Histopathologic changes in the testes of rats exposed to dibromoacetic acid

ELSEVIER

Reproductive Toxicology, Vol. 1 I, No. I, 47-56, 1997 Copyright 0 1997 Elsevier Science Inc. Printed in the USA. All rights reserved

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HISTOPATHOLOGIC CHANGES IN THE TESTES OF RATS EXPOSED TO DIBROMOACETIC ACID

RALPH E. LINUER,* GARY R. KLINEFELTER,” LILLIAN F. STRADER,*

D.N. RAO~EERAMACHANENI,-/- NAOMIL.ROBERTS,* and JUAND.SUAREZ* *National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency,

Research Triangle Park, North Carolina; and tDepartment of Physiology, Colorado State University, Fort Collins, Colorado

Abstract - The present report details histopathologic changes in the testis and epididymis of rats gavaged daily for 2 to 79 d with a by-product of water disinfection, dibromoacetic acid (DBAA). On treatment day 2 abnormal retention of Step 19 spermatids was observed in animals given the highest dosage of 250 mg/kg. Additional changes on day 5 included the fusion of mature spermatids and the presence of atypical residual bodies (ARB) in the epithelium and lumen of Stage X-XD seminiferous tubules. By day 9, ARB were seen in most stages of the seminiferous epithelial cycle and in the caput epididymidis. On day 16 distorted sperm heads were recognized in Step 12, and older spermatids, and luminal cytoplasmic debris was found throughout the epididymis. On day 31, there was vacuolation of the Sertoli cell cytoplasm, extensive retention of Step 19 spermatids near the lumen of Stage IX and X tubules, and vesiculation of the acrosomes of late spermatids. Marked atrophy of the seminiferous tubules was present 6 months after 42 doses of 250 mg/kg. ARB and retention of Step 19 spermatids were observed after 31 and 79 doses of 50 mg/kg and increased retention of Step 19 spermatids was seen in several rats dosed with 10 mg/kg. No abnormalities were detected at the dosage of 2 mg/kg. The changes suggest that the testicular effects of DBAA are sequelae to structural and/or functional changes in the Sertoli cell. 0 1997 Elsevier Science Inc.

Key Wur-dx: dibromoacetic acid; disinfectant by-product; subchronic; male rat; histopathology; testis; epididymis; sequential changes.

INTRODUCTION

Various disinfection by-products are formed via the re- actions of chlorine, ozone, and other disinfectants with organic and inorganic substances present in drinking water (16). Halogenated acids including many brominated species are among commonly occurring by-products. Of the bromoacetic acids, dibromoacetic acid (DBAA) is usually found in greater concentrations than the mono- substituted form (4,7,8). Accurate quantification of hu- man exposure to DBAA via drinking water is difficult at present, because DBAA in water supplies is a complex issue dependent on factors such as bromine ion concen- tration, temperature, and pH. In 35 water utilities, quar- terly median values for DBAA ranged from 0.09 to 1.5 p,g/L; in the utility with the highest influent bromide levels, DBAA concentrations ranged from 7.8 to 19 p&L

(4).

Address correspondence to Lillian F. Strader, Reproductive Toxi- cology Division, National Health and Environmental Effects Research Laboratory (MD 72), Research Triangle Park, North Carolina 27711.

Received 15 May 1996; Revision received 30 September 1996; Accepted IO Octnher 19%.

47

Although considerable information on the chlorinated acetates is available, data on the toxicity and health effects of the bromoacetates is minimal. The reported testicular effects of dichloroacetic acid (9,lO) prompted us to investigate the male reproductive toxicity of two brominated acetic acids (11,12). Bromoacetic acid produced no spermatotoxic effects, however, DBAA administered in a single oral dose (1250 mg/kg), or in 14 daily doses of 90 to 270 mg/kg produced abnormal sperm motility and/or morphology in male rats. Histopathologic changes in these short duration tests included altered spermiation, distortion of late spermatids, formation of atypical residual bodies, and the fusion of spermatid flagella; mild effects on spermiation occurred in some animals at dosages as low as 10 mg/kg/d. In a subsequent 80-d study we examined the time course and dose- response effects of DBAA on reproductive outcome and sperm quality (13). Early effects on male fertility, which appeared due to altered sexual behavior, occurred during the first breeding (days 8 to 14) in rats exposed to 250 mg DBAA/kg/d. However, subsequent antifertility effects were attributed primarily to marked alterations in sperm morphology and motility and a decline in the numbers of epididymal sperm. At a lower dosage (50 mg/kg), sperm

48 Reproductive Toxicology Volume 11, Number I. 1997

morphology, sperm motility, and epididymal sperm numbers were moderately affected when measured after 31 and 79 daily doses.

substantial-effect levels would likely fall within this dos-

age range ( 12).

