Effects of developmental exposure to indole-3-carbinol or2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring

TOXICOLOGYAND APPLIED PHARMACOLOGY 141, 68--75 (1996) ARTICLENO. 0261

Effects of Developmental Exposure to Indole-3-carbinol 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Reproductive Potential of Male Rat Offspring

or

CLYNN WILKER,* LARRY JOHNSON,* AND STEPHEN SAFEt *Department of Veterinary Anatomy and Public Health and +Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466

Received December 26, 1995; accepted July 1, 1996 as o,p '-DDT [ 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethane], kepone, hydroxy-polychlorinated biphenyls (PCBs), dieldrin, and several other organochlorine compounds exhibit estrogen receptor agonist activities (Tullner, 1961; Bitman et al., 1968; Welch et al., 1969; Bitman and Cecil, 1970; Hammond et al., 1979; Robinson et al., 1984; Korach et al., 1988; Soto et al., 1994). It has been hypothesized that human exposure to environmental estrogens may be responsible for the increased incidence of male reproductive problems including decreased concentration of sperm in the ejaculate (Sharpe and Skakkebaek, 1993). Sharpe and Skakkebaek (1993) have also hypothesized that in utero exposure to environmental estrogens and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds may be responsible for decreased sperm counts and other male reproductive tract disorders. Based on results from animal models, it was proposed that in utero and lactational exposure to TCDD may also be responsible for reproductive problems in human males. For example, in utero and lactational exposure to a single dose of TCDD resulted in a number of responses consistent with demasculinization of male offspring including decreased anogenital distance, delayed testicular descent, decrease in testicular parenchymal weight, reduction in spermatogenesis and epididymal sperm reserves, and decreased accessory gland weights (Mably et al., 1992a,b,c; Peterson et al., 1993; Gray et al., 1995). The consistent site of effects by perinatal TCDD exposure appears to be at the epididymis as reduced epididymal weight and decreased number of sperm in the epididymis are always reported following perinatal exposure to TCDD while a reduction in daily sperm production is occasionally reported (Mably et al., 1992a; Gray et al., 1995). The potential human risk from in utero exposure to TCDD and related compounds may also include risks from exposures to natural TCDD-like compounds which also bind to the aryl hydrocarbon (Ah) receptor. These compounds include polynuclear aromatic hydrocarbons and aromatic amines formed on cooking (Sinha et al., 1994) and indole3-carbinol (I3C) and related hetero-PAHs which are present

Effects of Developmental Exposure to Indole-3-carbinol or 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Reproductive Potential of Male Rat Offspring. WILKER, C., JOHNSON, L., AND SAFE, S. (1996). Toxicol. Appl. Pharmacol. 141, 68-75. Treatment of pregnant female Sprague-Dawley rats on Gestational Day 15 with a single oral dose of 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) (0.5, 1.0, or 2.0 #g/kg) or indole-3-carbinol (I3C, 1.0 or 100 mg/kg), an aryl hydrocarbon (Ah) receptor agonist which is found in cruciferous vegetables, resulted in reproductive abnormalities in the male offspring (three to five litters in each treatment group). Anogenital distance and crown to rump length were altered by both compounds; however, the timing of the effects (Day 1 or 5) was variable and the responses were not necessarily dose-dependent. In 62-day-old offspring, seminal vesicle (24 to 26%), prostate (32 to 44%), testicular parenchymal (14%), and epididymal weight (19%) were decreased by one or more doses of TCDD. In contrast, I3C at one or more doses decreased daily sperm production/g testicular parenchyma (13 to 20%) and daily sperm production/testis (22%). Total number of sperm in the epididymis was significantly decreased (30 to 33%) in rats perinatally exposed to TCDD and this was due to a decreased (49 to 51%) number of sperm in the tail of the epididymis. Perinatal exposure to I3C did not affect any of these parameters. TCDD did not affect epididymal transit time of sperm through the complete epididymis at any of the doses (0.5 to 2.0 #g/kg). However, at the two highest doses (1.0 and 2.0/~g/kg), TCDD increased epididymal transit rate of sperm through the tail of the epididymis by 33 and 37%, respectively. In contrast, primarily due to decreased transit rate (39%) of sperm through the head plus body of the epididymis, I3C (1 mg/kg) significantly increased total epididymal transit time by 31%. In conclusion, perinatal exposure of pregnant rats to I3C, an Ah receptor agonist similar to TCDD, causes reproductive abnormalities in male rat offspring; however, I3C and TCDD elicited both common and different responses. ©1996AcademicPress,Inc.

