-DDT p,p’-DDE

Estradiolpg/ml

0 4 ng 40 ng 400 ng 4 gm

* * * *

**

*

* *

*

* *

*

**

Fig. 1. The dose-dependent effect of DDT and its metabolites on estradiol secretion by co-culture of granulosa and theca cells isolated from medium-size follicles (MF). Initially the cells were cultured in serum-containing (10% calf serum) M199 for 24 h to allow for cell attachment to the plates. After 24 h, serum-containing M199 was discarded and cells were cultured for an additional 24 h in M199 supplemented with 5% calf serum; 4, 40, 400 ng/ml or 4mg/ml o,p’- DDT and DDT - p,p’and metabolites: o,p’-DDE, p,p’- DDE and o,p’-DDD were added to experimental groups. After 24 h of culture, the media were collected and frozen (–20°C) for estradiol analysis. Each histogram represents the mean ± SEM derived from three different experiments (n = 3) each in quadruplicate. *p <

0.05; **p < 0.01

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estriol, progesterone, testosterone, and corticoster- one) showed less than 1% cross-reaction.

Statistical analysis

Each treatment was repeated three times in quadruplicates and, thus, the total number of repli- cates was 12. Since the variations between the experiments were small, those 12 results were aver- aged and analyzed by analysis of variance followed by Duncan’s new multiple range test. All data (n = 12) are expressed as the means ± SEM.

RESULTS

Dose-dependent action of o,p’-DDT and p,p’- DDT and their metabolites o,p’-DDE, o,p’-DDD, p,p’-DDE on estradiol secretion

In control cultures, estradiol secretion into the medium for 24 h resulted in its final concentration of 2.28 ± 0.9 pg/ml.

Parent o,p’-DDT induced a significant decrease in estradiol secretion at all tested concentrations.

The amount of estradiol in the medium of o,p’- DDT-treated cells was on average 1.08, 1.1, 1.02

Single exposure

0 1 2 3 4 5 6 7 8 9

K o’,p’-DDT o,p’-DDE o,p’-DDD p,p’-DDT p,p’-DDE o,’p’-DDT o,p’-DDE o,p’-DDD p,p’-DDT p,p’-DDE

Estradiolpg/ml

4 ng/ml a

* * *

*

Repeated exposure

0 2 4 6 8 10 12

K o’,p’-DDT o,p’-DDE o,p’-DDD p,p’-DDT p,p’-DDE o’,p’-DDT o,p’-DDE o,p’-DDD p,p’-DDT p,p’-DDE

Estradiolpg/ml 4 ng/ml

b

** ** **

* *

*

Fig. 2. The effect of a) single and b) repeated exposure to DDT and its metabolites on estradiol secretion by co-culture of granulosa and theca cells isolated from medium-size follicles (MF). Initially the cells were cultured in serum-containing (10% calf serum) M199 for 24 h to allow for cell attachment to the plates. At 24 h, serum-containing M199 was discarded and cells were cultured for an additional 24 h or 72 h in M199 supplemented with 5% calf serum; 4 ng/ml (minimal concentration) and 4 mg/ml (maximal concentration) o,p’- DDT and DDT - p,p’ and metabolites: o,p’-DDE, p,p’ - DDE and o,p’-DDD were added to experimental groups. Each histogram represents the mean ± SEM derived from three different experiments (n = 3) each in quadruplicate. * p < 0.05; **p < 0.01.

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and 1.39 pg/ml vs. 2.28 pg/ml in the control cul- ture. (Fig. 1), o,p’-DDE at a dose of 4 ng/ml decreased estradiol secretion (p < 0.01) at doses of 40 and 400 ng/ml was without effect, while at a dose of 4 mg/ml significantly increased estradiol secre- tion (p < 0.01) (Fig. 1).

o,p’-DDE only at a dose of 4 ng/ml was without effect on estradiol secretion. At all other doses used in the experiment, it significantly increased secretion of estradiol into the medium (3.73, 3.73 and 4.74 ng/ml, respectively, after 40 ng, 400 ng (p < 0.05) and 4mg/ml (p < 0.01). (Fig. 1).

Both parent p,p’-DDT and its metabolite p,p’- DDE showed estrogenic action at all doses used (Fig. 1).

The effect of single and repeated exposure to o,p’-DDT and p,p’-DDT and their metabolites o,p’-DDE, o,p’-DDD, p,p’-DDE on estradiol se- cretion

After single exposure to o,p’-DDT and both metabolites at a lower dose (4 ng/ml), a decreased estradiol secretion was noted while at a greater concentration (4 mg/ml), they showed estrogenic action (2.4-fold and 2-fold increase in estradiol secretion, respectively, under the influence of o,p’- DDE and o,p’-DDD, p < 0.05). Parent p,p’-DDT and its metabolite p,p’ -DDE at both the lowest and the highest doses had estrogenic action (Fig. 2a).

After repeated exposure to both isomers of DDT and their metabolites, a decrease in estradiol secretion was noted (Fig. 2b).

