The Fungicide Benomyl (Methyl 1 -(Butylcarbamoyl)-2-benzimidazolecarbamate) Causes Testicular...

FUNDAMENTAL AND APPUED TOXICOLOGY 17,733-745 (1991)

The Fungicide Benomyl (Methyl 1-{Butylcarbamoyl)-2-benzimidazolecarbamate) Causes Testicular Dysfunction by Inducing the

Sloughing of Germ Cells and Occlusion of Efferent Ductules 1

REx A. HESS,· B. J. M<X>RE,· JANET FORRER,· RALPH E. LINDER,t

AND AHMED AWAD ABUEL-A IT A:j:

·DepartmenJ of Veterinary Biosciences. University of Illinois, 2001 SOulh Lincoln, Urbana, Illinois 61801; tReproductive Toxicology Branch, Health Effects Research Laboratory, u.s. EnVlronmenJaJ Protection Agency,

Research Triangle Park, North Carolina; and *Facu1ty of Veterinary Med,Cine, Zagazlg, Egypt

ReceIved December 18, 1990; accepted June 21. 1991

The Fungicide Benomyl (Methyl I-(Butylcarbamoyl)-2-benzimidazolecarbamate) Causes Tes­ticular Dysfunction by Inducing the Sloughing of Germ Cells and Occlusion of Efferent Ductules. HESS, R. A., MOORE, B. J., FORRER, J., LINDER, R. E., ABUEL-ATTA, A. A. (1991). Fundam. Appl. Toxieol 17, 733-745. Adult male Sprague-Dawley rats (100 days of age) were given single oral doses of the fungicide benomyl (methyl I-(butylcarbamoyl)-2-benzimidazolecarbamate) in dosages ranging from 25 to 800 mg/kg body weight. The testis and excurrent ducts were examined at 2 and 70 days posttreatment to determine the chemical effects on spermatogenesis and the epididymis. Primary effects seen at 2 days postexposure were testicular swelling and occlusions of the efferent ductules. Mean testis weight peaked with 400 mg,lkg. Premature release of germ cells (sloughing), detected even with the lowest dosage (25 mg/kg), was the most sensitive short­term response to benomyl. Sloughing was found primarily In Stage VII of the spermatogenic cycle at the lower dosages, but at higher dosages sloughing extended into all stages except for Stages VIII-XI. Occlusions of the efferent ductules of the testis were dose-dependent and correlated with testis weight. Sperm and sloughed germ cells were compacted in the ductal lumen of occluded ductules, which were surrounded by two to four layers of polymorphonuclear leukocytes and other inflammatory cells. Although the caput epididymidis contained sloughed germ cells and appeared swollen, evidence of permanent occlusion was not found. The long-term (70 days) effects ofbenomyl were decreased testis weight (at 400 mg/kg), dose-dependent increases in sem­iniferous tubular atrophy, and increases in the number of reproductive tracts containing occluded efferent ductules. Fibrosis, granulomas, and abnormal growth were long-tenn consequences of occluded ductules, which were present 100% of the time in testes containing 26-100% seminiferous tubular atrophy. Only a few testes were found to be completely regressed. Occlusion of efferent ductules and disruption of the seminiferous epithelium by sloughing of germ cells are overlapping dose-dependent mechanisms responsible for short- and long-term effects of benomyl on the rat testis. C 1991 Society of T oxicolosY

The fungicide benomyl (methyl l-{butylcar­bamoyl}-2-benzimidazolecarbamate) has been used extensively as a systemic agent for broad-

1 This article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not coll5titute en­dorsement or recommendation for use.

733

spectrum application on food crops and or­namental plants. This chemical is a known toxicant to the male'reproductive system in rodents and dogs, producing hypospermato­cytogenesis and multinucleated germ cells (Torchinsky et ai., 1976; U.S. Environmental Protection Agency, 1979; Carter and Laskey, 1982; Barnes et ai., \983; Linder et ai., \988).

0272-0590/91 $3.00 Copyriabl C 1991 by the Society ofT oxiroIoiY. AD "",IS of reproduction in any form raervod

734 HESS ET AL.

Past studies of benomyl have emphasized the effects resulting from multiple exposures. Chronic (12.5 and 62.5 mg/kg!day) and sub­chronic (15,45, and 500 mg/kg!day) exposures decrease testicular and epididymal weights, reduce epididymal sperm counts"and fertility, and induce seminiferous tubular atrophy (Torchinskiy el aI., 1976; Ivanova-Chemis­hanska and Antov, 1980; Linder el al., 1988). Multiple acute exposures (200-1000 mg/kg! day) in prepubertal animals produced no tes­ticular effects (Carter and Laskey, 1982); how­ever, pubertal and postpubertal animals re­sponded with hypospermatogenesis and dif­fuse seminiferous tubular atrophy, even though testes weights were decreased only slightly (Carter el al., 1984).