It has recently been suggested that the release of "endocrine-disrupting" industrial compounds into the environment has resulted in developmental, effects in exposed wildlife populations (Colborn et al., 1993; Davis et al., 1993). A large number of these environmental contaminants such 0041-008X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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EFFECTS OF I3C AND TCDD ON RAT OFFSPRING REPRODUCTION in cruciferous vegetables such as cauliflower, Brussels sprouts, and broccoli (Bjeldanes e t al., 1991; Jellinck e t al., 1993). I3C exhibits relatively low b i n d i n g affinity for the Ah receptor; however, I3C undergoes acid-catalyzed selfcondensation reactions to give polycyclic heteroaromatic c o m p o u n d s which are more potent A h receptor agonists (Bjeldanes e t al., 1991). Thus, the activity of I3C as an A h receptor agonist or antagonist is dependent, in part, on the extent of conversion into more active c o m p o u n d s in animal species and their subsequent uptake by target organs. The effects of I3C and other structural classes of A h receptor agonists on male reproductive d e v e l o p m e n t have not been investigated. This study reports the comparative effects of a single gestational administration (Gestational Day 15) of T C D D (0.5 to 2.0 #g/kg) or I3C (1 and 100 mg/kg) to pregnant S p r a g u e - D a w l e y rats on reproductive developm e n t of male offspring using protocols which have previously b e e n described (Mably e t al., 1992a; Gray e t al., 1995). The results show that both T C D D and I3C cause reproductive abnormalities in male rat offspring; however, the spectra of effects of both c o m p o u n d s were different. MATERIALS AND METHODS Timed pregnant Sprague-Dawley rats (Harlan Sprague-Dawley; Indianapolis, IN) on Day 15 of gestation were given a single oral dose of TCDD (0.5, 1.0, or 2.0 1~g/kg) or I3C (1.0 or 100 mg/kg), or an equal volume of vehicle (corn oil/acetone, 19/1, v/v). Each treatment group was composed of 3 to 5 litters of male offspring. After whelping (Day 0 = day of birth), litter size was standardized at 10 pups per litter. Anogenital distance and crown-to-rump length was determined on Days 1 and 5 of age. Pups were weighed on Day 1 and then every 5 days throughout the study. Pups were weaned at Day 21 and terminated at Day 62. Following termination the weights of the following organs were obtained: right and left testes and epididymides,prostate, and seminal vesicles. The right testis and epididymis were then frozen. The left testis was processed for stereologic evaluation of Sertoli cell number. Daily sperm production and epididymal transit time. Following thawing the weights of the right testicular parenchyma and total epididymis along with the weights of the tail or combined head plus body of the epididymis were determined. The tissues were homogenized separately in a Waring blender for 2 rain in homogenizing fluid (150 mM NaC1, 0.05%, v/v, Triton X-100, and 3.8 mM NAN3)(Amann and Lambiase, 1969; Amann et aL, 1976; Johnson et al., 1980, 1984a, 1992). Homogenates were evaluated by hemocytometer counts under phase-contrast microscopy to determine the number of homogenization-resistantspermatids or sperm from the testis or region of the epididymis, respectively (Amann and Lambiase, 1969; Amman et aL, 1976). Daily sperm production per gram testicular parenchyma (DSP/g) was calculated by dividing the number of spermatids in the homogenate by the product of 6.3-day life span of these spelrnatids and the weight of the testicular parenchyma homogenized (Robbet al., 1978; Johnson et aL, 1980). The life span was based on the duration of Stages IV-VII of a 12.9-day spermatogenic cycle length (Clermont and Harvey, 1965). Daily sperm production per testis (DSP/testis) was calculated by multiplying DSP/g by the weight of the testicular parenchyma of the right testis. Number of spermatozoa was determined for the combined head plus body and also for the tail of the epididymis. Epididymal transit time was estimated by dividing DSP/testis into the number of sperm in the total, head plus body, or tail of the epididymis (Robb et aL, 1978; Johnson and Varner, 1988).

