Carbendazim-induced alterations of reproductive development and function in the rat and hamster

FLJNDAMENTALANDAPPLIEDTOXICOLOGY

15,28l-297(1990)

Carbendazim-induced Alterations of Reproductive Development Function in the Rat and Hamster’ L.EARLGRAY,JR.,JOSEPHOSTBY,RALPHLINDER,JEROME GEORGIAREHNBERG, ANDRALPHCOOPER

and

GOLDMAN,

Developmental Reproductive Biology, Endocrinology & Gerontology, and Gamete Biology Sections, Reproductive Toxicology Branch, DTD, Health Efects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 2771 I

Received November 27, 1989; accepted April 17, 1990 Carbendazim-Induced Alterations of Reproductive Development and Function in the Rat and Hamster. GRAY, L. E.,JR., OSTBY,J., LINDER, R., GOLDMAN,J., REHNBERG, G., AND COOPER, R. (1990). Fundam. Appl. Toxicol. 15,281-297. We are developing a data base that will allow us to select endpoints that would be useful in the detection of reproductive toxicity in a multigenerational test. In this effort, carbendazim (MBC), a known reproductive toxicant, was administered to male and female rats from weaning, through puberty, gestation, and lactation. A similar study was conducted with hamsters. In rats, MBC was administered at 0,50, 100,200. or 400 mg/kg/day. Hamsters were dosed at 0 or 400 mg/kg/day. In the parent (PO) generation, landmarks of puberty were measured. In females, estrous cyclicity, litter size, the number of implants, organ weights, and histology were assessed.Our assessment of the male rat included organ weights, testicular and epididymal sperm counts, a quantitative measure of sperm motility, sperm morphology. testicular histology, and endocrine measures. The growth, viability, and reproductive function of the offspring (Fl) were observed during a 4-month period of continuous breeding. In the PO of both species, MBC did not alter pubertal development, growth, or viability. The reproductive potential ofthe rats treated with MBC at 200 and 400 mg/kg/day was reduced due to effects on sperm production and fetal viability. In the male rat, MBC treatment markedly altered sperm morphology, testicular and epididymal weights, and sperm numbers and testicular histology. Fertility, sperm motility, and hormonal levels were altered, primarily in the males with very low sperm counts. The ability to conceive did not appear to involve a female factor. In PO female rats, MBC administration caused postimplantation losses in the high-dosage groups and a few malformed rat pups were found in the litters from the 100 and 200 treatment groups. MBC was less toxic to the hamster than the rat. The only reproductive effects induced by MBC treatment were on sperm measures. Fertility of the PO generation and fetal and neonatal (Fl) viability were not decreased by MBC administration. In the male rat, testis weight, sperm numbers in the cauda epididymis and testis and sperm morphology were sensitive to the effects of MBC. In females, counting implantation scars at necropsy was useful, as this information allowed us to confirm pregnancy and identify postimplantation losses induced by MBC administration. o 1990 Society of Toxicology,

’ Although the research described in this article has been supported by the USEPA, it does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 281

Carbendazim (MBC) and benomyl, which degrades to MBC, are fungicides with activity against a number of plant pathogens. The fungicidal properties of both of these compounds are the result of the binding of MBC (methyl 2-benzimidazolecarbamate) to tu0272-0590190 $3.00 Copyright Q 1990 by the Society ofToxicology. All rights of reproduction in any form resewed

282

GRAY ET AL.

Puberty

I

young

Adulthood

I Breeding

FIG. 1. The protocol used in the present investigation to compare endpoints of reproductive function in the male and female rat. Male and female rats were dosed by gavage from weaning, through puberty, young adulthood, breeding, gestation, and lactation with Carbendazim (MBC) at 0,50, 100, 200, or 400 mg/kg/ day. Males (the PO generation) were necropsied after breeding while the females were necropsied at the end of lactation, Some of the offspring from the 0 and 100 mg/kg/day dosage groups, the Fl, were placed in breeding pairs at weaning and breeding was monitored for about 4 months.

bulins, an effect which disrupts microtubule formation and mitosis (Dustin, 1984; Burland, 1984; Clemons and Sisler, 197 1). MBC also binds to a lesser extent (Ireland et al., 1979) to mammalian tubulin and, presumably, this mechanism accounts for the adverse effects of benomyl and MBC on mammalian development (Kavlock et aE., 1982) and reproduction. These compounds reduce testicular sperm production in rats and mice (Linder et al., 1988; Evenson et al., 1987) with adult rats being more sensitive to the antispermatogenic effect of benomyl than prepubertal males (Carter et al., 1984). The fact that benomyl has little effect on the testes of young rats, as compared to adults, was one of the bases for the current study. We wanted to determine if MBC would produce testicular lesions in males, using our protocol, which initiates exposure at weaning. Our long range goal is to develop a reproductive data base, using the protocol shown in Fig. 1, that will enable us to determine the endpoints that would be useful in the identification of reproductive toxicants in an alternative/additional reproduction test (ART) protocol. Our approach would have little utility if a toxicant, like MBC, failed to induce testicular effects as a consequence of the age at which exposure is initiated. Our protocol

incorporates indices of puberty along with other morphological and physiological indices of reproductive health (Gray et al., 1988). Pubertal measures are included because these secondary sex characteristics develop in a predictable sequence and alterations of this process are easily identified. The need for ART protocols was recently recognized by the USEPA and guidelines for their development were presented (Francis and Kimmel, 1988). They stated that ART protocols should be at least two generations, their goal is to detect male and female reproductive and developmental toxicity, and they should include histology of the reproductive organs along with other endpoints. ART protocols would be used to identify reproductive toxicants and prioritize them for additional testing, if warranted. It was also noted that a critical data gap existed because there are very few multigenerational reproduction studies in the literature. Such protocols are needed because it has become apparent that fertility, litter size, and litter viability, the endpoints that are normally utilized for risk assessment, are often insensitive indices of reproductive health. For example, studies using male rats or mice have found that fertility is normal even when sperm counts or testis weight is reduced by