Although previous studies have demon­strated the testis to be a specific target of be­nomyl toxicity, they have not uncovered the mechanism ofbenomyl's activity that induces long-term permanent effects such as infertility and seminiferous tubular atrophy. To deter­mine the mechanism of toxicity it is necessary to compare the early postexposure events to the long-term changes in the testis. Our pre­liminary study found that a single dose of be­nomyl (400 mg/kg) induced testicular swelling 2 days after exposure, suggesting that the ex­current ductal system had been occluded. Thus, the present study of benomyl was de­signed to determine the testicular dose-re­sponse at both early (2 days) and late (70 days) time periods postexposure and to correlate changes in the testis with events in the prox­imal excurrent ducts of the male reproductive tract.

MA TERIALS AND METHODS

Animals. dosing and design. Male Sprague-Dawley rats (Charles River Breeding Laboratories Inc., Kingston, NY) between 97 and I 10 days of age were housed two per cage under normal laboratory conditions (12 hi lightjl2 hr dark cycle, 72 ± 2°F, 50 ± 10% relative humidity) and were given ad libitum access to Purina Laboratory Chow and tap water. Benomyl (95% technical standard), provided by E. I. Du Pont de Nemours and Co., (Wilmington, DE),

was suspended in corn oil and single treatments were given by oral gavage in total volumes between I and 2.5 ml. Food was removed 15 hr before treatment and then re­placed after each animal was gavaged.

Animals were terminated at either 2 days (8 animals per group) or 70 days (12 animals per group) postexposure. Treatment groups included benomyl dosages of 0, 25, 50, 100, 200, 400, and 800 mg/kg. The 70-day group did not include an 800 mg/kg dosage because the 2-day group exhibited maximum effects at 400 mg/kg.

Histological methods The rats were anesthetized with sodium pentobarbital, and their testicles were fixed by vascular perfusion with 3% glutaraldehyde in 0.05 M cac­odylate buffer (pH 7.3). The fixed testes were separated from the efferent ductules and epididymis, weighed and stored as thin transverse samples (cut 4 mm apart), and stored in neutral buffered formalin prior to embedding tissue in glycol methacrylate for light microscopy. Testic­ular cross sections and longitudinal samples of the excur­rent ducts were sectioned at a thickness of 1.5 to 2 I'm and stained with hematoxylin and eosin. To aid in the discrimination of stages of spermatogenesis (Hess, 1990) other testicular sections were stained with periodic acid­Schiff reaction and counterstained with hematoxylin.

Quanti/alion. Quantitation of testicular parameters was obtained using a Zeiss photo microscope with a 20X ob­jective and a light tube through which an LED lighted cursor (Jande I Scientific) is viewed (diameter of the cursor dot, 0.1 I'm). Data were coUected by computer using Sigma Scan Image Analysis software (Jandel Scientific) interfaced with a Numonics Graphics Digitizer. Diameters of 50 nearly circular semi niferous tubule cross-sections per testis were determined from measurements to the nearest mi­crometer of tubular circumferences. The average height of the seminiferous epithelium was determined from four measurements within each tubule. For animals sacrificed 2 days posttreatment the percentage tubules (100 tubules per testis) with sloughed epithelium (germ cells missing from the apical edge of the epithelium) was determined. For animals at 70 days posttreatment, the tubules were classified as normal, regressive, regenerative, or atrophic (Hess e/ al .. 1988). To ensure that focal lesions were not missed or overly emphasized, evaluations of seminiferous tubules were made randomly within aU four quarters of each testis cross section.

The ductuli efferentes were evaluated for the presence of occlusions, as depicted by compaction of the luminal contents and increase in the luminal diameter. The duc­tules were also examined for the presence of fibrosis, min­eralization, and granulomas. After it was determined that some testes appeared to recover from benomy! treatment, histologic samples of the caput epididymides were eval­uated for sperm density and rated as 0 (azoospennic), I (few number of sperm), 2 (moderate number of sperm), or 3 (heavy concentration of sperm). Percentage data were transformed by the arc sine and all data were evaluated

BE'iOM'I L-I'iDl'CED TESTICUL-\R A TROPH'I 73"

~ 2 5 -~ w 2.0 ~

~ I- , .5 VI w I-

1 0

" " ..

o 25 50 '00 200 400 800 BENOMYL 'MG/KG)

FIG. I . The effect of benomyl (single dose) on testis weight at 2 days postexposure (means ± SEM). Significant differences from control are indicated by different letters, p < 0.05.

first by ANOV A . If an overall difference was found (p < 0.05), then a multiple range analysis was used to deter­mine significance among the means by dosage.

