on Male Rats' Reproductive System. R. CHAYOTH, I. SHAKED and J. KAPLANSKI. U.A. SOD-MORIAH, U. SROR, D. SHEMI, G. POTASHNIK,. Summary: Adult ...
Received September 2,1987
andrologia 20 (1): 60-66 (1988)
Department of Biology, Infertility Unit Department of Obst. and Gynecol. and Clinical Pharmacology Unit, Ben Gurion University of the Negev, Beer SheValIsrael
Long Term Effects of Dibromochloropropane (DBCP) on Male Rats' Reproductive System U.A. SOD-MORIAH, U. SROR,D. SHEMI, G. POTASHNIK, R. CHAYOTH, I. SHAKED and J. KAPLANSKI
Summary: Adult male rats were injected S.C. once a week for 3 weeks with DBCP, 20 mg/kg B.W. Animals were sacrificed 5 , 9 , 13, 17, 25 and 50 weeks after last injection. Body weight was recorded once a week. Prior to sacrifice each male was presented with proestral females in order to determine the male's mating behaviour and fertility. Testes were removed, weighed and taken for standard histological examination. DBCP treatment caused a reduction of body weight which reverted back to control levels ,some 17 weeks post injection. Testes weights were reduced and remained low despite the recovery of body weight. Generally, all males showed normal mating behaviour but most of them were infertile. Testicular histology showed a correlation between decreasing testicular weight and increasing percentage of degenerated seminiferous tubules, which was on the other hand correlated with decreasing tubular diameter. Serum levels of FSH and LH were significantly increased in the infertile DBCP treated males while values for the fertile ones were similar to those of controls. There were no differences in serum testosterone levels between DBCP treated and control animals. It is concluded that in DBCP treated rats testicular degenerative damages are associated with increased circulating gonadotrophin levels and with normal testosterone levels. Although mating behaviour is unaffected fertility is depressed and does not recover for at least 50 weeks post injection. It is suggested that DBCP treatment affects mainly the activity of the Sertoli cells while the Leydig cells are affected to a much lesser degree. Der Langzeiteffekt von Dibromchlorpropan (DBCP) auf das Fortpflanzungssystem der mannlichen Ratte
Zusammenfassung: Erwachsenen mannlichen Ratten wurde uber drei Wochen einmal wochentlich DBCP, 20 mg/kg Korpergewicht, injiziert. 5 , 9 , 13, 1 7 , 2 5 und 50 Wochen nach der letzten Injektion wurden Tiere getotet. Das Korpergewicht wurde einmal wochentlich verzeichnet. Vor der Totung jeder Ratte wurde diese mit einer laufigen weiblichen Ratte zusammengebracht, um das Paarungsverhalten und die Fertilitat der Ratte zu bestimmen. Die Hoden wurden entfernt, gewogen und einer standardisierten histologischen Untersuchung zugefiihrt. Die DBCPBehandlung verulsachte eine Reduktion des Korpergewichtes, welches 17 Wochen nach der Injektion wieder zum Ursprungswert anstieg. Das Hodengewicht war herabgesetzt und verblieb niedrig, ungeachtet des Wiederanstiegs des Korpergewichtes. Im grol3en und ganzen zeigten alle Ratten ein normales Paarungsverhalten, aber die meisten von ihnen waren infertil. Die Hodenhistologie zeigt eine Korrelation zwischen Reduktion des Hodengewichtes und Zunahme des Pro-
Key words: DBCP, rat testes - dibromochloropropane, body weight
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FSH, DBCP - LH, DBCP andrologia 20 (1988)
Prolonged Effects of DBCP
61
zentsatzes an degenerierten Tubuli seminiferi, welche anderewits mit einem verminderten Tubulusdurchmesser korrelierten. Die Serumspiegel von FSH und LH waren bei den infertilen DBCP-behandelten Ratten signifihnt erhoht, wiihrend die Werte der fertilen Ratten der Kontrollgruppe ihnlich waren. Es gab keine Unterschiede des Serum-Testosteronspiegels zwischen DBCP-Behandelten und den Tieren der Kontrollgruppe. Wir schliefen daraus, daf die degenerative Hodenschadigung der DBCP-behandelten Ratten mit ansteigenden zirkdierenden Gonadotropinspiegeln und mit normalen Testosteronspiegeln verbunden ist . Obgleich das Paarungsverhalten unbeeinfluft ist, ist die Fertilitat herabgesetzt und erholt sich nicht vor Ablauf von 50 Wochen nach’der Injektion. Es deutet darauf hin,d P die DBCPBehandlung sich hauptsachlich auf die Aktivitat der Sertolizellen auswirkt, wtihrend die Leydigzellen in geringerem M P e beeinfluft werden. Introduction The use of the nemotocide dibromochloropropane (DBCP) was banned following reports of infertility and hormonal changes developed in workers involved in its production (Biava et al. 1978; Morton et al. - 1977, 1979; Potashnik et al. - 1978,1979; Cortez-Gallegos and Castanada - 1970). Long term exposure of laboratory animals to DBCP resulted in damages to the kidney and liver (Torkelson et al. - 1961). The most impressive damage in these animals was noted in the testes (Torkelson et al. - 1961; Faydysh et al. - 1970). In