No information on the genesis of the testicular le-

sions produced by haloacetates is available; therefore, several early necropsy time points were incorporated into

the above study to characterize the progression of histologic changes. The current report presents the histopathologic findings in the testes and epididymides of male

rats dosed with 0 or 2.50 mg DBAA/kg for 2, 5,9, 16, or 3 1 d, and in rats from lower dosage groups (0,2, 10, and 50 mg/kg) dosed for 3 1 and 79 days. Also included is the histopathology of animals that were killed 6 months after the last of 42 doses of 250 mg/kg to obtain preliminary information on the reversibility of testicular damage.

Necropsy

MATERIALS AND METHODS

Animals and dosing Procedural details for the animal exposures have

been previously described (13). Briefly, proven breeder

male (105-d-old) Sprague-Dawley rats (Harlan Sprague-Dawley Inc., Indianapolis, IN) were ranked by body weight and randomly distributed into five groups of

10 rats each (for breeding and final necropsy) and 13 groups of six rats each (for interim necropsies). The animals were caged individually with room conditions of

12-h light/dark, 22 f 1 “C, and 50 + 10% relative humid- ity. Custom-synthesized dibromoacetic acid (99+% pu- rity) was obtained from Aldrich Chemical Co., Milwau- kee, WI. The compound was dissolved in distilled water and the solutions were adjusted to approximately pH 6.5 with NaOH for oral dosing by gavage. Dosing solutions were prepared every 4 to 6 d and dosages of 0 (water control), 2, 10, 50, or 250 mg DBAA/kg were administered once a day in a dose volume of 5 mL/kg adjusted weekly for body weight. The dosages used were based on short duration studies that suggested both no-effect and

The necropsy schedule is given in Figure 1. Nec- ropsies of six rats dosed with 0 or 250 mg/kg were performed 24 h after the last of 2,5,9, or 16 daily doses. Six

rats from each dosage group (0, 2, 10, 50, 250 mg/kg) were necropsied after a mean of 31 doses (the rats were

killed over a 3-d interval, i.e., days 30 to 32). Breeder rats in the 0, 2, 10, and 50 mg/kg dosage groups were killed after a mean of 79 doses. The time points for the time course evaluation of the testicular lesion were based on earlier studies indicating that observable histologic changes were likely to occur between 2 and 16 d (11,12). The interim necropsies on day 31 and the final necropsies on day 79 of the lower dosage groups provided additional time points and data to evaluate the dose response. Some animals dosed with 250 mg/kg lost weight and developed transient diarrhea during the second and third treatment weeks. The toxicity became more severe including marked weight loss, atypical posturing, and abnormal movement of limbs; therefore, dosing of this group was prematurely terminated after 42 doses. These animals were necropsied following a 6-month posttreatment recovery period to obtain preliminary information on the reversibility of the lesions.

All rats were anesthetized with Halothane U.S.P. (Halocarbon Laboratories, North Augusta, SC). In 4 to 6 DBAA-treated animals and 2 to 6 controls at each necropsy time point, the right testes and epididymides were fixed in situ by vascular perfusion (Ringer’s solution followed by 5% glutaraldehyde in 0.05 M cacodylate buffer with 0.1 M sucrose, pH 7.4). The right testes and epididymides of nonperfused animals were immersion- fixed in Bouin’s solution. The left testes and epididymides of all rats were excised and used for previously reported sperm studies (13). Immersion-fixed tissues were

Necropsy Schedule

1 I 1 I 1 1 1 I 02 5 9 16

Experiment Day

Fig. 1. Necropsy schedule of male rats dosed with DBAA. Numbers along the horizontal axis are experiment days corresponding to the number of doses. Interim necropsies utilized six rats per group. The final necropsies (days 78 to 80) utilized 10 rats per group. Dosing was discontinued after 42 doses because of toxicity in rats given 2.50 mg/kg; these rats were necropsied after a recovery period of 6 months.