RESULTS

Day 2 Post-treatment

A single dose ofbenomyl induced testicular swelling and efferent ductal occlusions at 48 hr postexposure. Figure 1 shows a significant increase in mean testis weight after 200-800 mg/kg dosages. One male exposed to 100 mg! kg also had swollen testicles. Some enlarged

70

60

50

I- 40 Z tj ")0 0:: W 20 a..

10

o

c

b

.:. 1 G 2" 50 100 200 400 800

PP. V'L IMG/~r:

FIG. 2. The effCct ofbenomyl (single dose) on the per­centage of seminiferous tubules exhibiting epithelial sloughing at 2 days postexposure (means ± SEM). Signif­icant differences between dosages are indicated by different letters, p < 0.05.

475

4')C c

425

400 I,m

375 a

350 UI 325

300 ,.. 0 25 50 1 0 200 4

BENOMYL (MG/KG)

FIG. 3. The effect of benomyl (single dosc:) on the di­ameter of semihiferous tubules at 2 days postexposure (means ± SEM). Significant differences between dosages are indicated by different leners, p < 0.05.

testicles would not perfuse with Ringer's s0-

lution until the tunica albuginea was cut, thereby relieving back pressure. Testis weights in 75-85% of the animals in the 200-800 mg! kg dosage range exceeded the mean control weight by three to four times the standard de­viation. Beginning at 100 mg/kg, swelling of the testis, typified by an increase in testicular weight and tubular diameters, was accompa­nied by sloughing of the germinal epithelium and a reduction in seminiferous epithelial height (Figs. 1-4). The diameter of seminif­erous tubules showed a negative correlation with the height ofthe seminiferous epithelium (R "" -0.305).

120

100

11m 80

c $'

60 III 25 200 400 800

40 ~

BENOMYL (MG/KG)

FIG. 4 . The effect of benomyl (single dose) on the epi­thelial height of seminiferous tubules at 2 days postex­posure (means ± SEM). Significant d iffereo<:eS between dosages are indicated by different letters, p < 0.05.

736 HESS ET AL.

Premature release of germ cells (sloughing) was the most sensitive response to benomyl. Sloughing of the seminiferous epithelium was not detected in control testes (Fig. 5) but was seen beginning with the 25 and 50 mg/kg dos­ages, although in only 1 % and 2.8% of the tu­bules, respectively (Figs. 2 and 6). Sloughing at these lower dosages was not shown to be statistically significant but remains biologically important due to its specificity. At the lower dosages (25-100 mg!kg), sloughing of germ cells was more subtle than at the higher dos­ages, consisting of small areas of epithelial dis­organization or missing cells, specifically in Stage vn (Fig. 6) and Stage XIV. Missing round spermatids in Stages I and II did not appear to have been sloughed, but rather were evidence of meiotic pachytene cell degenera­tion earlier in time.

Sloughing of an entire layer of cells was ob­served regularly after 200-800 mg!kg dosages (Fig. 7). Maximum sloughing (54.5% of the seminiferous tubules) was reached with the 200 mg/kg dosage. At higher dosages (200-800 mg/kg), most testicles were swollen (as noted by tubular dilation (Fig. 3) and increases in testes weights (Fig. I» and sloughed ma­terial, including spermatids, round cyto­plasmic bodies, and spermatocytes, accumu­lated in dilated rete testes. Also at these higher dosages, germ cell sloughing extended into all stages except for Stages VIII-XI. Stages VIII­XI remained resistant to sloughing despite a significant thinning of their epithelial height. Sloughing did not include Sertoli cells. Other degenerative changes included cellular disor­ganization, p,hagocytosis of step 19 spermatid heads, chromatin margination, multinuclea-

FIG. 5. Control testis (Day 2 poste,;posure) with cross sections of normal seminiferous tubules. Stage VII is indicated.

BENOMYL-INDUCED TESTICULAR ATROPHY 737

FIG. 6. Benomyl 50 mg/kg (Day 2 postexposure). Sloughing of the step 19 spermatids is pointed out in a late Stage VII tubule (arrows). Stage XIV appears normal in this testis.

tion, and eosinophilic staining of late pachy­tene spermatocytes.