69

Sertoli cell number. The left testes from seven rats in the control, !.0 #g/kg TCDD, and 100 mg/kg I3C groups were fixed by vascular perfusion via the testicular artery on the surface and immersion in 2% glutaraldehyde in sodium cacodylate buffer (Johnson et al., 1980, 1984b). Three or four pieces of testicular tissue were embedded in Epon, sectioned at 0.5 #m, stained with toluidine blue, and observed under bright field microscopy. Specimens were evaluated by stereology for the volume density (percentage of the testis) of nuclei of Sertoli cells. Volume density was determined by Chalkley's point counting method employing 10,000 points obtained on a test of two histologic sections of two blocks for each rat (Chalkley, 1943; Johnson et al., 1980). The volume of an individual Sertoli cell nucleus was approximated by the formula for the volume of a sphere from the average maximum diameter (based on nuclear height and width). A correction factor (0.6297 + 0.029) was applied to circumvent the overestimationof nuclear volume of individual as the Sertoli cell nucleus is not spherical (Johnson and Nguyen, 1986; Johnson et al., 1996). Diameters were measured on a computerizeddigitizing system connected to a microscope with Nomarski optics using 20-#m Epon sections (Johnson and Nguyen, 1986). The number of Sertoli cells per testis was calculated as the product of the volume density of Sertoli cell nuclei in the parenchyma, parenchymal volume (parenchymal weight/ 1.05 sp grav), and a histologic correction factor for section thickness and nuclear diameter divided by the volume of an individual Sertoli cell nucleus (Weibel and Paumgartner, 1978; Johnson and Nguyen, 1986). Statistics. Analysis of the data was conducted using a one-way analysis of variance (ANOVA) with mean separation by Duncan's procedure corrected for unequal treatment groups (SAS Institute, 1985). Data were analyzed on a litter rather than an individual basis with the number of observations in each group representing the number of litters rather than offspring.

RESULTS Gestational administration of T C D D or I3C did not cause maternal death or decrease maternal weight gain (data not shown). Average litter size was not affected by maternal treatment (control, 11.6; TCDD, 12.3 to 14; I3C, 12 to 13.3 pups/litter). Only 4 total pups did not survive to w e a n i n g (control, 1 pup; 1.0 #g/kg T C D D , 2 pups; 1 mg/kg I3C, 1 pup); the 1.0 #g/kg T C D D treatment group had the lowest percentage of survival (96%) and this was similar to the control value of 98%. The results in Table 1 summarize the effects of perinatal exposure to T C D D (0.5, 1.0, or 2.0 #g/ kg) on the male offspring. Anogenital distance was significantly decreased on Days 1 and 5 in most of the treatment groups, whereas, the anogenital distance/body weight ratio was decreased significantly only in the 1.0 #g/kg group on Day 5. In animals perinatally exposed to I3C (100 mgfKg) anogenital distance and c r o w n - r u m p length were decreased significantly only on Day 1 (Table 2). Crown-to-rump lengths were also decreased in animals (Days 1 and 5) perinatally exposed to TCDD, whereas crown-to-rump length/ body weight ratio was significantly increased in only some of the treatment groups. T C D D significantly decreased body weight in the male offspring (Days 1 - 6 2 ) , whereas perinatal exposure to the high dose of I3C (100 mg/kg) resulted in increased body weight in the offspring on Day 20. Seminal vesicle and prostate weights were lower in 62day-old male rats perinatally exposed to T C D D (1.0 and

WILKER, JOHNSON, AND SAFE

70

TABLE 1 Effects of Perinatal Exposure to T C D D (0.5, 1.0, a n d 2.0 #g/kg) on Reproductive Potential of Male Rat Offspring" TCDD (#g/kg) Parameterb Control Anogenital distance (mm) Day 1 Day5 Anogenital distance/BW Day 1 Day 5 Crown-rump leng~ (cm) Day 1 Day5 Crown-rump length/BW Day 1 Day 5 Body weight (g) Day 1 Day5 Day20 Day45 Day62 Seminal vesicle weight (mg) Prostate weight (mg) Testicularparenchymalweight(g)

DSP/g (10 6) DSP/testis (10 6) Epididymal weight (mg) Sperm(head + body) Sperm(tail) Sperm (total) Epididymal transit time (head + body) (days) Epididymal transit time (tail) (days) Epididymal transit time (total)(days) Number of Sertoli cells/testis (10 6) Number of Sertoli cells/g (10 6) Number of elongated spermatids/Sertoli cells

Control

4.6 6.5

± ±

0.1 a 0.1 a

0.5

4.2 6.0

± ±

0.065 _+ 0.002 0.051 ± 0.002"

0.070 ± 0.053 ±

4.42 5.36

0.05 ° 0.06 a

4.16 5.16

0.020 a 0.013 a

0.692 ± 0.458 ±

± ±

0.627 ± 0.419 ± 7.1 12.9 49.1 243.9 358.5 508.9 818.9 1.38 20.0 27.8 366.8 76.5 81.7 158.2 2.8 3.0 5.8 40.0 27.5 4.6

± 0.3 a ± 0.5a ± 0.9a ± 4.3 ~ ± 8.8 ± 25.2 a ___ 54.7" ± 0.04 a ± 0.5 ± 1.4 -- 3.5~ ± 5.6 X 10 6 ± 4.2~× 106 ± 8.7a × 10 6 ± 0.1 ± 0.1 ° ± 0.2 ± 2.3 ± 1.6 _± 0.3 ~

6.0 11.3 45.3 226.8 343.5 502.2 726.2 1.29 17.6 22.7 341.2 83.3 68.2 150.5 3.6 2.9 6.5

1.0

0.1 ab 0.1 b

4.0 5.9

0.001 0.004 "b

+ 0.03 ~ _+ 0.01 bC 0.006 °b 0.004 °b

± ±

0.1 b 0.1 b

+.

