Mutation Research, 261 (1991) 181-191 0 1991 Elsevier Science Publishers B.V. All rights reserved ADONIS 0165121891001475

MUTGEN

181 0165-1218/91/$03.50

01714

Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse L. Pylkkanen, K. Jahnukainen,

M. Parvinen and R. Santti

Institute of Biomedicine, Department of Anatomy, Uniwrsify of Turku, Turku (Finland) (Received 13 December 1990) (Revision received 16 May 1991) (Accepted 22 May 1991)

Keywords: Oestrogens;

Development;

(Mice;)

Micronuclei;

Sperm-head

abnormalities;

Morphology

Summary

The mutagenicity and toxicity of diethylstilbestrol (DES), 17P-estradiol and zeranol on the male mouse germ cells were investigated with meiotic micronucleus assays in vivo and in vitro, sperm-head abnormality test and morphometry. Further, the developmental effects of DES on the testicular morphology were explored. Micronucleus induction was observed at lo-’ M concentration of DES and 17/3-estradiol in vitro, but other treatments yielded negative results. The micronucleus assay in vivo revealed a small number of micronuclei in early haploid spermatids 17 days after a single subcutaneous injection of DES 50 mg/kg, whereas estradiol and zeranol gave negative results. The sperm-head abnormality rates were significantly elevated 5 weeks after treatments with high doses of DES, 17@estradiol and zeranol, and testicular morphometry revealed transient changes in the volume densities of testicular tissue components. Prenatal and neonatal estrogen administration resulted in permanent alterations in seminiferous epithelium and dilatation of the rete testis, but did not affect micronucleus or sperm-head abnormality rates. The mutagenicity and toxicity of hormones in the mouse testis paralleled the hormonal activity of these compounds. Early estrogenization was the most sensitive toxicity test, followed by in vitro meiotic micronucleus induction, whereas the sperm-head abnormality assay and morphological analysis did not reveal subtle changes.

Many chemically different compounds have estrogenic biological functions. Besides natural estrogens (e.g., 17/3-estradiol), there are environmental estrogens, which involve various chemicals

Correspondence: Dr. L. Pylkkinen, Institute of Biomedicine, Department of Anatomy, University of Turku, Kiinamyllynkatu 10. SF-20520 Turku (Finland).

including many estrogenic substances in plants (e.g., coumestrol and equal) and mycotoxins produced by fungi (e.g., zearalenone), as well as synthetically processed chemicals used as hormones (e.g., diethylstilbestrol), or substances originally prepared for non-hormonal purposes (e.g., DDT). Humans can be exposed to environmental estrogens via their food. Previously, and in some countries still, both steroidal and non-steroidal

estrogens were widely used in beef and cattle breeding as growth promoters. Exposure to naturally occurring zearalenones in the form of moldy feed grains is possible. The best known example of the adverse effects of cstrogenic compounds on the genital tract is diethylstilbestrol (DES), which is an established transplacental carcinogen in humans causing clear-cell adenocarcinoma in females exposed prenatally (Herbst, 1981) in addition to diverse developmental disturbances in the female genital tract (Haney, 1987). Prenatal DES exposure of males has also been linked to various structural alterations ranging from epididymal and spermatocele cysts to testicular hypoplasia, cryptorchidism and hypospadias (Bibbo et al., 1977; Gill et al., 1979). Moreover, infertility problems have been suggested among DES-exposed males (Stillman, 19821, but no direct evidence for elevated cancer rates has been presented so far. In experimental animals genital tract alterations qualitatively comparable to those described in humans have been induced after exposure to DES prenatally or neonatally (McLachlan et al., 1975; Arai et al., 1977). In mutagen tests estrogens appear to be nonmutagenic in prokaryotic and in almost all eukaryotic cells. Under certain conditions DES has been shown to increase unscheduled DNA synthesis, sister-chromatid exchanges and chromosomal aberrations. Aneuploidy and cell transformation have also been observed in different mammalian cells and in yeast after in vitro administration of DES (see Degen and Metzler, 1987 for review). However, little is known about the direct effects of sex hormones on the male germ cells. The aim of this study was to investigate the mutagenicity and toxicity of structurally different estrogens in the mouse testis employing the micronucleus and sperm-head abnormality tests as well as morphological methods. The natural steroidal estrogen (17@estradiol), the synthetic non-steroidal estrogen (diethylstilbestrol) and a commercially produced non-steroidal anabolic agent (zeranol, derived from a class of natural metabolites of mold, zearalenones) were chosen as test substances in toxicity studies. DES was selected as a reference compound in experiments of developmental toxicity.