The lowest effective dosage to induce ductal occlusions was 50 mg/kg (Table 1). Starting with 100 mg/kg, the number of reproductive tracts containing ductal occlusions increased dramatically from 60% of the reproductive tracts to 93% at the 400 mg/kg dosage (Table 1). Occluded efferent ductules were identified by compacted luminal contents, swollen duc­tules, and the presence of granulomas, which were absent in control tissue (Figs. 8 and 9). After occlusion, the ductal epithelium was disorganized by the luminal compaction of sloughed testicular material and the smooth muscle layers were stretched or displaced by polymorphonuclear leukocytes (PMNs). In occluded regions, the ductules were sur­rounded by one to four layers of PMNs (Fig.

9) and in some instances the PMNs penetrated ·the epithelium. Other inflammatory cells, in­cluding eosinophils, lymphocytes, monocytes, and macrophages, were present, although fewer in number than the PMNs. The ductal occlusions appeared to be located primarily in the proximal region (nearest the rete testis), but it was difficult to determine their precise location from histologic sections. Determining a precise location of occlusions was further complicated by the fact that distal regions of the efferent ductules as well as the caput epi­didymidis often were dilated although sperm were not compacted within the lumen. In these dilated regions, sloughed cells from the sem­iniferous epithelium were present in abun­dance and included step 19 spermatids with

FIG. 7. Benomyl 800 mg/kg (Day 2 postexposure). Sloughed cells of the seminiferous epithelium (S) are in the lumen of Stages XIII and XIV (arrows). In Stage VI, the epithelium is thin and round spennatids are missing (arrows).

--

738 HESS IT AL.

TABLE I

PERCENTAGE OF MALE REPRODUCflVE TRACTS CONTAINING OccLUSIONS

OF THE EFFERENT DvcruLES AFTER BENOMYL EXPOSURE

2 Days 70 Days

Dosage (mg/kgf N b % Occluded N % Occluded

0 12 0 24 0 25 12 0 24 0 50 10 10 24 0

100 15 60 23 39 200 12 83 23 57 400 15 93 22 68 800 13 92 NT" NT"

a A single oral dose. b Samples with Incomplete sections of efferent ductules not included. C Not treated.

attached cytoplasmic lobes, round spermatids, and occasional spermatocytes. The caput, corpus, and cauda epididymides did not ap­pear to be severely affected by benomyl treat­ment.

Day 70 Post-treatment

The long-term effects of a single exposure to benomyl were seen in both the testis and the excurrent ducts. At 70 days, mean testis weight and tubular diameters were reduced only at the highest dosage, but seminiferous tubular atrophy and efferent ductal occlusions were found as low as the 100 mg/kg dosage (Figs. IO and II; Tables 1-3). Testes and ef­ferent ductules of animals treated with 25 and 50 mg/kg dosages were normal in appearance. Although tubular atrophy was progressively greater in the 100-400 mg/kg dosages, the mean testis weights did not differ from control values at the 100 and 200 mg/kg dosages. Also, a small percentage of seminiferous tubules were disorganized in appearance at the 100, 200, and 400 mg/kg dosages (1.1 ± 1.8%; 2.6 ± 3.9%; 2.9 ± 4.2%, respectively) or showed evidence of the epithelium in the process of regeneration (0.1 ± 0.3%; 1.4 ± 3.1 %; 0.9

± 1.0%, respectively). Seminiferous tubules containing compacted sperm and mineralized matrix were commonly found at the 200 and 400 mg/kg dosages. For example, at the 400 mg/kg dosage, 17% of the testes showed min­eralization in 1-10 tubule cross sections. Only a few testes were found to be totally regressed (90-100% atrophic tubules). Atrophic tubules with only Sertoli cells remaining in the epi­thelium were surrounded by a thickened base­ment membrane (400 mg/kg dosage) with a mean thickness of 4.83 ~m, compared to 2.05 ~m for normal tubules.

Many of the occlusions in efferent ductules that appeared on Day 2 were resolved by Day 70, although a dose-dependent number of re­productive tracts still retained occlusions after 70 days (Table 1). Associated with ductal oc­clusions were numerous fibrotic lesions (Fig. 11), some granulomas, and reduced numbers ofsperrn in the caput epididymides (Table 3). These pathologic changes were present more frequently in ductules from testes exhibiting higher percentages of seminiferous tubular atrophy (Table 2). Fibrosis appeared as regions of dense connective tissue either surrounding a regressed ductule or completely obliterating the ductule (Fig. 11). In rare ductules the lu-

BENOMYl-INDUCED TESTICULAR ATROPHY 739

-... \ . \ .