0.6 ab

± 3.0ab __+ 4.2 +_ 18.0~ ± 12.5a ± 0.07 ~b ± 0.7 ± 1.4 ± 9.3 ~b ± 4.6 ± 9.8" ± 10.9~ ± 0.2 ± 0.3 ~ ± 0.4 ND ND ND

± ±

2.0

0.1 b 0.1 b

4.3 5.9

0.069 ± 0.057 ±

0.003 0.001 a

0.064 ± 0.002 0.052 +_ 0.002 "b

4.12 5.02

0.02 b 0.02 c

4.15 5.24

0.024 b 0.025 b

0.627 __+ 0.031a 0.469 _± 0.014 ab

± ±

0.709 ± 0.485 ± 5.8 10.5 43.4 204.0 307.1 376.9 558.2 1.31 15.9 20.7 317.2 69.3 41.6 110.9 3.4 2.0 5.4 44.2 31.6 3.3

± 0.2 b _-2 0.6 b ± 0.7 c ± 1.0b ± 3.8 ± 3.3b ± 25.3 b + 0.0&b ± 0.5 + 1.1 _ 10.8~a ± 5.9 + 4.2 b --+ 8.9b ± 0.3 ± 0.1 b - 0.3 --- 4.7 ± 2.7 ± 0.3b

6.7 11.3 44.2 204.5 311.2 385.1 454.5 1.19 16.5 19.9 295.9 65.9 39.7 105.6 3.6 1.9 5.5

± ±

± _

0.1 ab 0.1 b

0.06 b 0.08 ~b

_ 0.3 "b ± 0.5 b ± 2.5~b ± 16.7b ± 24.4 ± 42.2 b _ 57.9 b ± 0.03 b ± 2.6 ± 3.6 ± 28.9 b ± 4.5 ± 9.4b ± 13.7a + 0.5 ___ 0.2 b ± 0.3 ND ND ND

Pregnant female rats were exposed on Gestation Day 15 to a single dose of TCDD and various parameters were measured in the male offspring as described under Materials and Methods. b Results are expressed as means ± SEM; means with different letters are different (p < 0.05).

2.0 # g / k g ) . I 3 C a l s o d e c r e a s e d s e m i n a l v e s i c l e a n d p r o s t a t e

m a l f o r m e d e p i d i d y m i d e s revealed that no s p e r m were pres-

weights but these w e r e not statistically significant using A N -

ent in these regions, indicating that regions o f the rete testes

O V A a n d D u n c a n ' s a n a l y s i s . T e s t i c u l a r w e i g h t w a s signifi-

and/or efferent ductules m a y also h a v e b e e n m a l f o r m e d . In

c a n t l y d e c r e a s e d a f t e r p e r i n a t a l e x p o s u r e to T C D D (2.0 # g /

one case, there was also m a l f o r m a t i o n o f the seminal vesicles

kg); h o w e v e r , a s i g n i f i c a n t d e c r e a s e w a s n o t o b s e r v e d at a n y dose of I3C. E p i d i d y m a l w e i g h t was d e c r e a s e d significantly

along with the abnormality of the epididymal anatomy. The weights of the testes, e p i d i d y m i d e s , and a c c e s s o r y sex glands

b y T C D D at t h e 2.0 # g / k g d o s e , w h e r e a s n o s i g n i f i c a n t effects w e r e o b s e r v e d in the I3C-treated animals. A d d i t i o n -

f r o m t h e s e 3 rats w e r e n o t u s e d in t h e c a l c u l a t i o n o f m e a n w e i g h t s f o r this t r e a t m e n t g r o u p ; h o w e v e r , t h e y w o u l d h a v e

ally, 3 o f t h e 11 ( 2 7 . 3 % ) m a l e rats i n t h e 2.0 # g / k g T C D D dose group had significant anatomical m a l f o r m a t i o n s of the

increased treatment differences. Daily sperm production per gram of testicular parenchyma