Materials

and methods

Animuls and treatment protocols Outbred Han:NMRI mice aged IO-14 weeks were used. For the in vitro micronucleus assay male mice of strain CS7Bl aged IO-16 weeks were used, because this strain is technically more suitable for isolation of seminiferous tubule segments. Animals were housed with a l4/lO light/dark cycle, 5 in each cage after weaning at the age of 22-24 days. Standard laboratory chow (Hankkija Oy, Finland) and water were given ad libitum. At the end of each experiment animals were killed by cervical dislocation. In prenatal studies timed pregnant NMRI mice were treated with DES 100 pug/kg of maternal body weight on days IO- 12 or on day IS of gestation. DES given in higher doses or after the 15th day of gestation prevented normal delivery in many animals. In neonatal studies animals were treated on either day 1, days l-3 or days 1-S after birth with DES 2 pg/pup/day in 20 ~1 corn oil. Controls were treated with a corresponding volume of corn oil. The male progeny were investigated at the age of 2 months. Diethylstilbestrol (Sigma, St. Louis, MO, U.S.A.), 17P-cstradiol (Sigma). zeranol (Nzearanolol, kindly provided by International Mineral and Chemical Corporation, Terre Haute, IN) (DMBA, and 7,12-dimethylbenzanthracene Sigma) were dissolved in ethanol (1% of injection volume) in corn oil (Laaketukku Oy. Finland). and clear solutions were prepared with a magnetic mixer. Volumes for subcutaneous injection of test substances were 0.3 ml except for zeranol 750 mg/kg, that was 0.6 ml. For in vitro studies, the hormones were dissolved in ethanol (final concentration 0.1 %I. In riL.0 micronucleus assuy Micronuclei induced by mutagen exposure arise from chromosomal fragments and chromosomes that are not incorporated into daughter nuclei at the time of cell division (Heddle, 1973; Schmid, 197.5). In the male mammalian germ cells, micronuclei formed during meiotic division can be scored after exposure in vivo (Lahdetie and Parvinen, 1981) or in vitro (Toppari et al., 1986) in early postmeiotic germ cells, which can

be enriched by transillumination-assisted microdissection (Parvinen and Vanha-Perttula, 1972) combined with identification of the exact stages by phase-contrast microscopy (Lahdetie and Parvinen, 198 1). Germ cells exposed to chemicals at the onset of meiosis can be harvested in stage 1 as early round spermatids 17 days after treatment (Oakberg, 1956b). Micronucleus formation at this time point reflects genotoxicity, which requires replicative DNA synthesis. Our aim was to get a sample where some cells of stage 12 (meiotic division figures) could be seen together with early round spermatids of stage 1 (Parvinen and Hecht, 1981). From isolated segments (about 0.5 mm) of this zone squash preparations were processed and stained as described elsewhere (Lahdetie and Parvinen, 1981). The cell associations were then identified according to the criteria described by Oakberg (1956a). Quantitation of micronucleated spermatids was done from coded slides under oil immersion at lOOO-fold magnification with a Leitz fluorescence microscope using a K430 barrier filter and UG-1 and BG-38 exciter filters.

suggested to reflect the mutagenic potential of these chemicals (Wyrobek and Bruce, 197.5). The dimensions of the sperm heads are highly heritable and are postulated to be polygenetically determined (Wyrobek, 1979). Likewise, a good correlation between dose-response curves of abnormal sperm heads and chromosomal abnormalities after irradition has been demonstrated, further suggesting a role for genetic damage in induction of sperm-head abnormalities (Bruce et al., 1974). For the sperm-head abnormality assay, the total dose of test substances was divided into 5 consecutive injections, which were given subcutaneously. 5 or 10 weeks after the onset of exposure to hormones, the preparation of the smears from caudae epididymides, the processing of the slides, and the analysis of the morphology of 1000 spermatozoa recording the type of abnormality were performed according to Wyrobek and Bruce (1975). Analyses were carried out from coded slides with a Leitz light microscope at lOOO-fold magnification under oil immersion using green filters to enhance the contrast.

In vitro micronucleus assay Segments (1 mm> from stages 9-l 1 of mouse seminiferous tubules containing late pachytene and diakinetic primary spermatocytes were separated and cultured for 3 days as described by Toppari et al. (1985). Careful examination under phase-contrast optics of OS-mm segments adjacent to each selected sample was done to exclude any meiotically dividing cells in the selected sample at the onset of the culture. In these culture conditions, late pachytene and diakinetic primary spermatocytes from stages 12 and 13 of the rat seminiferous tubular segments have been shown to be capable of complete meiosis (Toppari et al., 1985). In this study micronuclei were analyzed in early haploid spermatids formed in vitro during 3 days in culture conditions. Culturing and processing of the samples, and quantitation of micronucleated spermatids were carried out as described elsewhere (Toppari et al., 1986).