. '8 ; • FIG. 8. Control ductuli efferentes (Day 2 postexposure). Nonnal ductules are small in diameter and

contain few spennatozoa (S) in the ductal lumen (l). Connective tissue is relatively free of migrating leukocytes.

men was also mineralized. Granulomas con­sisted of aggregates of white blood cells (pri­marily lymphocytes and plasma cells) ass0-

ciated with occluded ductules and sometimes containing stagnant sperm. Regressed ductules did not contain sperm and the epithelium was irregular in appearance (Fig. II).

Although only a subjective assessment was made of caput sperm density per histologic cross-sectional area, there was a dramatic de­crease in the apparent sperm numbers as sem­iniferous tubular atrophy became more de­tectable (Table 3). Testicular atrophy only rarely produced azoospermia, but when pres­ent it was always unilateral, occurring four times with the left and twice with the right testicle. Azoospermia was found only when greater than 80% of the seminiferous tubules were atrophied, which occurred most fre­quently (4 of 12 testes) at the 400 mg/kg dos-

age. Of particular interest was the occasional presence of normal sperm density with 100-and 200-mg/kg dosages, when 50-75% of the seminiferous tubules were atrophied, which suggests a compensatory increase in sper­matogenesis by the remaining normal tubules. Also, when one testis exhibited considerable atrophy, the contralateral testis showed com­pensatory increases in weight, which masked the detection of testicular atrophy based upon weight alone at the 100 and 200 mg/kg dosages (Table 2).

DISCUSSION

A single dose ofbenomyl induces long-term, diffuse seminiferous tubular atrophy in the rat, which appears to be caused by occlusions of the efferent ductules of the testis within 2 days

740 HESS ET AL.

FIG. 9. Benomyl 400 mg/kg (Day 2 postexposure). The efferent ductu1es are distended and occluded by material in the lumen (L) that stains eosinophilic. An abundance of white blood cells fills the connective tissue (Ct) and PMNs line the ductal wall (arrows).

of exposure. Blockage of the excurrent duc­tules is directly associated with an increase in testis weight and dilation of seminiferous tu­bules at 2 days and with increases in seminif­erous tubular atrophy at 70 days postexposure.

Only two studies of acute benomyl exposure (either 5 or 10 doses) in the postpubertal male have been reported (Carter and Laskey, 1982; Carter et ai., 1984). Both studies failed to de­tect transient changes in testis weight but did report significant decreases in epididymal sperm counts with 200-1000 mg,/kg/day. No effect was reported on testis weights in one study (Carter et ai., 1984) and only a slight decrease in weight was found at 400 mg/kg/ day in the other study (Carter and Laskey, 1982). In contrast, we found an increase in testis weight at 2 days postexposure (with a single dose between 200-800 mg,lkg) and a

decrease in weight at 400 mg/kg on Day 70, with both responses associated with occlusions of the efferent ductules. These early and late effects of benomyl on the male reproductive system were missed in the previous studies be­cause testes were examined only during the period 14-31 days following treatments, and efferent ductules of the testis were not exam­ined (Carter and Laskey, 1982; Carter et ai., 1984).

The mechanism of benomyl toxicity in the testis has been a subject of speculation for some time (U.S. Environmental Protection Agency, 1979). One mechanism proposed in the past was the inhibition of gonadotropin stimulation. This hypothesis, however, is not supported experimentally, as hCG-stimulated serum testosterone levels are not altered by benomyl (Carter and Laskey, 1982), and

BENOMYL-INDUCED TESTICULAR A TROPHY 741

FIG. 10. Benomyl 400 mg/kg (Da} 70 postexposure). Greater than 500/, of the seminiferous tubules in this testis were atrophic (A). containing Sertoh cells and rare germ cells. The fev. tubules exhibiting sper­matogenesis were normal in diameter and appearance.

serum FSH concentrations actually become elevated (Carter et ai., 1984). Because benomyl inhibits microtubule formation in fungi, it has also been suggested that testicular hyposper­matogenesis in the rat may be caused by the impediment of cell division and other micro­tubule-dependent processes of spermatogen­esis (Parvinen and Kormano, 1974; Torchin­skiy et al., 1976; Carter and Laskey, 1982; Da­vidse and Flach, 1977; Ireland et a/., 1979).