epididymis including absence of the head and/or body of

was significantly d e c r e a s e d in m a l e offspring perinatally ex-

t h e e p i d i d y m i s (Fig. 1). C a r e f u l d i s s e c t i o n a n d r e m o v a l o f e p i d i d y m a l f a t w a s p e r f o r m e d to e n s u r e t h a t s m a l l r e g i o n s

p o s e d to b o t h d o s e s o f I 3 C (1 a n d 100 m g / k g ) , w h e r e a s d a i l y s p e r m p r o d u c t i o n p e r t e s t i s w a s o n l y r e d u c e d at t h e 100

o f the e p i d i d y m i s w e r e not m i s t a k e n l y r e m o v e d . In addition, h o m o g e n i z a t i o n o f t h e tail a n d p u t a t i v e h e a d r e g i o n s o f t h e

m g / k g d o s e o f I3C. N o s i g n i f i c a n t e f f e c t s o n d a i l y s p e r m p r o d u c t i o n w e r e o b s e r v e d in the T C D D treatment groups.

71

EFFECTS OF I3C AND TCDD ON RAT OFFSPRING REPRODUCTION TABLE 2 Effects o f P e r i n a t a l E x p o s u r e to I 3 C (1 or 100 m g / k g ) o n R e p r o d u c t i v e P o t e n t i a l o f Male R a t Offspring~ I3C (mg/kg) Parameter b Anogenital distance (ram) Day 1 Day 5 Anogenital distance/BW Day 1 Day 5 C r o w n - r u m p length (cm) Day 1 Day 5 C r o w n - r u m p lengtlffBW Day 1 Day 5 Body weight (g) Day 1 Day 5 Day 20 Day 45 Day 62 Seminal vesicle weight (mg) Prostate weight (rag) Testicular parenchymal weight (g) DSP/g (106) DSP/teestis (106) Epididymal weight (mg) Sperm (head + body) Sperm (tail) Sperm (total) Epididymal transit time (head + body) (days) Epididymal transit time (tail) (days) Epididymal transit time (total) (days) Number of Sertoli cells/testis (106) Number of Sertoli cells/g (106) Number of elongated spermatids/Sertoli cells

Control

4.6 6.5

1

4.4 6.1

_+ 0.1 a .+ 0.1

100

,+ 0.1 ab + 0.1

4.1 6,5

.+ 0.1 b _+ 0.1

0.065 _ 0.002 0.051 _+ 0.002

0.058 _+ 0.003 0.051 __ 0.003

0.059 _+ 0,004 0.048 _+ 0.002

4.42 5.36

4.41 5.43

4.!6 5.52

_+ 0.05 ° _+ 0.06

0.627 +_ 0.020 0.419 _+ 0.012 7.1 12.9 49.0 243.9 358.5 507.9 818.9 1.38 20.0 27.8 366.8 76.5 81.7 158.2 2.8 3.0 5.8 40.0 27.5 4.6

-- 0.3 ± 0.5 + 0.9 .+ 4.3 ,+ 8.8 + 25.2 _+ 54.7 _+ 0.04 _+ 0.5 ° ± 1A ° _+ 3.5 _+ 5.6 x 106 ± 4.2 × 106 ,+ 8.7 x 106 .+ _+ _+ .+ .+ ,+

0.1 a 0.1 0.2 ~ 2.3 ~ 1.& 0.3 ~

± ±

0.04 a 0.06

0.584 .+ 0.020 0.451 _+ 0.018 7.6 12.1 47.2 222.8 330.9 447.6 636.2 1.31 17.4 23.1 346.3 83.6' 76.8 160.4 3.9 3.7 7.6

.+ 0.2 _+ 0.4 _+ 1.2 _+ 5.8 _+ 15.4 ,+ 32.9 _+ 22.7 ,+ 0.05 ,+ 1.9b .+ 3.3 °b _+ 11.1 _+ 1.0 _+ 4.7 ± 5.7 _+ 0.6 b -+ 0.8 _+ 1A b ND ND ND

,+ 0.07 e _+ 0.16

0.596 + 0.033 0.411 _+ 0.009 7.1 13.5 51.3 238.6 344.2 485.4 731.6 1.38 15.9 21.8 376.1 76.0 80.6 156.7 3.6 3.8 7.3 52.5 36.2 2.8

.+ 0.5 _+ 0.6 _+ 1.5 _+ 13.2 .+ 19.6 _+ 32.7 _+ 84.9 .+ 0.02 .+ 0.5 b ,+ 0.5 ~' + 22.9 + 3.8 _+ 2.9 ,+ 3.9 _ + _ + _ ,+

0.2 ~b 0.1 0.2 ~b 2.8 b 1.7b 0.2 b

Pregnant female rats were exposed on Gestation Day 15 to a single dose of TCDD and various parameters were measured in the male offspring as described under Materials and Methods. b Results are expressed as means _ SEM; means with different letters are different (p < 0.05).