MBC AND REPRODUCTIVE

90% (Me&rich, 1982; Gray et al., 1984; Gray et al., 1989a). In the female, alterations of puberty and estrous cyclicity may occur at doses below those that alter fertility (Gray et al., 1988). Some consideration has been given to using more sophisticated assessments of testicular histology (Lamb and Chapin, 1985; Russell, 1983) and sperm function, (Linder, 1986; Overstreet, 1984) but effects on other reproductive organs in the male (e.g., pituitary and endocrine function) and additional reproductive measures in females (such as estrous cyclicity) are rarely reported. The protocol used in the current investigation intentionally exceeds the requirements proposed for ART protocols. In order to develop this critical data base, rats are exposed to known reproductive toxicants in a doserelated manner, as in Fig. 1. When sufficient data have been collected, we will then be able to determine which endpoints should be included in an ART protocol and which others should be reserved for more specialized evaluations, We have previously published data on methoxychlor (Gray et al., 1989) and the present study describes our data on MBC. In the present study, MBC was administered to male and female rats by gavage starting at weaning, through puberty, young adulthood, breeding, gestation, and lactation (at 0, 50, 100, 200, and 400 mg/kg/day). Their offspring, exposed only during pregnancy and lactation, were bred continuously for 4 months after weaning, and then necropsied. In a similar study, male and female hamsters were dosed with MBC at 400 mg/kg/day to determine if there were species differences in the toxicity of this compound. The reproductive toxicity of MBC in the hamster has not been previously described. METHODS-RATS Animals. Long-Evans hooded rats (Charles River Breeding Laboratory, MI) were housed, maintained, assigned to treatments, and dosed as previously described (Gray et al., 1989b). Laboratory grade corn oil was the vehicle and the MBC was technical grade carbendazim

DEVELOPMENT

283

(at least 95% pure, provided courtesy of E. I. DuPont deNemours and Co., Wilmington, DE). In this study, a total of 88 rats were used in the PO generation. The groups consisted of 24 control animals ( 12 of each sex) and 16 rats (8 per sex) in each of the four MBC-treatment groups. Treatments were initiated at weaning (Day 2 1) and were administered by gavage at 0, 50, 100, 200, or 400 mg/kg/day. In females, dosing was continued through Postpartum Day 20, while males were dosed daily from weaning until necropsy (Fig. 1). A second experiment was designed to reexamine the effects of low doses of MBC (50 and 100 mg/kg/day) on sperm morphology. In this experiment males (15 controls and 12 in each MBC treated group) were dosed from weaning, as above, until necropsy at 80 to 85 days of age. Pubertal indices. Pubertal indices, measured in female rats, included the ages at vaginal opening, the first comified smear, and the onset of regular estrous cycles. Vaginal estrous cycleswere monitored daily until parturition. The age at puberty in the male was considered to be the age at preputial separation (PPS) (Korenbrot et al.. 1977). Muring oJP0. Male and female rats from the same group were paired for 16 days, starting at 84 days of age. The male and female sperm in the vaginal smear or copulatory plugs confirmed mating, while blood in the smear was often associated with fetal death. PO necropsy. Males were necropsied at 104- 106 days of age (for details see Gray et al., 1989b) and blood was collected for hormonal analysis oftestosterone, androgen binding protein, FSH, LH, TSH. and prolactin (for details of the hormone analyses and the results see Rehnberg et al., 1989 and Goldman et al., 1989). Body, testes, liver, kidney, adrenal, seminal vesicle plus coagulating glands (with fluids), the epididymis, and pituitary were weighed. One testis, was fixed in Bouins for 24 hr, stored in 70% alcohol, embedded in paraffin, stained with H and E or PAS, and examined by Pathco, Inc., for histopathological lesions. Similar methods were used for all histological evaluations. Caudal sperm motility was estimated in Dulbecco’s phosphate-buffered saline, 37°C at 100X. Debris or abnormal sperm were noted if present. A quantitative assessment of progressive sperm motility was also conducted using the methodology described in detail by Linder et al., (1986). While the latter measure is quantitative, the group means can be less accurate than the estimate when low sperm counts or clumps of sperm or debris preclude quantitative analysis. The second cauda epididymis was used for the determination of total sperm content (Gray et al.. 1989). Cauda epididymal sperm were fixed in approximately 5% formalin for assessment of sperm morphology by phase contrast microscopy. Two-hundred sperm heads, or intact sperm, per animal were categorized as normal intact cells with no abnormalities or as abnormal. The abnormalities include: (1) isolated heads; (2) misshapen

284

GRAY Cl--

heads; or (3) intact (head plus tail) cells with degenerating flagellae. In the pituitary, prolactin, FSH, TSH, and LH were measured (Goldman et al., 1989). Testicular interstitial fluid (IF) was collected, and the IF and a caput epididymis was used for an androgen binding protein and testosterone determinations. After IF collection, the testis was used for hCG-stimulated testosterone synthesis in vitro (Rehenberg et al.. 1989) and then for sonication resistant spermatid head count (Gray et al.. 1989b: as modified from Cassidy et al., 1983). When the Fl rat pups reached 25 days of age the PO dams were killed using CO2 and necropsied. Body weight, weights of the liver, kidney, adrenals, pituitary, and ovaries and the number of implantation scars were determined and the ovaries were examined for histopathological lesions. Rat Fl. Litter sizes and weights were taken at I, 15, and 2 1 days of age. At 35 days of age rats were randomly selected for fertility assessment. Breeding was monitored continuously for 4 months using 20 pairs of control and 22 pairs of MBC-treated (100 mg/kg/day) offspring, the highest dose with surviving pups. F2 pups were counted and removed on Postnatal Day 1. Shortly after placing the Fl into breeding pairs, the temperature control failed in the animal room and a number of rats died. reducing the n to about 16/dosage group. At about 5 months of age, the males were necropsied and the liver, kidney, adrenals, testis, cauda epididymis. pituitary, and seminal vesicleswere weighed. One testis was saved for histological evaluation. Two weeks later a number of females were also necropsied and body. liver. kidney, adrenal, and ovarian weights were taken.