DBAA testis histopatholo~y * R. E. LIHDER ET AL. 49

embedded in paraffin and perfusion-fixed tissues were embedded in glycol methacrylate (GMA). Paraffin sec-

tions (5 to 6 rJ,m) and GMA sections (1.5 to 2 pm) were stained with H&E or PAS counterstained with hema- toxylin for light microscopy. The histologic processing was performed by American Histolabs Inc., Gaithers- burg, MD. For electron microscopy, portions of glutaraldehyde-fixed testicular parenchyma from selected animals dosed with 0 or 250 mgfkg were postfixed in 1%

aqueous osmium tetroxide for 1 h on ice and dehydrated in graded, ascending concentrations of ethanol on ice. Tissue was then processed at room temperature through 100% ethanol and propylene oxide, and embedded after overnight infiltration in Poly/Bed 812 Epon. Thin sections (silver and gold) were stained with uranyl acetate and lead citrate according to the procedure of Sato (14) and were examined with a Phillips 410 electron micro- scope. Staging of seminiferous tubules was done according to the criteria of Leblond and Clermont (15) and Hess (16). Differences in the histology of the testis and morphology of epididymal sperm were evaluated

qualitatively.

RESULTS

The reader is referred to the earlier publication for additional details of the sperm measures and reproductive studies (13). Briefly, daily treatment with the highest dosage of DBAA (250 mg/kg) compromised male fertility prior to deterioration of caudal epididymal sperm

quality and quantity and before the onset of marked signs of toxicity, which were primarily apparent during and

after the third week of treatment. This appeared to be the result of alterations in mating ability or libido. Later, marked effects on epididymal sperm morphology and motility developed and sperm that were artificially in- seminated failed to produce litters. Animals that were allowed to recover for 6 months after receiving 42 doses had substantially decreased testis weights and sperm counts. The lower dosages did not elicit gross signs of toxicity during the 80-d treatment period; compared to the higher dosage group, there were only moderate effects on sperm morphology, motility, and epididymal sperm counts. The males remained fertile, but, compared to controls, there were fewer inseminations, fewer copu- latory plugs, and fewer multiple litters in DBAA-treated rats, suggesting that some alteration in sexual behavior compromised male reproduction at dosages as low as 10

mg/kg.

Histopathology The various histologic changes in the testis and ep-

ididymis of DBAA-treated rats are summarized in Table

1 and major stage-specific effects over time are further depicted in Figure 2.

Day 2,250 mglkg. The testis and epididymis of rats given two daily doses of 250 mg/kg appeared qualitatively similar to control animals. However, compared to controls, three of six DBAA-treated rats were judged to

have moderately increased numbers of retained Step 19 spermatids (no~ally released in Stage VIII) in Stage IX of the cycle of the seminiferous epithelium.

Day 5, 250 mglkg. After five doses, atypical residual bodies (ARB) were numerous near the luminal border of the germinal epithelium in Stage IX, and less frequent in Stage X (Figure 2). These residual bodies were relatively large and irregular in shape compared to

controls and gave the appearance of coalescing or un- condensed forms. In Stages X-XII many of these irregular residual bodies appeared to become more rounded

and form large ARB (diameter 13 to 35 pm) that were observed both within the epithelium and free within the tubular lumen (Figure 3a). In addition, substantial retention of Step 19 spermatids (primarily near the lumen but also near the basement membrane) was observed in Stage IX. A number of basally located Step 19 nuclei were present in Stages X, XI, and occasionally XII. Fur- ther reflecting the failed sperm release were increased numbers of partially resorbed residual bodies near the basement membrane in Stages X and XI (residual bodies are normally resorbed in Stage IX).

Electron microscopy revealed the presence fused flagella of mature spermatids in the testis by day 5 (Fig- ure 3b). Two or more intact flagella within a common

membrane were separated by a thin rim of cytoplasm. Fusion was commonly observed at the level of the mid- piece; there appeared to be no fusion of the fibrous sheath. These fusion products were observed only in retained mature spermatids in Stage IX and later tubules; however, the observations did not rule out the possibility of fusion of younger cells.

No immature germ cells, testicular debris, or other abnormalities were detected in tissue sections of the epididymis.