DISCUSSION

Estrogen mimicry is one of the most commonly reported effects of endocrine disrupters [24, 28], so it is important to focus on estrogen sensitive tissues in the female reproductive system. The present experiments using co-culture of Tc and Gc whose ra- tio was the same as that existing in vivo in the ovar- ian follicles, showed an increase in estradiol secretion after single exposure to all tested agents but o,p’-DDT. Oppositely, a huge antiestrogenic action of all investigated chemicals was noted after repeated exposure. The strongest estrogenic action was observed after exposure to p,p’-DDE which is a very stable and highly lipophilic metabolite of p,p’-DDD, acting as an estrogen [4], an antiestrogen [39], or antiandrogen [6] depending on the studied system. Clark et al. [4] investigated the ef-

fect of DDT and p,p’-DDE on the development of amphibian gonaducts and showed that DDT an- tagonized the estrogenic action of the steroid treat- ments, while p,p’-DDE acted as an estrogen on the Müllerian duct of females. Danzo [6] examined binding properties of [³H] physiological ligands (present at a concentration of 7 nM) to the androgen and showed that DDT, p,p’-DDT, p,p’-DDE and o,p’- DDT caused a statistically significant inhibition of specific binding of [³H] 5a-DHT to the androgen receptor. A very important question con- cerns the mechanism(s) of this stimulatory effect.

Based on the current literature on estrogenicity of the investigated compounds, three mechanisms can be envisioned: (i) ability to react with the estrogen receptor thereby changing intracellular signaling and causing a stimulation of estradiol synthesis [1, 20, 36], (ii) induction of the activity of enzymes involved in estradiol biosynthesis [5, 38], (iii) simi- larly to PCBs they can inhibit the breakdown of estradiol, resulting in an increased bioavailability of this steroid [10, 22, 23].

The follicles of the ovary are major producers of estrogen in the body, a function that is influ- enced by compounds such as dioxin [12, 13, 17, 18, 31] and higher chlorinated PCBs [11, 14–17, 25–28]. Thus, it is possible that lower chlorinated PCBs might also interfere with estrogen production. Indeed, one major effect of PCBs is the induction of metabolizing enzymes, especially the cytochromes P450s. Several cytochrome P450 iso- zymes induced by xenobiotics are active in steroid hormone hydroxylation [2], therefore, xenobiotics that are capable of altering steroid hormone hydroxylation can perturb endogenous steroid me- tabolism and cause disturbances in endocrine ho- meostasis. DDT could possibly increase estrogen production by increasing the activity of the rate limit- ing enzymes that are involved in estradiol synthesis, for example by activating P450 aromatase, which metabolizes androgens to estrogen, as it was suggested by Wójtowicz et al. [40, 41] for PCB action.

Finally, Kester et al. [22, 23] suggested that the estrogenic action of some compounds could be in- direct, by increasing estradiol bioavailability in target tissues through an inhibition of the breakdown and inactivation of estrogen. In this case, DDT it- self does not mimic estrogen, but rather alters the levels of endogenous estrogen by inhibiting the breakdown and inactivation of estrogen by inhibiting the formation of inactive E2 sulfate.

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Relatively little is known about the presence of individual cytochromes P450 in the ovarian follicles. To understand the differences in action of DDT and its metabolites on ovarian steroidogenesis and to elucidate the mechanisms involved in these effects, further studies on the induction of CYP izo- symes by these compounds are needed.

Taking into consideration various data concern- ing the antiandrogenic action of pesticides [21, 30, 35, 37], it seems that the drastic decrease in estradiol secretion observed in the medium and large follicles after repeated exposure to DDTs could be due to the insufficient testosterone production which is a substrate for estrogen production. Andersen et al. [1]

tested twenty four pesticides for interactions with the estrogen receptor (ER) and the androgen receptor (AR) in transactivation assays on human placen- tal microsomes and documented that dieldrin, endo- sulfan, methiocarb, and fenarimol, acted both as estrogen agonists and androgen antagonists. Those observations confirm what was suggested in the present study that in the future studies it would be necessary to examine possible action of DDT as an antiandrogen in particular stage of follicular development.

DDT may be responsible for inadequate estradiol secretion by follicles, what accounts for the anti-fertility effects. Estrogen mimicry is one of the most commonly reported effects of exposure to endocrine disrupters [28]. However, in addition to estrogenic potency, those xenobiotics may act as anti- androgens and, thus, produce “estrogenic environ- ment” [32] but also they can decrease the amount of a substrate available for estrogen production in the follicles and so act as an antiestrogen [16, 39]. The observed decrease in estradiol secretion after repeated exposure to DDT and its metabolites could be due to the stimulation of progesterone production by follicular cells. Progesterone is known inhibitor of aromatase [8, 11].

In summary, both estrogenic and antiestrogenic action of DDT and its metabolites depending on time and doses suggests complex mechanism of their action.

Acknowledgments. The authors would like to thank M. Mika, PhD, from the Department of Animal Physiology, Academy of Agriculture, Kraków, Poland, for radioimmunological determinations of steroid hormones. The work was supported by Jagiellonian University grant DS/IZ/Fz/2004.

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Received: January 23, 2004, in revised form: June 2, 2004.