AL. ’-

females were necropsied. In the dams, pituitary and kidney weights were taken and the numbers of implantation scars were counted. The ovaries, the uterus, the pituitary, and the kidney were saved for histopathological evaluation (by Experimental Pathology Labs, Inc, Research Triangle Park, NC). PO male reproductive indices. In the male hamster, the flank gland (an androgen-dependent external sebaceous gland) and preputial separation were measured as indices of pubertal development. At 60 days of age, the sexual behavior of the males was observed (Gray, 1982). At 8 l85 days of age the males were necropsied and body, liver, kidney, testis, cauda epididymis, seminal vesicle, paired adrenal, and pituitary weights were taken. One testis was utilized for histology, while the other testis was used to determine the sperm content. A sample of the caudal sperm was examined to estimate sperm motility, and caudal sperm content was determined. The epididymis, vas deferens, pituitary, and kidneys were also saved for histological examination (by EPL). Fl hamster. The F 1 pups were weaned at 2 1 days of age and housed in unisexual groups with littermates throughout the study. Estrous cyclicity was observed over two cycles from 120 to 140 days of age. The females were necropsied at about 260 days of age and body, liver, kidney, adrenal, pituitary, and ovarian weights were taken. The sex behavior of the male offspring was observed at about I52 days of age and they were necropsied at I86- 190 days. In the males, body, testis, epididymal, liver, kidney, adrenals, Harderian gland, pituitary and seminal vesicle weights were measured and the total sperm content of the cauda epididymis was determined. Both Species-Statistical

METHODS-HAMSTER Animals. Syrian hamsters were obtained from Engle Labs and housed and maintained as previously described (Gray, 1982). Weanling male and female pups were randomly assigned to either the control group ( I5 hamsters per sex) or the MBC group (12 hamsters per sex). The hamsters were housed in unisexual groups with three animals per cage. Oral dosing began at 22 days of age. The controls were dosed with corn oil while the treated group was administered 400 mg/kg/day of MBC (98.1% pure). Males were dosed until necropsy, while the females were dosed up to parturition. POfemale reproductive indices. In the female hamster. the age at the first behavioral estrus was utilized as an index of pubertal development. Behavioral estrous cyclicity was monitored over two cycles from 47 to 58 days of age. At 68-7 1 days of age the females were mated once for 1 hr to males from the same treatment group. The Fl pups were counted and weighed on Postnatal Days 1, 5, and 21. At weaning. the pups were housed in unisexual groups with 4 to 5 pups/cage while the PO

Analyses

The parametric data were analyzed using the general linear models procedure (PROC GLM) available in the 1985 edition of the Statistical Analysis System (SAS User’s Guide, 1985). When significant (p < 0.05) effects were detected then two-tailed t test comparisons were made using the LSMEANS options of the PROC GLM. When significant effects on organ weights were noted in the Fl generation, using individual values, the data were reanalyzed using litter means. Percentage data (e.g., fertility) were analyzed using Fisher’s Exact test.

RESULTS

PORats PO rat-growth, viability, and development. Growth was not markedly affected by MBC administration at any dose level and there was no treatment related mortality.

MBC AND REPRODUCTIVE

285

DEVELOPMENT

TABLE 1 THE EFFXYS OF SUBCHRONICADMINISTRATION OF MBC ON GROWTH AND REPRODUCTIVE DEVELOPMENT IN THE FEMALE RAT (PO, PARENT GENERATION) Dose of MBC (mg/kg/days) 0

50

100

200

400

Pubertal indices Vaginal opening (days) Weight at V.O. (g) First estrous smear (days) First estrous cycle (days) Average cycle length prior to mating (days) Percentage of smears with leucocytes (%)

31 104 32 33 4.7 56

28 87 30 31 5.0 61

31 102 32 32 5.1 55

32 103 33 33 5.0 59

32 103 33 33 4.7 52

Necropsy data weights Body (4 Liver(g) Kidney (g) Adrenals (mg) Pituitary (mg) Ovaries (mg)

288 14.4 1.24 74 I1 120

298 16.5 1.23 68 11 116

298 16.3 1.19 69 11 124

279 13.8 1.07 55 12 115

285 15.2 1.13 71 12 112

In females, the estrus, and the weight at vaginal by treatment at

ages at vaginal opening, first onset of cyclicity and the opening were not affected any dose level (Table 1).

In addition, estrous cycles were normal throughout the premating period and no organ weight changes were present at necropsy.

TABLE 2 THE EFFECTS OF SUBCHRONIC

ADMINISTRATION OF MBC ON THE GROWTH IN THE MALE RAT (PO GENERATION)

AND REPRODUCTIVE

DEVELOPMENT

Dose of MBC in mg/kg/day 0

50

100

200

400

Age at puberty Preputial separation (days) Weight at PPS (g)

42 206

40 190

40 187

41 193

42 179

Necropsy weights BUY (g) Liver(g) Kidney(g) Adrenals (mg) Seminal vesicle + fluid (g) Testis(g) Pituitary (mg) Cauda epididymis (mg)

444 17.3 1.69 52 1.76 1.75 9.4 311

452 19.7 1.57 49 1.77 1.72 9.9 288

434 17.5 1.59 46 1.69 1.61 9.5 284

415 18.7 I .45 45 1.64 1.18” 9.7 219”

396 16.7 1.55 45 I .60 1.O” 9.7 162”

Op < 0.00 I, different from the control value

286

GRAY

ET AL.