Day 9, 2.50 mglkg. After nine doses of DBAA, a number of ARB were still evident at the periphery of the lumen in Stages IX and X. Numerous ARB were seen largely within the epithelium (some of these appeared to be ~dergoing dissolution) in Stages X-XII and mostly in the lumen in Stages X-XIV and I-VII (Figure 2). Mild to moderate retention of Step 19 spermatid nuclei near the lumen was seen in Stages IX and X; a few basally located remnants of Step 19 nuclei were seen in Stages X-XII. A number of ARB were present in the proximal caput epididymidis at this time.

50 Reproductive Toxicology Volume 11, Number I, 1997

Table 1. Summary of histopathologic changes in the testis of rats dosed daily with DBAA

Day

2 5

9

16

31

421186b

31

79

31 79

31 79

Dosage mtig/d

250 250

250

250

250

250

SO

SO

10 10

2 2

Principal changes

Moderate retention of Step 19 spermatids near lumen in Stage IX in three rats. Many ARBa in Stages IX-XII; substantial retention of Step 19 spermatids near lumen in Stage IX; basally located

remnants of Step 19 nuclei in Stages IX-XII; fused Step 19 spermatid flagella in Stage IX. Many ARB in Stages IX-XIV and a few in Stages I-VII; moderate retention of Step 19 spermatids near lumen in

Stages IX, X; basally located remnants of Step 19 nuclei in Stages X-XII; fused spermatid flagella in Stage IX; ARB in caput epididymidis.

Moderate numbers of ARB in Stages X-XIV and I-VIII; substantial retention of Step 19 spermatids near lumen in Stages IX, X; basally located remnants of Step 19 nuclei in Stages IX-XII; distorted acrosomes and/or heads of Step I2 and later spermatids; maloriented late spetmatids; fused spermatid flagella in Stage IX; ARB and other cytoplasmic debris throughout epididymis.

Rare ARB; extensive retention of Step 19 spermatids near lumen in all Stage IX, X tubules; luminal and basal retained Step 19 spermatids in Stages XI-XIV; abnormally shaped nuclei and vesiculated acrosomes of Step 12 and older spetmatids; maloriented late spermatids; vacuolation of Sertoli cell cytoplasm; fused spermatid flagella in Stage IX; extensive cytoplasmic debris throughout epididymis.

Extensive tubular atrophy (Sertoli cells only or rare germ cells in 95% of tubules); disorganization of sperm producing tubules and de~encration of mature and round spermatids.

A few ARB observed in most stages; moderate to substantial retention of Step 19 sperm&s near lumen in Stage IX; basally located remnants of Step 19 nuclei in Stages X-XII; rare ARB throughout epididymis.

ARB seen in most stages; substantial retention of Step 19 spermatids near lumen in Stages IX, X; basally located remnants of Step 19 nuclei in Stages X-XII; cytoplasmic debris throughout epididymis.

Moderately increased numbers of retained Step 19 spermatids near lumen in Stage IX in three rats. Moderately increased numbers of retained Step 19 spetmatids near lumen in Stage IX in three rats; basally located

relnn~ts of Step 19 nuclei in Stages IX-XI in three rats. None None

“ARB Atypical residual bodies. ‘186 days after the last of 42 doses of DBAA.

Dq 16, 250 ~zg~~~. The histologic changes in the testis after 16 doses were more variable among animals than the changes seen on days 5 and 9. ARB ranged from few to moderate in number and were found in Stages X-XIV and I-VIII. Substantial retention of Step 19 spermatids near the tubular lumen was observed in Stage IX and occasionally in Stage X. Remnants of Step 19 spermatid nuclei were seen basally in Stages IX-XII. In Stage XII-XIV and I-VIII tubules, late spermatid heads appeared distorted (Figure 2). Distortion of spermatid heads was particularly evident in Stage I, in which the ventral and dorsal aspects of the acrosomes of Step 15 spermatids appeared irregular in shape and/or undeveloped. In particular, the characteristically shaped dorsal fin normally observed in these cells was not apparent (Figure 4). Distortion of Step 16 to 19 spermatids was less obvious, but was also recognized in some tubules. ~alorientation of a number of Step 19 spermatids was apparent in Stage VII and VIII tubules. A few to many variably staining ARB as well as more uniformly staining eosinophilic globules were present throughout the lumen of the epididymis.