a study camed out in our laboratory, male rats were injected subcutaneously with DBCP, 20 mg/kg body weight once a week for 3 weeks. Degenerative histological changes were noticed in the testes which apparently recovered 6 months later (Shemi et al. - 1982). There is no information available whether such experimental animals were still fertile despite of the histological testicular damage. The present study was set to determine if DBCP treated animals are fertile and to detect hormonal changes that may result from the treatment. Materials and Methods Chemicals used: DBCP - 1,2-dibromo-3chloropropane, supplied by “Bromine Compounds”, Ramat Hovav, Israel. DMSO - dimethylsulfoxide, BDH, England, used to dissolve the DBCP. AnimZs: Adult male rats from the colony of the department of Biology, Ben Gurion University, descendents of Wistar, weighmg 300-350 g were injected subcutaneously with DBCP, dissolved in DMSO, 20 mg/kg body weight (BW) once a week for 3 consecutive weeks. Control animals were similarly injected with the vehicle alone (DMSO). Animals were kept in a room maintained at 23 f 2” C and 35%-55% relative humidity. Light schedule was 14:lO LD. Animals were kept 4 per cage on wood shavings and were offered standard rat chow and tap water ad libitum. All animals were weighed once a week. Groups of animals were sacrificed by decapitation 5, 9, 13, 17, 25 and 50 weeks after last injection. Prior to sacrifice each male was presented with two proestral females to determine the male’s fertility. Mating was visualized, the presence of vaginal plug was checked and the delivery of young by the females determined whether the male was fertile or infertile. Upon sacrifice, body and testes weights were recorded and blood was collected, serum was separated and stored at - 20” C until hormone assays were performed. Testes were processed by standard histological techniques for examinations. Estimation of testicular damage was carried out by counting the number of tubular cross sections per microscope field (1.1 3 mm diameter at a magnification of x 125) and at the same time determining the percentage of total of damaged tubules, in 5 fields per testis. andrologia 20 (1988)
62
U.A. Sod-Moriah et al.
Blood serum FSH was determined by RIA according to the method of Daane and Parlow (1971) with homologeous kits kindly supplied by Dr. Raiti from NIAMDD. The standardprotein used was RP-1 and I-5-R-FSH was used for labelling. The antibody used was anti-R-FSH. Serum LH was determined by RIA as described by Nieswender et al. (1968) with kits kindly supplied by Dr. Raiti from NIAMDD. The standard protein used was RP-1 and r-LH(1-7) was used for labelling. Anti-o-LH 15, kindly supplied by Dr. Nieswender was used as antibody. Testosterone level was assayed by RIA using the method described by Canfi and Teitelbaum (1983) in untreated plasma using testosterone-3carboxymethy1-oxime-iodohistamine-125-1 (Nuclear Research Center Negev, Israel) as tracer and rabbit anti-testosterone 7carboxy-methylthioether: BSA at a working dilution of 1 :20000. The sensitivity of the assay was 5 pg/ml. The within assay C.V. were 5.9% for x = 1.13 ng/ml (n = 3) and 9.8% for x = 2.59 ng/ml. The between assay C.V. was 23.8% for x = 3.28 ng/ml (n = 7). Statistical evaluations were carried out by using the “Student-t” test and correlation analysis. Results In all groups of DBCP injected rats testicular weights, corrected for differences in body weights, were significantly reduced as compared with their respective controls (Fig. 1). Already 5 weeks after last injection of DBCP meah testicular weight was reduced by 59% as compared with controls, reaching 23.5% of controls by week 17 post injection. The percentage of total of damaged seminiferous tubules’ cross sections was determined in the htological preparations. In addition, the total number of tubules’ cross sections per microscope field was also recorded. Concomitantly with increase in the percentage of damaged tubules with advancing time, 66.2% on the 5th week post injection to 100% on week 19, the mean number of tubules per microscope field increased significantly as compared with that of the respective controls (Table 1). There was a significant correlation (p < 0.05; r = 0.830) between the percentage of damaged tubules and the number of tubular cross sections per microscope field (Fig. 2). The percentage of damaged tubules was also significantly correlated (p < 0.01; r = 0.954) with testicular weight (Fig. 3). Prior to sacrifice each male was presented with two proestral females. Some animals in the various DBCP treated groups were found to be fertile (5 out of 31 in one set and 5 out of 27 in another). Sperm counts were not carried out for either fertile or infertile rats. The non-fertile did mate, however, the vaginal plugs they produced were devoid of sperm. Expressing the previously described