TABLE

3

THE EFFECTSOF SUBCHRONICADMINISTRATION OF MBC ON THE REPRODUCTIVE POTENTIAL OF THE RAT (PO) Dose of MBC in mg/kg/day

No. pairs No. that mated No. of pregnant No. live litters No. dams died No. litters resorbed No. without implants No. malformed litters No. implants/preg dam Maternal weight gain during pregnancy (g) Postnatal litter size Day 1 Day 5 Day21 Pup weight Day 1 (g) Day 5 k)

Dav 2 1(a)

0

50

100

200

400

I1 11

8 8

8 8

7 I

1 1

119 1 I 0 0 14.0 125

88

I8

3” 3

;a

0 0 0 0 14.2 119

1 0 0

0 0

0

10.3 9.3 1.5 5.1 10.2 45.5

13.9 11.1

2

13.0 115 9.1

4”

4” 3”

1 10.8

14.0

89

41’

8.5

9.0 6.6

2.0‘ 2.0‘ 0.8’

0’ 0’ 0’

5.7 8.9 43.6

6.1 10.3 46.5

4.3d 8.4 41.9

-

Note. PO male and females were treated from weaning, through puberty, breeding, gestation, and lactation with MBC orally. ’ p < 0.05 different from control value by Fisher’s Exact test. ‘p < 0.01 different from control value by Fisher’s Exact test. ‘p < 0.001 different from control value by a t-test. dp < 0.05 by a t-test.

The indices of puberty in the male rat were also unaffected by treatment with MBC (Table 2). The age and weight at preputial separation were similar in all groups. Mating was not affected by MBC administration (Table 3). All the females were sperm positive and most males produced copulatory plugs. There was also no indication that mating or parturition were delayed by MBC treatment. However, in the 200 and 400 mg/kg/day MBC groups a number of the mated females were pseudopregnant rather than pregnant, lacking implantation sites at necropsy. It was subsequently determined that these females had been paired with males with severe treatment-induced testicular atrophy and very low sperm counts. In those high-dose group MBC-treated females that did become preg-

nant, postimplantation fetal viability was reduced. All four of the pregnant females at 400 mg/kg/day resorbed their litters (p < 0.05). As a result of MBC’s adverse effects on spermatogenesis and fetal viability, there were no viable litters at 400 mg/kg/day and only 3 at 200 mg/kg/day. One of the three litters at 200 mg/kg/day and 2/7 at 100 had malformed pups (visibly hydrocephalic or kinky tail, p = 0.3) (Table 3). Necropsy data and histology. In the female rat, body and organ weights and ovarian histology were not altered by subchronic administration of MBC (Table 1). In males, body weight was slightly reduced at 400 mg/kg/day (Table 2). This effect was not significant by ANOVA, a > 0.15. Liver, kidney, adrenal, seminal vesicle, and pituitary weights were not affected by MBC administration. Testis

MBC AND REPRODUCTIVE

287

DEVELOPMENT

TABLE 4 A SUMMARY OF THE HISTOPATHOLCXXCAL EFFECTSOF SUBCHRONICADMINISTRATION OF MBC ONTHETESTESOFTHE POMALERATS Dose of MBC in mg/kg/day 0 Histopathological findings No. rats No. normal testes No. abnormal testes p-Value (Fisher’s exact) Severity of the testicular pathology Minimal to mild atrophy Minimal to mild atrophy and degeneration Moderate degeneration and atrophy Marked degeneration and atrophy Note.

12 12

50

100

200

8 6 2

8 5 3

0.14

0.05

0

2

3

1

0

0 0 0

0 0 0

0 0 0

2 3

2 4 1

0 -

8 0 8

400


2

7 0 I


MBC was administered from Day 2 I up to necropsy at about Day 105.

and cauda epididymal weights were both re- males from the 50 mg/kg/day dose group had duced by about 40% of that of control at 200 debris and 5/8 had broken sperm in the cauda1 semen sample. This decrease in the perand 400 mg/kg/day (t, < .OO1). Histologically, minimal to mild testicular centage of normal sperm (97.8 vs 91.6; conatrophy was noted at 50 and 100 (p < 0.05) trol vs 50) did not repeat in the second experimg/kg/day, while some males at 200 and 400 ment. However, the percentage of abnormal (p < 0.0001) mg/kg/day had moderate to se- sperm was increased at 100 mg/kg/day in vere degeneration and atrophy (Table 4). both experiments (Table 5). In the two highMinimal and mild atrophy were characterdose groups most males had debris and broized as the presence of multifocal seminiferken sperm in their cauda epididymal sperm ous tubules which lacked spermatogenesis, sample and less than 50% of the sperm were while the remaining tubules appeared nor- normal. These samples contained many bromal. Degeneration, leading to atrophy, was ken sperm (isolated heads) and sperm with characterized by eosinophilia and karyorabnormal heads. The estimation of sperm motility indicated rhexis of the spermatocytes, with spermatogonia being affected in the final stages of tu- that motility was reduced at 200 and 400 mg/ bular damage. Multinucleated giant cells and kg/day. Low motility was particularly apparsloughed cellular debris were also observed in ent in samples with very low counts and this the damaged testes. often precluded the determination of motility Testicular sperm head count was signifiby the quantitative methodology. For this cantly reduced at 200 and 400 mg/kg/day reason, the quantitative measure of motility (Table 5), while cauda epididymal sperm was only reduced at 400 mg/kg/day. count was slightly reduced at 50 mg/kg/day The endocrine data from the males in this (p < 0.06) and was significantly reduced at study are presented in parallel papers by 100 and above. Rehnberg et al. (1989) and Goldman et al. Semen quality and sperm morphology ( 1989). A summary of the effects seen in these were altered at all doses. We found that 7/8 studies is presented in Fig. 2. Significant alter-