Dny 31,250 mgikg. After 3 1 daily doses of DBAA, the histologic changes in the testis were unifomny more severe than at the earlier time points. Vacuolation of the Sertoli cell cytoplasm was present to varying degrees in all DBAA-treated animals (Figure 5a). The vacuoles were stage-specific in that they appeared only in Stages

XIV and I-VII. but they were found predominantly in

Stage I (Figure 2). Exaggerated phagocytic vacuoles

were prominent near the basement membrane in Stage

IX (Figure 5b). Abnormal retention of Step 19 spermatids was extensive with an apparent full complement of

Step 19 spetmatids and associated residual bodies pres-

ent near the lumen in virtually every Stage IX and X tubule (Figure 6a). These retained Step 19 spermatids

were obviously degenerating in Stage XI as evidenced by

their presence near both the lumen and basement membrane in Stages XI-XIV {Figure 6b). Remnants of con-

densed nuclei (presumed to be Step 19 nuclei) were oc-

casionally recognized near the basement membrane as late as Stage V. Compared to earlier time points, there

was little distortion of residual bodies in Stage IX, and

ARB were seen in the various stages only rarely. The sperm heads and acrosomes of late spermatids appeared

distorted. A number of maloriented Step 19 spermatids were usually apparent in Stages VII and VIII.

EM indicated acrosomal vesiculation in late sper-

matids (Figure 7) and increased numbers of fused sperm in Stages IX and X compared to earlier time points. Cells with shared acrosomes were occasionally seen in devel- oping spermatids and epididymal sperm.

Extensive cytoplasmic debris of testicular origin was present throughout the epididymis on day 31. This

debris consisted primarily of eosinophilic globules, residual bodies, and ARB. There were also many mal-

DBAA testis histopa~ology 0 R. E. LENDER ET AL. 51

16

IX x XI XII XIII XIV I II Ill IV v VI VII VII1 EPID

stagra

Fig. 2. Principal histopathologic changes during days 2 to 3 1 in rats dosed with 250 mg DBAA/kg/d. The changes occurring in the various stages are illustrated by the shaded bars. The effects are descriptive only and do not reflect the degree of change. LR = luminal retention of Step 19 spermatids; BR = basal retention of Step 19 spermatids; ARB = atypical residual bodies; FS = fused sperm; DH = distorted late (Step 12 to 19) spermatid heads; M = maloriented late spermatids; V = Sertoli cell vacuoles; CP = caput epididynlidis~ CD = cauda epididymidis.

formed late spermatid nuclei embedded in globules of excess cytoplasm, suggesting faulty elimination of cytoplasm and/or premature sloughing. In two animals a mild increase in the number of round germ cells was observed.

Recovery, 250 mglkg. The rats dosed with 250 mg/ kg for 42 d were killed after a recovery period of 186 d. Most (7 out of 9) animals at this time had small testes and epididymides. Most the seminiferous tubules were char- acterized as Sertoli cells only, with the occasional presence of germ cells. A few tubules with spermatogenesis (estimated at less than 5% of total tubules in most animals) were present in all animals (Figure 8a). A number of tubules with spermatogenesis exhibited abnormalities including disorganization, degenerating spermatids, intraepithelial vacuoles, and symplasts of round spermatids (Figure 8b).

Day 31, 50 mg~kg. Animals dosed with 50 mg/kg were examined after 31 and 79 doses. After 31 doses, ARB at the periphery of the tubular lumen and moderate to substantial retention of Step 19 spermatids near the lumen were observed in Stage IX. Moderate numbers of condensed spermatid nuclei were present near the basement membrane in Stages X-XII. ARB were seen only rarely in Stage X and subsequent stages, but were present

in all six animals. ARB were also seen rarely throughout the epididymis in all DBAA-treated animals.

Day 79, 50 mglkg. The changes observed after 79 doses of 50 mg/kg were similar to those described after 31 doses. ARB were occasionally present in Stage IX and various other stages. The abnormal retention of Step 19 spermatids was more pronounced than on day 3 1 with animals exhibiting subst~tial retention near the lumen in Stages IX and X (Figure 9) and near the basement membrane in Stages X-XII.

Variable amounts of cytoplasmic debris were seen throughout the epididymis. Also, increased numbers of condensed spermatids embedded in excess cytoplasm were observed in all DBAA-treated animals.

Day 31,lO mgikg. Relative to control, three animals dosed with 10 mg/kg had moderately increased numbers of retained Step 19 spermatids at the lumen of Stage IX tubules. No changes were observed in the epididymis.