The early testicular effects of benomyl (sloughing of germ cells) may be due to the inhibition of microtubule formation in the Sertoli cell and in the mitotic apparatus of di­viding germ cells, as seen with single doses of other microtubule poisons (Russell et ai., 1981). Direct effects of toxicants on Sertoli cell function have been shown to produce epithe­lial disorganization and subsequent tubular

atrophy, apparently independent of efferent ductal involvement (Chapin et al., 1983; Hess et ai., 1988; Blackburn et ai., 1988). The Ser­toli cell contains an abundance of microtu­buIes in its apical cytoplasm (Russell et al., 1981; Christensen, 1965) which appear to be involved in the control of spermiation (Rus­sell, 1984), and when disturbed, cause the seminiferous epithelium to become disorga­nized. In the present study, there is evidence to support the contention that the early sloughing of germ cells is not caused by efferent ductal occlusion, but rather by another mech­anism such as the inhibition of microtubule formation. In some males with occluded ef­ferent ductules, sloughed germ cells were seen in zones 2 and 3 of the caput epididymidis, but the initial segment of the epididymis was empty. This finding indicates that ductal oc-

742 HESS ET AL.

FIG. II. Benomyl 400 mg/kg (Day 70 postexposure). Persistence of ductal occlusions was evident by the presence of fibrotic lesions (F). Circular patterns of thick collagen bundles replace the previous location of efferent ductules. Thick regions of dense connective tissue also surround regressed ductules that exhibit irregular growth of the epithelium (G).

clusions occurred after the sloughed germ cells had passed the initial segment. It will be of interest to determine if repeated doses of be­nomyl, at dosages that do not induce ductal occlusions, will also cause long-term hypo­spermatogenesis and focal tubular atrophy. After a single dose, it is doubtful that micro­tubule inhibition alone can explain the long­term atrophy, because some stages of sper­matogenesis are resistant to sloughing and all effects at lower dosages are repaired by 70 days. Thus, long-term atrophy of seminiferous tu­bules, particularly when the testes are in­creased in weight due to swelling, must involve occlusions of the efferent ductules.

The literature is quite clear regarding the effects of efferent ductal occlusions. The effer­ent ductules are a vital link between the testis

and the epididymis and in the rat consist of two to eight tubules that merge into a single narrow ductule for the transport of newly re­leased sperm from the rete testis to the caput epididymidis (Reid, 1959; Talo, 1981). The ductuli efferentes remove nearly 90% of the fluid leaving the testis (Jones and Jurd, 1987) and if blocked by ligation (Anton, 1979), vas­cular stenosis (Macmillan, 1953), or chemical trauma (Jones, 1978) create a back-pressure of luminal fluids (Kuwahara, 1976). In the present study, testicular swelling by fluid back­pressure was created by benomyl's induction of occlusions in the efferent ductules, which then led to seminiferous tubular atrophy, in a manner similar to that produced by ductal li­gation (Harrison, 1953; Smith, 1962; Anton, 1979).

BENOMYL-INDUCED TESTICULAR A TROPHY 743

TABLE 2

TESTICULAR EfFECTS OF BENOMYL AT 70 DAYS PosTEXPOSURE

Percentage of testesl' Testis Tubule

Dosage weigh~ Weight Weight diarnetrr % Number of testes (mg/kg) N (g) < 1.62g > 2.0g (pm) Atrophied' > 50% atrophy

0 24 1.86 ± 0.03' 0.0 15.4 345.8 ± 1.1' 0.0' 0 25 24 1.91 ± 0.04' 3.8 38.5 354.5 ± 1.1' 0.0' 0 50 22 1.91 ± 0.02' 0.0 30.8 343.9 ± 1.0' 0.0' 0

100 24 1.84 ± 0.09' 23.1 50.0 336.6 ± 1.5' 11.5 ± 5.52 3 200 22 1.89 ± 0.08' 11.5 34.6 331.3 ± 1.7' 16.5 ± 5.62 4 400 24 1.59 ± 0.082 42.3 23.1 320.3 ± 2.<f 31.9±7.13 to

• Mean ± SEM; 100 tubules per testis, two testes per animal. Significant difference from control indicated by different numbers (p ... 0.05).

b 1.62 g was the lowest and 2.12 g was the highest testicular weight in the control group. , Mean percentage ± SEM. Seminiferous tubules with Sertoli cells only or rare germ cells; 100 tubules per testis, two

testes per animal. Significant differences between dosages indicated by numbers (p ... 0.05).