288

GRAY ET AL. TABLE 5 MBC ONTHESPERMMEASURESINTHEPOMALERATS

THEEFFECYTOFSUBCHRONICADMINISTRATIONOF

Dose of MBC in mg/kg/day 50

0

Testicular sperm head count (millions) Cauda epididymal sperm count (millions) Motility estimate (%) Quantitative motility (%) No. of males with debris in sample No. of males with broken sperm in sample Sperm morphology EXP I Percentage normal (%) Isolated heads(%) Degenerate tails(%) Misshapen heads (%) Transformed sperm morphology data EXP 1* Abnormal sperm Isolated heads Degenerate tails Misshapen heads Sperm abnormalities EXP 2 Percentage normal (%) Isolated heads (%) Degenerate tails (%) Misshapen heads (%)

199 141 58 58 l/12 o/12 97.8 0.6 0.8 0.7

185 113’ 58 47 7/8d 5/8d 91.6 6.8 0.8 0.7

0.47 0.29 0.36 0.30 98.5 0.7 0.6 0.5

0.64 0.66’ 0.39 0.27 96 1.8 0.6 1.4

100 179 I lob 54 45 8/8d 418’ 93.5 2.2 2.0 1.7 0.83” 0.76h 0.64b 0.57 92” 2.7” 2.7” 2.9”

200

400

63” 17” 16” 44 7/8d 7/8d

61” II” 13” 2(jb 8/8d 8/8d

38.5 27.3 9.2 25.0

48.5 26.6 6.5 18.0

1.69’ 1.54” 1.10” 1.53” -

1.69” 1.66” 1.09” 1.54”

-

NW. MBC was administered from Day 21 until necropsy on about Day 105. ’ p < 0.001 different from control. b p < 0.05. ‘p < 0.03, one tailed t test. dp
ations of testicular and pituitary endocrine function were noted at 200 and 400 mg/kg/ day. In addition, it was determined that most of the endocrine alterations were seen in the infertile males with severe hypospermatogenesis, while the endocrine measures of the fertile males in these dosage groups were less affected. Rat-F1 ofspring. After weaning, the growth, viability and reproductive performance of the rats indirectly exposed to MBC at 100 mg/kg/day during gestation and lactation did not differ from the controls (Table 6). In addition, the body and organ weights at

necropsy did not differ between the control and treated rats. Hamster PO hamster growth and reproductive development. MBC administration at 400 mg/kg/ day did not effect the growth of the females or body weight at necropsy (Table 7). The age at the first behavioral estrus (puberty) was not delayed, estrous cyclicity was not altered, and all of the treated females mated successfully, became pregnant, and had pregnancies of

MBC AND REPRODUCTIVE

DEVELOPMENT

289

290

GRAY ET AL. TABLE 6

THE

EFFECTS

OF INDIRECT

ING GESTATION AND GROWTH. VIABILITY OF THE Fl RAT Pups

EXPOSURE

TO

LACTATION OF THE AND REPRODUCTIVE

MBC

DUR-

POSTNATAL FUNCTION

Dose of MBC in mg/kg/day 0 Fl males No. males necropsied Body weight(g) Liver weight (g) Kidney weight (g) Adrenals (mg) Testis(g) Cauda epididymis (mg) Pituitary (mg) Seminal vesicles Fl females No. females necropsied Body weight (g) Liver weight(g) Kidney weight(g) Adrenals (mg) Ovaries (mg) Pituitary (mg) F 1 fertility No. breeding pairs No. F2 pups born per dam Mean litter size Percentage Fl breeding F2A F2B F2C F2D

17 485 19.3 1.98 49.4 1.70

296 10.5 1.91

100

16 486 21.0 1.96

46.5 1.69 293 10.8 1.82

4 336 11.8 1.12 68.0 95.5 12.7

312 11.3 1.08 70.3 79.9 13.9

15 42.0 11.6

16 36.8 11.3

100 100 93 67

I

94 88 81 56

Note. Both parents were exposed to MBC by gavage from weaning, through breeding, and the dams were also dosed during gestation and lactation.

normal length. The numbers of implants and live pups on Postnatal Days 1,5, and 2 1 were also not altered by MBC exposure, but pup weights were slightly reduced. There were no histopathological lesions in any of the organs examined (ovaries, uterus, pituitary, and kidney). In the male hamster, MBC administration at 400 mg/kg/day did not retard growth (Table 7) or flank gland development. In addi-

tion, the age at preputial separation was not delayed. At necropsy body, testicular, epididymal, Harderian gland, adrenals, seminal vesicle, and pituitary weights did not differ between the control and treated groups and there were no histopathological lesions in the testis, epididymis, vas, pituitary, or kidney in the treated males. The MBC-treated male hamsters had significantly larger livers and kidneys as compared to the untreated males. The only adverse reproductive effect of MBC on the male hamster was on sperm counts. Sperm counts in the testis and cauda epididymis were significantly lower (about 2 1%) in the MBC-treated males than in control males (Table 7). Sperm motility was not affected, but 4/ 12 treated males had debris in the semen as compared to O/ 15 controls (p < 0.03). FI hamster results.MBC treatment did not permanently effect the growth of the F I hamsters. In females, estrous cyclicity and body and organ weights did not differ between the control and MBC-treated groups (Table 8). In the Fl males, testis and seminal vesicle weights and epididymal sperm counts were significantly reduced by prenatal exposure to MBC at 400 mg/kg/day (Table 8). Male sex behavior and body weight and other organ weights were not significantly affected. GENERAL

DISCUSSION

In the present study, we evaluated the effects of MBC on pubertal development and reproductive function in the rat and hamster as a component of our effort to develop an ART protocol (Fig. 1). The purpose of the current investigation was to compare the sensitivity of multiple reproductive measures in the male and female rat to fertility following subchronic exposure to the known reproductive toxicant MBC. Such information will be useful in the selection of endpoints for an ART protocol. The second study was designed to determine if MBC produced reproductive effects in the hamster at a dose that was toxic to the rat.