Day 79, 10 mgikg. After 79 doses, moderate retention of Step 19 spermatids at the lumen and near the basement membrane in Stages IX-XI was present in three animals. One animal had a number of disorganized and partially atrophic tubules, retained spermatids in Stages IX-XII, and many round germ cells throughout

Reproductive Toxicology Volume I I. Number I, 1997

Fig. 3. (A) Stage XI-XII tubule with several ARB in the lumen and within the epithelium (arrows) and smaller residual bodies (arrowheads) near the basement membrane. (B) Electron micrograph of retained fused Step 19 spermatid flagella within a common membrane (arrows). The flagella were separated by a thin rim of cytoplasm suggesting symplasts of mature spermatids. They were present as early as day 5 and were more prominent on day 3 1 (shown). They were observed only in Stages IX and X. x10.200.

the epididymis. Because tubular disorganization was not seen at 50 mg/kg, the changes in the latter animal were probably not treatment related.

Days 31 and 79, 2 nzglkg. No histopathologic changes in the testes or epididymides were apparent after 31 or 79 doses of 2 mg/kg.

DISCUSSION

A major manifestation of the action of DBAA on the testis was the malmorphogenesis of maturation-phase spermatids. Previous studies identified abnormal sperm morphology as one of the earliest and most sensitive responses to DBAA (1 l-13); fused sperm and abnormalities in the shape of the sperm head were noted in the caput epididymidis as early as day 5. Examination of the nuclei of epididymal sperm after staining with a fluoro-

Fig. 4. (A) Step 1.5 spermatids from a control rat. Note the prominent acrosomal dorsal fins (arrows) (bar = 20 pm). (B) Step 15 spermatids of rat on day 16 that received 250 mg/kg. The spermatid heads are misshapen and acrosomal material is scant and distorted (bar = 20 km).

chrome (Hoescht) indicated a somewhat flattened curva- ture in the nuclei of sperm from DBAA-treated rats com-

pared with controls (unpublished observation). In the present studies aberrations in the shape of the sperm head were recognized also in cross-sections of the testis. These changes were readily apparent in Stage I in which the prominent acrosomes of Step 15 spermatids appeared

misshapen and/or undeveloped. In particular, there appeared to be little acrosomal material at the caudal pole of the nucleus, which normally exhibits a prominent acrosomal crest. Thus, DBAA appears to affect both

nuclear and extranuclear constituents of the sperm head. Another effect of DBAA on late spermatids was the

apparent fusion of two or more sperm cells. These fused cells first appeared in the caput epididymidis on day 5 (13). In the present study, electron microscopy revealed multiple flagella within a common membrane in prepa- rations from the testis as early as day 5. These flagella appeared intact and usually appeared to be separated by a thin rim of cytoplasm suggesting that the multiple flagella shared only a common cytoplasmic matrix and

DBAA testis histopathology l R. E. LINDER ET AL. 53

Fig. 5. (A) After 31 doses of 250 mg/kg, vacuoles (arrows) in the Sertoli cell cytoplasm have formed. The vacuoles were seen in Stages XIV and I-VII, but were present predominantly in Stage I (bar = 160 pm). (B) Electron micrograph indicating prominent phagocytic vacuoles and resorption of fused sperm (arrows). xl 1,880.

plasma membrane. The fused cells were observed only in

abnormally retained Step 19 spermatids in Stages IX and X; however, their occurrence in earlier stages was not ruled out. The fused sperm observed in the testis in stages subsequent to Stage VIII (and in the epididymis) on day 5 indicate that the fusion of these particular cells could not have involved spermatids younger than Step 18. Step 18 (Stage VI) require about 5 d to mature and undergo spermiation (17,18). Therefore, the presence of fused sperm in Stage IX tubules and caput epididymidis on day 5 indicates that the possible cell types involved in the fusion process have to include, but are not necessarily restricted to, Step 18 and 19 spermatids. Kaido and co- workers (19) reported similar fusion products in rats treated with vitamin B,.