Morphometric analysis of seminiferous tubules For light microscopy, the testes were fixed by immersion in Bouin’s fluid for 2 days. Specimens were then dehydrated and embedded in paraffin. 5-Frn sections were prepared against the short axis of the testis in animals investigated 5 weeks after treatment (Table 4) and against the long axis of the testis in other experiments. The latter procedure proved to be more favorable, because thus most tubules were cut transversely. Staining was done with the periodic acid Schiff and hematoxylin methods. Morphometric analysis was performed with a Reichert microscope, the projecting head of which was equipped with Weibel’s multipurpose screen with 21 test lines and 42 test points (Weibel, 1966). The length of each line was 1.5 cm. The final magnification was 200 X . From a representative section of each testis 10 systematically selected fields (starting from the upper left corner) were analyzed from coded slides. The test points located on seminiferous epithelium, tubular lumina and interstitial tissue were scored and the volume densities of seminiferous epithelium, tubular lumen and interstitial

Sperm-head abnormality assay Elevated sperm-head abnormality duced by several model mutagens,

rates, inhave been

tissue were estimated according to the principles of point-counting stereology (Weibel, 1966). Statistics Analyses for statistical significance were carried out by one-way analysis of variance. If significance was observed, pairwise comparisons to control values were made with Student’s t-test after Bonferroni’s correction. In micronucleus experiments the data were analyzed after square root transformation. Statistical processing was carried out using a BMDP computer program library (Dixon, 1985). Results IH rilso and in ritro micronucleus assays Diethylstilbestrol at 50 mg/kg significantly (p < 0.05) increased the in vivo micronucleus rates 17 days after exposure, whereas estradiol and zeranol yielded negative results (Table 1). 7.12Dimethylbenzanthracene, which has been demonstrated to induce meiotic micronuclei in rat testis at preleptotene stage (Lahdetie, 1983), induced micronuclei significantly also in this study at a dose of 40 mg/kg (p < 0.05). In vitro, DES and 17&estradiol induced micronuclei at 1O-7 M concentration, while other concentrations gave negative results (Table 2). Zeranol did not induce micronuclei significantly.

TABLE

I

THE NUMBER I SPERMATIDS

OF MICRONUCLEATED IN 1000 SPERMATIDS

Treatment I7 clq5 ufter trYUIment Experiment 1 corn oil DMBA 40 mg/kg DES 10 mg/kg DES 20 mg/kg DES SO mg/kg Experiment 2 corn oil EST 50 mg,‘kg ZER SO mg/kg * p < 0.05 compared

GOLGI (i SEM)

Number of mice

3.00 + 0.62 5.43 + 0.84 *

II 7

3.40i_ 1.29 4.fJo+ 1.17 8.25 -i: 1.7s *

5 5 8

2.20 t 0.66 2.00 t 0.40 2.67 + 0.57

5 ‘l 6 control

group.

2

THE NUMBER I SPERMATIDS Treatment

OF MICRONUCLEATED IN 500 SPERMATIDS Micronucleated spermatidh

PHASE

Number of samples

I

Experiment control

lO.S_+ I.8

DES IO-” M DES10 ‘M DES 1V” M Experiment control

GOLGI

C+ SEM)

18.0*5,2 32.4+6.8 14.6*X5

*

2 6.x+ 1.0

EST IO-’ M EST10 “M EST IO- M EST 10 -’ M

11.x*23 6.6 i I .o 15.Oi4.I *7.x * 1.x

ZER ZER ZER ZER

13.0+2.H 7. I + I .4

IO -’ IO-” IO ’ IO-”

M M M M

28 i IO 6

ll.h+3.7 6.x+ I.5

* p < 0.05

DES prevented the formation of early round spermatids at lop5 M concentration in vitro, whereas this concentration of estradiol or zeranol had no effect. Three different micronucleus types were induced after estrogen exposure in vitro, as illustrated in Fig. 1. High concentrations of estrogens also caused the formation of large spermatid-like nuclei containing several nucleoli (Fig. 1).