MBC AND REPRODUCTIVE TABLE 7 THE EWCXOFSUBCHRONIC ADMINISTRATIONOF MBC AT 0 OR 400 mg/kg/day ONTHE DEVELOPMENT OFTHE PO(PARENTGENERATION)MALEANDFEMALE HAMSTERS Dose of MBC in mg/kg/day 0 PO females/fertility Age of first estrus (days) No. bred Percentage fertile No. implants/pregnant dam Weight gain during preg (g) Litter size/preg dam Day 1 Day 5 Pup weight (g) Day 1 Day 5 Dams weight at necropsy (g) Pituitary (mg) Kidney (mg) PO males Age at preputial separation (days) Flank gland size (mm) Day 33 Day 40 Day 47 Day 54 Day61 Day 68 Day 75 At necropsy Necropsy weights Body kc) Liver(g) Kidney (mg) Harderian gland (mg) Adrenals (mg) Pituitary (mg) Seminal vesicle (g) Cauda epididymis (mg) Testis(g) Sperm counts (in millions) Testicular sperm heads Caudal sperm count Male sex behavior Percentage mating Latency to mount (set) No. mounts No. intromissions No. with debris in semen

400

30.0 15 73 11.7 24.6

30.6 12 100 13.3 23.1

11.0 8.5

10.8 7.8

2.8 5.3 118 5.6 571

2.4b 4.5” 114 5.3 589

35

36

0.8 3.6 4.4 4.5 6.3 6.8 6.4 5.6

0.5 3.1 3.0 4.3 5.1 5.8 5.9 6.1

125 4.89 414 205 24.6 3.17 1.72 359 1.79

131 6.42” 469” 208 26.9 3.5 I .84 341 1.72

286 118

239b 97h

100 89 1.9 8.7 l/15

91 99 2.5 8.3 4112’

Note. Males were dosed from weaning until necropsy. Females were dosed until parturition. op
DEVELOPMENT

291

Mule rat. In the male rat, MBC administration altered testicular histology, sperm morphology, and cauda epididymal sperm counts in males at doses below those that reduced fertility (50 and 100 mg/kg/day). Additional alterations were noted in the males from the two high-dose groups and the severity of these lesions varied with their fertility (Table 9). In the fertile males, testis weight (down 25%) testicular sperm count (reduced by 50%) cauda epididymal weight, and caudal epididymal sperm reserves (down 77%) were reduced. These males had at least 65 million sperm heads/testis and had epididymal reserves of at least 20 million sperm. In the infertile males in the 200 and 400 mg/kg/day dosage groups the testicular, epididymal and endocrine measures were more severely affected than in fertile males from these dose groups. Testicular sperm counts were reduced by 90%, cauda epididymal sperm reserves were reduced to 1% of control and testis weights were reduced by 50%. The sperm counts in these males ranged from 2.7 to 43.6 million in the testis and from 0 to 6 million in the cauda epididymis. The endocrine alterations were also more dramatically affected in the infertile than the fertile males (Fig. 2). Serum FSH was increased, testicular testosterone and androgen binding protein (ABP) levels were increased, while hCG-stimulated testosterone production in vitro was dramatically increased. The endocrine and pubertal data in the current study suggest that MBC acts on the hypothalamic-pituitary endocrine axis indirectly, through effects on the Sertoli cell. The lack of effect of MBC on pubertal development is indicative of normal hypothalamicpituitary-gonadal maturation and demonstrates that the hormonal changes associated with puberty were not affected. In addition, the profile of endocrine alterations seen in the males in the current study is typical of compounds that severely alter spermatogenesis and induce the “Sertoli cell only syndrome” (Jansz and Pomerantz, 1985), as does MBC.

292

GRAY ET AL. TABLE 8

THE EFFE~SOFADMINISTRATIONOF MBC ~~400 mg/kgJday TOTHE PO HAMSTERSONTHE POSTNATAL DEVELOPMENTOFTHEIROFFSPRING,THEFI Dose of MBC in mg/kg/day 0 F I male hamsters No. litters No. males Necropsy weights Body k) Liver(g) Kidney (mg) Adrenals (mg) Harderian gland (mg) Pituitary (mg) Cauda epididymis (mg) Testis(g) Seminal vesicle(g) Cauda epididymal sperm count in millions Male sex behavior Percentage mounting (%) Latency to mount (set) No. mounts No. intromissions F I female hamster No. litters No. females BUY (4 Liver(g) Kidney (mg) Adrenals (mg) Pituitary (mg) Ovaries (mg)

10 32

400 7 18

148 6.58 543 32.4 229 3.8 423 1.98 2.16

142 6.92 541 30.6 215 3.3 408 1.81” 1.92h

159

126h

100 123 2.9 1.4

100 122 3.3 6.5

10 10 111 6.72 721 20. I 7.3 14

10 10 167 6.13 690 20.5 6.6 70

Note. The means presented here are based on individual values. “p < 0.003 different from control using litter means. bp < 0.05.

It is likely that the increases in abnormal sperm and debris in the semen samples of male rats administered MBC at 50 and 100 mg/kg in the current study are indicative of subtle alterations of spermatogenesis and Sertoli cell function. A single oral dose of MBC causes marked disturbances of meiosis and massive sloughing of germ cells and Sertoli