Ligation of the ductuli efferentes in adult rats results in a rapid increase in testis weight, dilation of the seminiferous tubules, and eventual degeneration of the seminiferous ep­ithelium (Harrison, 1953; Smith, 1962; Anton, 1979). Other degenerative features common to both ductal ligation and benomyl exposure include the formation of multinucleated giant cells (Linder et aI., 1988), chromatin margin-

ation in round spermatids, and thickened basement membranes (Anton, 1979). How­ever, one major difference between these two treatments is that complete ductal ligation in­duces total testicular atrophy in which Sertoli cells and occasional spermatogonia are the only remaining cells of the seminiferous epi­thelium (Harrison, 1953). After benomyl ex­posure, only one testis exhibited total atrophy

TABLE 3

EfFECTS OF BENOMYL ON EfFERENT DucruLES OF THE TESTIS AT 70 DAYS PosTEXPOSuRE"

Histopathology of the excurrent ducts'"

Seminiferous tubule Occlusion Fibrosis Granuloma atrophyh (%) N' (%) (%) (%) Caput sperm'

0-25 51 42 42 16 2.96 ± 0.19' 26-100 17 100 88 47 1.24 ± 1.062

• Data taken from treatment groups 100,200, and 400 mg/kg; long-term effects were not found in treatment groups 0, 25, and 50 mg/kg.

b Percentage of seminiferous tubules with Sertoli cells only or rare germ cells. 'Number of testes evaluated. d Percentage of reproductive tracts containing one or more occlusions, fibrotic lesions, or granulomas in the efferent

ductules. 'Sperm content was rated as 0 (azoospermic), I (few number of sperm), 2 (moderate number of spenn), or 3 (heavy

density of sperm). Significant difference (p < 0.05) is indicated by different numbers.

744 HESS ET AL.

and 5 of 71 testicles contained 90-99% atrophic seminiferous tubules. Thus, benomyl does not have as severe an effect on the testis as does surgical ligation of the efferent duc­tules, which suggests that occlusions induced by benomyl are only temporary or that they do not include all ductules present within the male tract. Future studies must address the dose-dependent relationship of the number of occluded ductules to the severity of seminif­erous tubular atrophy. In a preliminary ex­periment, we found that benomyl appears to induce sloughing of germ cells as early as 2 hr postexposure, prior to ductal occlusions. Thus, it is possible that disruption of the seminifer­ous epithelium by the sloughing of germ cells and occlusion of efferent ductules are overlap­ping dose- and time-<iependent mechanisms that contribute to seminiferous tubular atro­phy. However, dosages that induce early ductal occlusions will produce effects that override all other mechanisms of testicular atrophy that are in progress.

The observation of efferent ductal occlu­sions after benomyl exposure is highly signif­icant to male reproductive toxicology. If oc­clusions occur during the early period of a subchronic or subacute test, then all subse­quent exposures to the toxicant will have little to do with the resultant testicular atrophy be­cause regression will be a direct result of the rapidly formed occlusions. In light of this mechanism of toxicity, when testicular atro­phy is found after chronic or subchronic ex­posures, the proximal segments of the excur­rent ducts (a region commonly overlooked by gross dissection) should be examined for evi­dence of ductal occlusions.

ACKNOWLEDGMENTS

The authors appreciate the contributions of Susan Healy and Robert Guttroff. This work was supported in pan by NIH Grant f.S..{)5214-0 I and Reproductive Hazards in the Workplace, Home, Community, and Environment Research Grant 15-140 from the March of Dimes Birth Defects Foundation.

REFERENCES

ANTON, E. (1979). Early ultrastructural changes in the rat testis after ductuli efferentes ligation. Fertil. Steri/. 31, 187-194.

BARNES, T. B., VERU.NGIERI, A. J., AND WILSON, M. C. (1983). Reproductive toxicity of methyl-I-{butylcar­bamoyl)-2-benzimidazole carbamate (benomyl) in male Wistar rats. Toxicology 28, 103-115.

BlACKBURN, D. M., GRAY, A. J., LLOYD, S. c., SHEARD, C. M., AND FOSTER, P. M. D. (1988). Comparison of the effects of the three isomers of dinitrobenzene on the testis in the rat. Toxieol. Appl. Pharmacol. 92, 54-64.

CARTER, S. D., HEIN, J. F., REHNBERG, G. L., AND lAs. KEY, J. W. (1984). Effect ofbenomyl on the reproductive development of male rats. 1. Toxieol. Environ. Health. 13,53-68.

CARTER, S. D., AND LAsKEY, J. W. (1982). Effect of be­nomyl on reproduction in the male rat. Toxieol. Le1l. 1,87-94.

CHAPIN, R. E., MORGAN, K. T., AND Bus, J. S. (I983). The morphogenesis of testicular degeneration induced in rats by orally administered 2,5-hexaned.ione. Exp. Mol. Pathol. 38, 149-169.