Sertoli cell number was increased only in the I3C group (Table 2). The increase in Sertoli cell number was observed for both Sertoli cells per testis and per gram of testicular parenchyma. The ratio of the number of elongated spermarids from the testicular homogenates to the number of Sertoli cells per testis was reduced by both TCDD and I3C treatments (Tables 1 and 2). No evidence of altered sperm released was seen during histologic evaluation of fixed testicular parenchymal samples from control, I3C, or TCDDtreated rats. Total number of sperm in the epididymis was significantly decreased in animals perinatally exposed to TCDD (1.0 or 2.0 #g/kg), whereas no significant changes were observed in animals exposed to I3C (1.0 or 100 mg/kg). The reduction

in sperm number in the total epididymis was a result of a reduction in sperm number in the tail of the epididymis at both the 1.0 and 2.0 #g/kg doses of TCDD. I3C did not alter the number of sperm in the total epididymis or in the head plus body or tail of the epididymis. TCDD did not affect epididymal transit time of sperm through the complete epididymis at any of the doses (0.5 to 2.0 #g/kg). However, this was not the case for I3C; total epididymal transit time was increased by almost 2 days in the 1 mg/kg treatment group. Neither TCDD nor the 100 mg/kg dose of I3C produced any effect on transit time of sperm through the head and body of the epididymis; however, transit time of sperm in this region was significantly increased by more than 1 day in animals perinatally exposed

72

WILKER, JOHNSON, AND SAFE

FIG. 1. Epididymidesfrom control, 0.5, 1.0, and 2.0 /zg/kg TCDDtreatedrats. The body of the epididymisfromthe 1.0/zg/kgTCDDtreatment group is smaller comparedto the epididymis from the control rat. TCDD treatment at a dose of 2.0 #g/kg produced anatomical malformationof the epididymis in 27.3% of the animals as seen in this figure by the absence of the body of the epididymis.

to 1 mg/kg I3C. Evaluation of epididymal transit time of sperm through the tail of the epididymis showed that T C D D at the two highest doses (1.0 and 2.0 #g/kg) increased transit rate, whereas I3C did not alter transit rate of sperm through this region of the epididymis. DISCUSSION Several studies have reported that in utero and lactational exposure of pregnant rats to TCDD resulted in femininization and demasculinization of the male offspring (Mably et al., 1992a,b,c; Peterson et al., 1993; Gray et al., 1995). For example, Gray and co-workers (1995) reported that in adult male Long Evans rats perinatally exposed to 1 #g/kg TCDD, there were significant decreases in testicular and epididymal weights, sperm counts in the tail of the epididymis were decreased by 30%, and ejaculated sperm counts were reduced by 58%. These results demonstrate that the epididymis is a sensitive target for TCDD exposure and this study has focused, in part, on the comparative effects of TCDD (0.5, 1.0, or 2.0 #g/kg) and I3C (1.0 or 100 mg/kg) on the epididymis. I3C is a relatively weak Ah receptor agonist which exhibits partial antagonist activity in human breast cancer cell lines. For example, I3C partially inhibits induction of CYP1A1 gene expression in the T47D human breast cancer cell line (Chen et al., 1996). However, under acidic conditions, I3C is converted into a diverse spectrum of condensation products, including indolo[3,2-b]carbazole, which exhibit more potent Ah receptor agonist activities (Bjeldanes et al., 1991). Thus, I3C was orally administered on Gestational Day 15 to pregnant Sprague-Dawley rats to ensure that fetuses were potentially exposed to acid-catalyzed con-