PHASE

Micronucleated spermatids

to corn oil-treated

TABLE

Sperm-head and testicular morphology The sperm-head abnormality assay and testicular morphometry (Tables 3 and 4, respectively) were carried out with the same animals treated 5 or 10 weeks before decapitation. Estradioi significantly induced sperm-head abnormalities at total doses of 250 and 375 mg/kg, while morphometric analysis revealed an increase in the volume density of interstitial tissue in all estradiol-treated groups and a decrease in that of seminiferous epithelium in the group given estradiol 250 mg/kg. DES already increased the sperm-head abnormality rates significantly in the lowest dose group of 125 mg/kg (p < 0.05), and the volume density of interstitial tissue was significantly in-

185

creased in groups given DES 250 and 375 mg/kg. Zeranol did not elevate the sperm-head abnormality rates at a dose range of 125-375 mg/kg. A total dose of 750 mg/kg was needed for a significant elevation of sperm-head abnormalities. The

volume density of interstitial tissue was increased at the dose levels of 2.50, 375 and 750 mg/kg. These hormone treatments did not induce any special type of sperm-head abnormality. The observed effects were transient, because sperm-head

Fig. 1. Different micronucleus types induced in vitro. (a) Two early round spermatids containing type 1 (pale) micronuclei (long arrow). The other spermatid also contains a type 3 (bright condensed) micronucleus (short arrow). Magnification 1200 x (b) Large spermatids containing type 2 (pale, bright spot inside) micronuclei. Only those micronuclei that were well separated from the main nucleus were recorded (one arrow), excluding the others (two arrows). Magnification 1200x. Cc)Early round spermatid containing type 2 (pale, bright spot inside) micronucleus at a higher magnification (3600 X ). This was the most common micronucleus type.

Fig. 2. Alterations height) 30x.

in the rete tratis

and seminil’erous

on the ISth day ot gestation. (a) Grossly

(b) Degcnrrative changes

(;lrrows).

Magnification the cpithelium

120x.

cpithelium

ai’tcr prenatal ~rdminiatratioll

of DES

dilated rctc testis combined with ~rcduced seminifcI-ous

in the testicular- parenchyma vary from

slightly

(c) Rcte testis area ~11a higher magnification

is mainly nol-mal. (d) Testicular

diaturhed

(I00

pg/hg

epitheliurn.

sprrmatogenesis

hod)

Magnltication

to acellulat- tuhulea

(120X 1. Rete testis i\ filled with aprrmatogenic

and rrte testis histology I’]-om ;I control animal. Magnification

abnormality rates and stereological parameters returned to normal in most cases 10 weeks after treatment (see Tables 3 and 4). Exposure to DES and 17P-estradiol reduced

matrrnal

wlls.

120 x

testicular weights (11 < 0.05) 5 weeks after treatment when the highest doses were used. The testicular body ratios remained normal. Zeranol did not have an effect on absolute or relative

1x7 TABLE

3

THE NUMBER OF ABNORMAL SPERM HEADS AFTER TREATMENT WITH CORN OIL, 7,12-DIMETHYLBENZANTHRACENE (DMBA), DIETHYLSTILBESTROL (DES), 17~-ESTRADIOL (EST)AND ZERANOL (ZER) (iSEM) Number of mice

Treatment

5 vt1eek.s ufrrrtreutment 16 corn oil 4 DMBA 80 mg/kg

Number of abnormalities/l000 spermatozoa k SEM

41.9* 2.6 Y&3* 14.0 ***

EST 125 mg/kg EST 250 mg/kg EST 375 mg/kg

6 6 6

47.8& 5.6 YY.8+ 20.3 * 106.5 k 26.5 *

DES 125 mg/kg DES 250 mg/kg DES 375 mg/kg

h 6 6

73.2i 12.4 * 53.5+ 7.6 72.5 + 8.9 *

ZER 125 mg/kg ZER 250 mg/kg ZER 375 mg/kg

Y Y 9

54.4+ 6.6 48.1 * 8.5 43.0+ 10.2

corn oil 0.6 ml ZER 750 mg/kg

5 4

10 weeks after treatment corn oil EST 375 mg/kg DES 375 mg/kg ZER 750 mg/kg

4 4 4 4

* p < 0.05,

54.4+ 4.9 136.0 * 30.2 *

37.3* 7.0 52.x * 12.5 37.0* 11.6 42.0+ 14.6

*** p

Testicular toxicity and mutagenicity of steroidal and non-steroidal estrogens in the male mouse.

The mutagenicity and toxicity of diethylstilbestrol (DES), 17 beta-estradiol and zeranol on the male mouse germ cells were investigated with meiotic m...
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