results separately for the fertile and the non-fertile DBCP treated animals is carried out in Table 2. It is obvious that DBCP treatment affected all injected animals, but the animals which remained fertile were affected to a significantly lesser degree as compared with controls than the infertile males. Blood serum hormones’ levels determinations revealed great differences between the fertile and infertile DBCP treated animals. As seen in Table 3 serum FSH and LH levels were significantly increased in the infertile DBCP treated animals as compared with controls. Similarly, the fertile DBCP treated animals’ serum LH and FSH levels differed significantly from those of the infertile animals and was similar to those of controls. Serum testosterone levels were similar in both fertile and infertile DBCP treated animals and that of controls (Table 3). Discussion
The male rats were injected once a week for 3 weeks with DBCP, 20 mg/kg. The toxicity of DBCP was expressed by a severe reduction of body weight as was already previously shown (Shemi et al. - 1982) compared with controls. With time, animals recovered and regained their weight reaching weights similar to their control counterparts some 40 weeks from last injection. andrologia 20 (1988)
63
Prolonged Effects of DBCP
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100-
Fig. 2
L
$ 803
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60-
'0
a 400
20-
n I
No. of tubular cross sectionslfield Fig. 1: Testes weights as percentage of body weight of DBCP treated and control animals on day of sacrifice. Means i S.E.M. *P < 0.05; **P < 0.001.
Weeks post treatment
loo-
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=I
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Fig. 3
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Fig. 2: Correlation between the number of tubular cross sections per microscope field (diam. 1.13 mm, magnification x 125) and percentage of damaged tu- 13 bules observed in the histological sections in DBCP treated rats.
40 -
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n Fig. 3: Correlation between testes weight and percentage of damaged tubules observed in the histological sections of control and DBCP treated rats.
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.5 I 1.5 2 2.5 3 3.5 4 Testes weight, g
Organs removed upon sacrifice, starting 5 weeks after last injection were examined histologically and except for the testes did not reveal any pathological changes. It seems that the toxicity that may have affected organs such as the kidney and liver (Torkelson et al. - 1961) either did not occur at the dose level used or there was a full recovery by the time the first group of animals was sacrificed. The testes however, were damaged and did not seem to recover by that short period of 5 weeks post injection. In a previous study (Shemi et al. - 1982) one testis was removed about 6 weeks post injection and examined while the second remained intact for additional 6 months. It was also noticed in this previous study that in some animals the testicular damage was not pronounced. Comparison of the degree of damage to the first testis and that in the second which was removed 6 months later suggested that recovery had taken place. There is no information whether the damage and recovery were associated with loss and regaining of fertility. The males in the groups to be sacrificed at increasing times after last DBCP injection were presented with proestral females. The results show that mating behaviour of the DBCP treated males was unaffected. They all mated and in most cases vaginal plugs were present. However, only a few of them were fertile as judged by the delivery of young. It is possible that most animals were infertile or if they did produce sperm cells the latter were unable to bring upon the development of normal pregnancy as was suggested by Teramoto et al. (1980). The histological examination of the testes revelaed however that the production of sperm cells was suppressed, excluding therefore, the possibility of infertility due to abnormal spermatozoa or oligospermia. In addition, examination of the vaginal plugs showed them to be devoid of sperm cells. andrologia 20 (1988)
U.A. Sod-Moriah et al.
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Table 1 Number of seminiferous tubules cross sections per microscope field (1.13MM; x 125) and the percentage of damaged tubules. Means f S.E.M. (average of 5 fields/testis) number of tubuleslfield weeks postinjection 5
control
DBCP
(4 1 13.8 f 1.0
(6 ) 66.2
(41 13.5 f 0.26
(5)
f 2.42
(4) 14.1 f 0.60
(61 62.0
c (5) 34.1 f 3.66
(4) 13.1 f 0.40
(6)
b 2.76
(3) 13.1 f 0.18
(6)
c
(6) 12.3 f 0.26
(6)
DBCP (6) 20.2
9
a f 2.02
b (5) 25.6 f 2.35
13
(6) 21.4
17 25
(3) 29.4 (4) 25.7
50
7% of damaged tubules
a
f
f 2.47
f
control
17.75
100.0 f
19.66
100.0
100.0 (4)
0
100.0
a =P