densation products formed in the gut. The in vivo half-life of I3C or its condensation products is unknown. However, increased estadiol 2-hydroxylase and ethoxyresorufin-O-deethylase (EROD) activity and induction of CYP1A1 and 1A2 have been reported at 20 hr following oral dosing of rats with I3C (Bradfield and Bjeldanes, 1987; Jellinck et al., 1993). No information is available regarding the transplacental or lactational transfer of I3C or its by-products to offspring. A comparison of the effects of perinatal exposure to TCDD and I3C (Tables 1 and 2) indicates that in utero exposure to both compounds resulted in some common adverse effects in the male offspring. One or more doses of TCDD or I3C resulted in decreased anogenital distance and decreased prostate and seminal vesicle weights and daily sperm production/testis. In contrast, epididymal weight was decreased by TCDD and not I3C. Testicular weights were decreased by TCDD at the highest dose level (2.0 #g/kg). DSP/g (a measure of efficiency of spermatogenesis) (Johnson, 1995) was affected only by I3C at both doses. Number of Sertoli cells in control rats in this study are within the range of other estimates using similar and different methods (Hochereau de Reviers and Courot, 1978; Russell et al., 1990; Johnson et al., 1996). The increase in Sertoli cell number per testis and per gram of testicular parenchyma following I3C exposure was intriguing given the reduction in testicular weight and DSP/testis. Given that the number of Sertoli cells is significantly correlated to DSP and testicular weight in various species including humans (Johnson et al., 1984b) and rats (Orth et al., 1988; Russell et al., 1990), one would expect that the large population of Sertoli cells should be accompanied by a large population of germ cells. However, testes from rats in the I3C group deviated by having a large population of Sertoli cells and fewer germ cells. This may be an indication of reduced Sertoli cell function (Johnson et al., 1984b). Perinatal TCDD treatment altered epididymal development and function as evident by morphological anomalies and altered transit rate. The high incidence (27.3%) of epididymal anomalies and morphological characteristics (segmental absence of regions of the epididymis) seen in the 2.0 #g/kg TCDD treatment group is similar to those reported for rats and mice followed gestational treatment with the antiandrogen flutamide (van der Schoot, 1992; Cain et al., 1994a,b). This indicates that TCDD may compromise testosterone-dependent differentiation of the Wolfian duct into the epididymis during gestation. One of the most striking effects in the epididymis of adult males perinatally exposed to TCDD is a reduction in the number of sperm stored in the tail of the epididymis (49%), compared to only a modest and insignificant reduction in the number of sperm produced daily in the testis (26%). The decreased number of sperm in the epididymis is consistent with an increased rate of

EFFECTS OF I3C AND TCDD ON RAT OFFSPRING REPRODUCTION

transit of sperm in the tail of the epididymis or sperm resorption in the epididymis. Resorption of sperm in the epididymis is usually associated with inflammatory conditions of the epididymis (i.e., sperm granulomas, epididymidis, or postvasectomy) in which large numbers of inflammatory cells accumulate in the epididymis (Flickinger et al., 1995; Working and Chellman, 1989). In addition, human infertility caused by degeneration and death of sperm in the epididymis is characterized by high numbers of sperm with poor motility and abnormal morphology (Wilton et al., 1988). Both motility and morphology of sperm from perinatally exposed rats have been reported to be normal (Mably et al., 1992a). Accumulation of sperm in the lumen of the seminiferous tubules is associated with abnormal epididymal function including sperm degeneration following exposure to the reproductive toxicant methyl chloride (Working and Chellman, 1989). Again, abnormal testicular histology has not been reported following perinatal exposure to TCDD and was not seen in this study. In light of the lack of evidence supporting sperm :resorption it is our conclusion that perinatal exposure to r C D D causes an increase in epididymal transit rate of sperm in the tail of the epididymis. However, recently Sommer and Peterson (1996) reported that perinatal exposure to TCDD ,:lid not alter epididymal transit rate. A conclusion of in,creased epididymal transit rate is supported by a number of :findings including: a modest and insignificant decline in daily sperm production/testis with a significant reduction in '~he number of sperm in the tail of the epididymis, decreased number of ejaculated sperm during mating (Gray et al., 1995), normal testicular histology, normal sperm morphology and motility (Mably et al., 1992a), and lack of evidence of an inflammatory process occurring in the epididymis. In contrast, perinatal exposure to I3C did not affect the number of sperm in different regions of the epididymis but significantly decreased transit rate through the total epididymis with the greatest decrease in transit rate seen in the head plus body of the epididymis. This initially appeared to be a contradiction since perinatal treatment with I3C reduced daily sperm production/testis (Table 2) in adult male rat offspring, yet die number of sperm in the epididymis was similar to that of control rats. However, the decreased epididymal transit rate of sperm provided an explanation for the apparent contradiction. Retention of sperm in the testis would have increased (not decreased) daily sperm production/testis, and was not seen histologically. Degeneration of sperm in the epididymis would have produced a reduced number of sperm in the epididymis rather than a similar number. Thus, prolonged epididymal transit time of sperm in adult rats following perinatal exposure to I3C is supported by the data. Both TCDD and I3C significantly affected rate of epididymal transit of sperm but in the opposite direction. The increased epididymal transit rate of sperm observed in rats perinatally exposed to TCDD may result in two im-