cell fragments (Parvinen and Kormano, 1974; Hess et al., 1985). Other compounds, like vinblastine and colchicine, which produce similar alterations of microtubule function and Sertoli cell structure (Russell et al., 198 1), cause similar alterations of spermatogenesis. It is interesting that the dramatic effects on testicular cell functions, seen in this study, occur in the absence of any signs of general toxicity, as somatic growth and nonreproductive organ weights were not affected by MBC-treatment. As indicated earlier, we elected to use MBC to determine if it would be a “false negative.” Our protocol initially uses immature males and it is known that benomyl, which degrades to MBC, has little effect in the immature male rat (Carter et al., 1984). However, our results were not negative and are similar to those obtained when benomyl is administered to adult male rats (Linder et al., 1988). Subchronic dosing of adults with benomyl (45 mg/kg/day) causes a slight increase in abnormal sperm, an increased incidence of mild testicular pathology, reduced caudal sperm content, and a slight reduction in testis and epididymal weights. The fact that our data agree well with those of Linder et al. (1988) indicates that initiating dosing in weanling animals does not compromise our ability to detect the adverse effects of a chemical which produces age-related toxicity. Female rat. In the treated female rats pubertal development and estrous cyclicity were normal, indicating that MBC did not adversely effect hypothalamic-pituitary-gonadal endocrine function. The lack of effect on the weight of the ovary, as compared to the testis, could be due, in part, to sex differences in gonadal development and function. First, the germinal epithelium with its spermatogenic elements comprise most of the testis, while in the ovary, the oocytes are a minor component with respect to the weight of the gonad. For this reason, a reduction in the number of oocytes could occur without immediately affecting ovarian weight. Secondly, in the female, the oogonia complete their mi-

MBC AND REPRODUCTIVE

293

DEVELOPMENT

TABLE 9 Dose of MBC/breeding

Testis weight (g) Caudal weight (mg) Caudal sperm count Testis sperm count Motility estimate (W) Litter size No. implants Testicular histopathology No. with marked No. with moderate No. with minimum to mild

status

O/fert

2OO/fert

4OO/fert

2OO/inf

4OO/inf

1.75 311 141 199 58 12.7 14

1.52b 294 33” 110” 33” 7.56 11

1.10” 189” 21” 83” 20” 0” 14

0.84” 144” 1.5” 16” 0.2” 0” 0”

0.90” 135” 2.3” 31” 3” 0” 0”

2 2 0

I 2 0

0 0 0

0

0

1 3

2 2

Note. The data from the males in the two highest dosage groups have been partitioned to show the differential testicular effects of MBC on the fertile and infertile pairs. The data show that fertility is highly related to testicular and epididymal sperm content. Treated-fertile males are lessaffected but still differ from controls, displaying reduced sperm production. The histopathological data are less useful in discriminating the fertile from the infertile pairs. a p < 0.0 1 different from control mean. bp < 0.05.

totic divisions during the perinatal stage of life and meiosis is arrested in the dicytate stage until ovulation and fertilization. In contrast, in the adult male rat, stem cells are continuously producting spermatogonia and meiosis in an ongoing process. Therefore, the male may be more susceptible to treatments like MBC during adulthood than the female, because new oocytes are not being produced in the adult female. Developmental toxicity-rat. In the rat, fetal viability was dramatically reduced at 200 and 400 mg/kg/day, and malformed pups were noted in the 100 mg/kg/day dosage group. These results agree well with those reported (Kavlock et al., 1982) for the fetal effects of oral administration of benomyl in rats. In their study, benomyl was administered to dams orally on Days 7 to 16 of gestation at 0, 15.6, 3 1.2,62.5, or 125 mg/kg/day. On Day 2 1 of gestation dams were killed and fetuses were examined for skeletal and visceral malformations. At 125 mg/kg/day 76% of the fetuses were dead and 35% of those alive had malformations, including encepha-

loceles or hydrocephaly. Nineteen percent of the fetuses at 62.5 mg/kg were malformed, while 3 1.2 was a NOEL. In the present study the pregnant females at 200 and 400 mg/kg/ day dosage group had normal numbers of implants, but all of the fetuses were resorbed at 400 mg/kg/day and most of the pups died in the 200 mg/kg/day dosage group. In addition, hydrocephalic pups were seen in litters from the 100 mg/kg/day dose group. The protocol used in the current study should be able to detect developmental toxicity, as does the standard multigenerational test, by assessing postnatal growth and viability. However, the assessment of developmental toxicity from any multigenerational test is limited to doses below those that cause infertility ifevents that precede implantation are altered. In addition, such tests may overestimate the NOEL for developmental toxicity if nonlethal malformations go undetected. Hamster. The hamster was much less affected by the continuous administration of MBC at 400 mg/kg/day than was the rat. PO males had slightly lower sperm counts in the

294

GRAY ET AL. TABLE 10 Control male rat data

Weights at necropsy Body weight(g) Liver(g) Kidney (g) Adrenals (mg) Seminal vesicle (g) Testis (g) Pituitary (mg) Cauda epididymis (mg) Pubertal landmarks Age at PPS (days) Weight at PPS (g) Sperm measures Caudal sperm count Testis sperm count Sperm motility (%) Normal sperm morphology (%) Serum hormones (rig/ml) Testosterone LH FSH TSH ABP Prolactin Pituitary hormones (ng/mg) LH FSH TSH Prolactin Testicular interstitial fluid measures Weight (mg) Testosterone (rig/ml) ABP (&ml) Seminiferous tubular fluid measures Weight (mg) Testosterone (rig/ml) ABP (rig/ml)

Mean

cv

LSD

444

17.3 1.69 52 I .76 1.75 9.4 311

14 16 36 21 19 9 12 11

52 2.4 0.51 9.6 0.28 0.14 0.9 30

12 14 31 18 16 8 10 10

42 200

5 10

1.75 17

4 9

143 199 58 98

19 18 34 1.4

23 31 17 1

16 15 29 1

2.0 0.53 5.3 5.5 222 4.8

62 66 25 29 15 64

1.1 0.30 1.1 1.4 28 2.6

53 51 21 25 13 54

%D

758 331 1659 3202

30 31 27 14

197 88 390 393

26 27 23 12

80 222 1039

31 43 23

21 82 208

21 37 20

164 815 3143

28 23 14

40 160 368

24 20 12

Control female rat data Necropsy weights Body Liver Kidney Adrenals Pituitary Ovaries (mg) Pubertal landmarks/estrous cyclicity Age-at vaginal opening Weight-vaginal opening Age-first estrous smear Age-onset of cychcity Estrous cycle length Leucocytic smears (%)