CHRISTENSEN, A. K. (1965). Microtubules in sertoli cells of the guinea pig testis. Anal. Rec. 151,335.

DA V1DSE, L. c., AND FlACH, W. (1977). Differential binding of methyl benzimidazol-2-yl carbamate to fun­gal tubulin as a mechanism of resistance to this antJm­itotic agent in mutant strains of Aspergillus niduJans. 1. Cell BIOI. 72,174-193.

HARRISON, R. G. (1953). The effect of ligation of the vasa efferentia on the rat testis. Proc. Soc. Study Feml. 5, 97-100.

HESS, R. A., UNDER, R. E., STRADER, L. F., AND PER­REAULT, S. D. (1988). Acute effects and long-term se­quelae of 1,3-dmitrobenzene on male reproduction in the rat. II. Quantitative and qualitative histopathology of the testis. 1. Androl. 9, 327-342.

HESS, R. A. (1990). Quantitative and qualitative charac­teristics of the stages and transitions in the cycle of the rat seminiferous epithelium: Light microscopic obser­vations of perfu,,;on-fix. d and plastic-embedded testes. BIOI Reprod. 42, 525-:;42.

IBACH, B., WEISSBACH, !.., AND HILSCHER, B. (1976). Stages of the cycle of the seminiferous epithelium in the dog. Andrologia 8, 297-307.

IREu.ND, C. M., GuLL, K., GlITfERIDGE, W. E., AND PoGsoN, C. I. (1979). The interaction of benzimidazole carbamates with mammalian microtubule protein. Biochem. Phanrwcol. 28, 2680-2682.

IVANovA-CHEMtSHANSKA, L, AND ANTOV, G. (\980). Changes in the gonads reproduction and generations of white rats under the effect of some pesticides. Arch. Toxieol. Suppl. 4, 459-462.

BENOMYL-INDUCED TESTICUlAR A TROPHY 745

JOHNSON, L, AND NEA VfS, W. B. (1983). Enhanced daily sperm production in the remaining testis of aged rats following hemicastration. J. Andral. 4, 162-166.

JONfS, A. R. (1978). The antifertility actions of a-chlo­rohydrin in the male. Life Sci. 23, 1625-1646.

JONfS, R. C, AND JURD, K. M. (1987). Structural differ­entiation and fluid reabsorption in the ductuli efferentes testis of the rat. Aust. J. Bioi. Sci. 40, 79-90.

KUWAHARA, M. (1976). The effects of ligation of vas def­erens, corpus epididymidis and vasa efferentia on the testicular function in rats. Tohoku J. Exp. Med. 120, 251-257.

LINDER, R. E., REHNBERG, G. L., STRADER, L. F., AND DIGGs, J. P. (1988). Evaluation of reproductive param­eters in aduh male WIStar rats after subchronic exposure (gavage) to benomyl. J. To_tical. EfTViron. Health 25, 285-298.

MAcMILLAN, E. W. (1953). Higher epididymal obstruc­tions in male infertility. Fertil. Stenl. 4, 101-129.

PARVINEN, M., AND KORMANO, M. (1974). Early effects of antispermatogenic benzimidazole derivatives U 32.422E and U 32.104 on the seminiferous epithelium of the rat. Andrologia 6,245-253.

REID, B. L (1959). The structure and function of the ep­ididymis. II. The histogenesis of the rat epididymis. A ust.

J. Zool. 7, 22-38. RUSSELL, L. D. (I 984). Spermiation-The sperm release

process: Ultrastructural observations and unresolved problems. In Ultrastructure of Reproduaion (J. Van Blerkom and P. M. Motta, Eds.), pp. 46--66. Martinus Nijhoff, Boston.

RUSSELL, L. D., MALONE, J. P., AND MAcCuRDY, D. S. (1981). Effect of the microtubule disrupting agents, col­chicine and vinblastine, on seminiferous tubule structure in the rat. Tissue Cell. 13,349-367.

SMITH, G. (1962). The effects ofligation of the vasa effer­entia and vasectomy on testicular function in the aduh rat. J. Endocrinol. 23,385-399.

TALa, A. (1981). In-vitro spontaneous electrical activity of rat efferent ductules. J. Reprod. Fert.63, 17-20.

TORCHINSKIY, A. M., ORLOVA, N. Y., SMlRNOVA, Y. B., AND MAKA vov A, L. F. (1976). Data on the toxicological and public health characteristics of the carbamate-group pesticide benlate. Gig. Sanit. 2, 35-41.

U.S. Environmental Protection Agency (1979). Benomyl Position Document 2/3, 1-79.