73

portant consequences on reproductive competence: decreased numbers of ejaculated sperm and decreased quality of ejaculated spermatozoa. Gray and co-workers (1995) have already demonstrated that perinatal exposure to TCDD significantly reduced the number of sperm that are inseminated into females during mating. Rats which have undergone hypophysectomy or castration to lower androgen levels have an increased transit rate of sperm through the epididymis and accompanied decreased fertility (Dyson and OrgebinCrist, 1973). Male mice exposed to estradiol benzoate also exhibited increased transit rate and decreased fertility (Meistrich et al., 1975). Other epididymal-specific toxicants, such as ethane dimethanesulfonate and chloroethylmethanesulfonate, accelerated epididymal transit of sperm and had profound effects on the fertility of epididymal sperm (Klinefelter et al., 1992, 1994a,b). A thorough evaluation of the effects of TCDD on sperm quality, including fertilizing ability, has not yet been reported and should be investigated in future studies. The results of this study also show that perinatal exposure to I3C increased epididymat transit time of sperm in the male offspring. Partial sympathetic denervation of the tail of the epididymis by surgical ablation of the inferior mesenteric plexus decreases epididymal transit rate and increases the number of sperm in the tail of the epididymis without altering testicular sperm number (Billups et al., 1990a,b). This prolonged epididymal exposure time following sympathetic denervation of the epididymis is similar in length to that seen in adult rats exposed developmentally to I3C and has been shown to reduce the fertility of sperm from the rats that had their epididymides denervated (Ricker et aI., 1995). Thus, the effects caused by I3C in this study could decrease the fertilizing ability of sperm; this should be further investigated. In addition, future studies on the effects of developmental exposure to I3C should include ejaculated sperm counts and mounting behavior studies. The results of these studies show that perinatal exposure to I3C and TCDD elicit both common and different toxic responses in male rat offspring. Although I3C and related condensation products are Ah receptor agonists, the results of this study indicate that some of the I3C-induced responses may be Ah receptor-independent. In addition, the ability of I3C to alter male reproductive development from a single gestational administration, given its short half-life compared to TCDD, is noteworthy (Bjeldanes et al., 1991). Based on the results previously obtained for TCDD in the rat model, it was hypothesized that exposure to TCDD and related compounds may be responsible for decreased male sperm counts and other male reproductive problems (Sharpe and Skakkebaek, 1993). There has been increasing concern regarding the potential adverse human health effects of pre- and postnatal exposure to TCDD and related compounds, and these concerns are also based on reproductive abnormalities in wildlife

74

WILKER, JOHNSON, AND SAFE

p o p u l a t i o n s and l a b o r a t o r y a n i m a l studies ( C o l b o r n e t al., 1993). T h e results r e p o r t e d in this study s h o w that p e r i n a t a l e x p o s u r e to I 3 C , a natural p r o d u c t p r e s e n t in c r u c i f e r o u s v e g e t a b l e s , also c a u s e s a b n o r m a l i t i e s in m a l e rat o f f s p r i n g . T h e r e f o r e , h u m a n h e a l t h risk a s s e s s m e n t o f T C D D and related c o m p o u n d s b a s e d o n l a b o r a t o r y a n i m a l e x p e r i m e n t s s h o u l d c o n s i d e r the effects o f I 3 C and o t h e r natural A h r e c e p t o r a g o n i s t s s u c h as a r o m a t i c a m i n e s (106 to 107 p g / day) and p o l y n u c l e a r a r o m a t i c h y d r o c a r b o n s (1.2 to 5 × 106 p g / d a y ) w h i c h can b e p r e s e n t at r e l a t i v e l y h i g h l e v e l s in c o o k e d f o o d s for h u m a n s ( V a e s s e n e t al., 1988; B j e l d a n e s e t al., 1991; L u t z and Schlatter, 1992; M e n z i e e t al., 1992; S i n h a e t al., 1994). T h e r e f o r e , risk a s s e s s m e n t o f A h r e c e p t o r a g o n i s t s on the d e v e l o p i n g m a l e r e p r o d u c t i v e system, as w e l l as g e n e r a l h e a l t h o f h u m a n s , s h o u l d take into a c c o u n t the p o t e n t i a l dietary c o n t r i b u t i o n s o f d i o x i n s s u c h as T C D D and n a t u r a l l y o c c u r r i n g T C D D - l i k e c o m p o u n d s (Safe, 1996). ACKNOWLEDGMENTS The financial assistance of the National Institutes of Health [(ES07273 and AGl1093-10(LJ)] and the Texas Agricultural Experiment Station is gratefully acknowledged. S.S. is a Sid Kyle Professor of Toxicology.

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