288 14.4 1.24 74 11 120

5 17 18 18 7 26

12 2.1 0.19 12 0.9 27

4 15 15 16 8 22

31 104 32 33 4.7 57

5 12 8 7 I1 8

1.5 10 2.0 2.0 0.4 3.8

5 IO 6 6 9 7

MBC AND REPRODUCTIVE

295

DEVELOPMENT

TABLE l&-Continued Control female rat data Fertility/postnatal Fl data Fertility (%) Pregnancy weight gain Litter size (Day 1) Litter size (Day 5) Litter size (Day 2 1) Pup weight (Day 1) Pup weight (Day 5) Pup weight (Day 2 I)

Mean

cv

LSD

%D

90 125 10.3 9.3 1.5 5.7 10.2 45.5

31 54 63 67 13 10 9

33 4.8 5.1 4.3 0.062 0.85 3.4

26 47 54 56 I1 8 I

Note. The means, the coefficients of variation, the LSD (least significant difference) for the control rats (n = 12) and a treated group (N = 8, 19 total df; t = 2.093), and an estimate of the percentage change required for statistical significance (% D = LSD/control mean).

testis and cauda epididymis but sperm production was not reduced enough to reduce fertility. MBC administration did produce signs of developmental toxicity in the Fl. Neonatal weight and testis weight at necropsy were reduced, but fetal and postnatal viability were not reduced as they were in the rat. Endpoint sensitivity. The sensitivity of an endpoint to toxicant-induced alterations is one of the criteria that should be used to design ART protocols. Sensitive measures are those that are significantly affected at low doses. It is important to distinguish sensitivity from the precision of an endpoint, because these are not necessarily related attributes of the data. The statistical precision of the data are also important and this is discussed later. In the present study, epididymal sperm counts, testicular histology, semen quality and sperm morphology were sensitive indicators of the effects of MBC, being altered at doses below those that affected fertility. However, the sensitivity of these endpoints varies from compound to compound, depending upon the chemical’s mechanism of action. For this reason, the measures described above that were sensitive to perturbation by MBC differ from those that were sensitive to alteration by methoxychlor treatment. In an earlier study, we found that methoxychlor alters pubertal development and estrous cyclicity at relatively low doses, endpoints that were in-

sensitive to treatment with MBC. Methoxychlor also differed from MBC in that it had little effect on testicular measures at doses that altered the sex accessory glands and cauda1 epididymal sperm reserves. Endpoint precision. Precision refers to the variability of the data around the mean. A summary of the statistical precision of the data from the untreated rats in the present investigation is shown in Table 10. These values should be stable from study to study and are useful in evaluating the quality of the data. While the estimation of precision in the controls is useful for quality control, it may be misleading to use only this information to predict the size to detect a specific percentage change as statistically significant (% D in Table 10). Some of the data from the present study highlight the shortcomings of this approach. The % Ds and LSDs (least significance difference) for our data (Table 10) are based on the variance within the control group. These values may be inaccurate if a treatment affects the variability of the data as well as the mean. In such a case, the pooled error variance used to test for treatment effects in an ANOVA may be quite different from the variance of the control group alone. For example, the variance associated with the parameter “sperm morphology” in Table 10 is relatively small (CV = 1.4; variance = 1.93) in the control group, while in the males

296

GRAY ET AL.

treated with MBC at 200 mg/kg/day the variante is much larger (CV = 97%; variance = 1414). For other parameters, like cauda epididymal and testicular sperm counts, MBC-treatment caused the variance to decrease along with the mean, as compared to the control group. Criteria for the selection of “useful” endpoints. In the preceding discussion, we indicated that sensitivity and precision are two of the important criteria to be considered in the selection of “useful” endpoints for ART protocols. Other factors to consider in this selection process, include (3) ease and cost of measurement; (4) accuracy, defined as the proximity of the estimated mean to the “true” value (is the measure unbiased?); (5) measures that are apical are more useful in “screening” than ones that are specific; and (6) the biological relevance of the alterations (do the changes adversely impact reproductive function?). Clearly, any ART protocol must include a battery of “useful” measures. Alternative reproductive test protocol development. It is our goal to continue to develop dose-response data on alterations of reproductive function induced by a variety of known reproductive toxicants. With this data we will be able to identify a battery of reproductive endpoints that should be included in an ART protocol. An ART protocol that included such endpoints would have advantages over standard multigenerational reproduction tests for a number of reasons. The inclusion of more sensitive endpoints would enable the risk assessor to establish more accurate NOELs. Such a protocol should also require fewer animals because the statistical precision of these quantitative measures is often greater than that of fertility (Table 10). From the investigations conducted to date, using the present protocol (Gray et al., 1988) we have found that reproductive organ weights, pubertal landmarks, estrous cyclicity, cauda epididymal and testicular sperm counts, testicular histology, and sperm morphology are useful endpoints. We feel that it is premature to conclude about the utility of

some other endpoints, like sperm motility or serum hormones, at this time because we have not tested compounds that directly alter these reproductive parameters. ACKNOWLEDGMENTS We acknowledge the extremely valuable contributions ofJ. Ferrell, L. Strader, K. McElroy, and J. Hein for tech-

nicat assistance.

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RUSSELL, L. D., MALONE, J. P., AND KILLIAN, G. F. ( 198 I). Effect of the microtubule disrupting agents, colchicine and vinblastine, on seminiferous tubule structure in the rat. Tissue Cell 13(2), 349-367. RUSSELL, L. D. ( 1983). Normal testicular structure and methods of evaluation under experimental and disruptive conditions. In Reproductive and Developmental Taxicity of Metals (T. W. Clarkson, G. F. Nordberg, and P. R. Sager, Eds.), Plenum, New York. SAS Institute, Inc. (1985). SAS User’s Guide: Statistics, Version 5 ed. SAS Institute, Inc., Cary. NC.