Cytochalasin D, an inhibitor of actin filaments, se- lectively causes certain intercellular bridges to open and form symplasts of round spermatids; interestingly, some syncytial cells accumulate excess cytoplasm at the ex- pense of other conjoined cells that become cytoplasm

Fig. 6. (A) Adjacent Stage IX and X tubules of rat that received 31 doses of 250 mg/kg. Essentially all tubules in these stages retained a full complement of Step 19 spermatids and residual bodies near the lumen. Note that in contrast to the early time points, there is little distortion or enlargement of residual bodies (bar = 50 km). (B) By Stage XI the Step 19 spermatids were overtly degenerating as evidenced by their presence also near the basement membrane (arrows).

poor (20). This suggests the possibility of an analogous response in DBAA-treated rats resulting in the fusion of mature spermatids and the formation of abnormally large lobes of residual cytoplasm, i.e., ARB, such as observed in the present study. However, some intercellular bridges narrow dramatically in late spermiogenesis just prior to sperm release (21); therefore, abnormal or delayed at- tenuation of bridges also could be a factor in the fusion of late spermatids. The fusion of sperm may indicate asynchrony in the processes controlling disengagement of sperm from the Sertoli cell.

The early histologic changes (days 5 to 16) included the formation of giant, atypical residual bodies (ARB). In an earlier study the incidence of ARB in the various stages of the seminiferous epithelium was determined in rats given 14 daily doses of 270 mg DBAA/kg; ARB were found in all stages except Stages VIII and IX (12). From the single time point examined in this short duration study it was not clear if the ARB were aberrant

Reproductive Toxicology Volume 1 I, Number 1. 1997

Fig. 7. (A) Stage I tubule of rat dosed with 250 mg/kg for 3 I d. Note the acrosomal vesiculation of Step 15 spermatids (arrows). x 1,100. (B) Late spermatid with acrosomal vesiculation (arrow). x12,800.

forms of the residual bodies normally occurring in Stages VIII and IX, or if they were structures similar to residual bodies that might have formed in various stages by abnormal loss of cytoplasmic organelles. The sequential observations in the present studies indicate that the ARB are forms of the residual bodies regularly seen in Stages VIII and IX. ARB were first noted in the testis on day 5; additional studies in rats dosed with 250 mg/kg revealed that ARB were formed by day 4, but were not yet present on day 3 (unpublished observation). In rats dosed with 50 mg/kg, the incidence of ARB appeared similar on days 31 and 79. In general, the timing and incidence of ARB and fused sperm coincided, further suggesting that ARB could be cytoplasmic organelles of fused sperm. How- ever, in rats dosed with 250 mg/kg, ARB were rare in the

Fig. 8. Testis from rat 186 d after the last of 42 daily doses of 250 mg DBAA/kg. (A) Preponderance of atrophic seminiferous tubules (asterisks). Spermatogenesis (stars) was present in only a few tubules and many of these tubules exhibited abnormalities (bar = 160 km). (B) A tubule with degenerate (chromatin margination) and multinucleate round spermatids (arrows).

testis on day 31 although fused sperm were still prominent. Thus, any direct relationship between these phe- nomena is confounded by this discrepancy.

Many ARB were either released into the tubular lumen or were degraded more slowly than residual bodies. Normally, residual bodies are degraded by the Sertoli cell in Stage IX (22); therefore, the ARB seen within the epithelium and free in the lumen of tubules in Stages X-XIV and I-VIII were apparently being phagocytosed more slowly than usual and/or were being transported through the seminiferous tubules to the excurrent duct system. Disposal of residual bodies probably involves both autolytic processes in the residual bodies and phagocytic and lysosomal activities of the Sertoli cell (22-24). Degenerating sperm and surplus apical Sertoli cell plasma membrane remaining after spermiation is also thought to be internalized by the Sertoli cell (25,26). Thus, the unresorbed ARB, residual bodies, and other cytoplasmic debris observed in the epididymides of DBAA-treated rats suggest impairment of the endocytic/ phagocytic mechanisms of the Sertoli cell.

DBAA testis histopathology l R. E. LINDER ET AL. 55

Fig. 9. Stage IX-X tubule from rat given 79 doses of 50 mg/kg. Extensive retention of Step 19 spermatids is seen near the lumen (arrow heads) and some residual bodies appear to be coalescing (arrows).

With the exception of ARB, the testicular changes in rats dosed with 2.50 mg/kg were more severe on day 3 1 than at the earlier time points. For example, on days 5 to 16 the retention of mature sperm beyond Stage VIII appeared rather variable. However, on day 3 1 the retention of Step 19 spermatids became extensive, and the re-

sponse was both stage-specific and uniform among animals. A full complement of Step 19 spermatids was retained near the lumen in essentially all Stage IX and X tubules. However, beginning in Stage XI, these Step 19 spermatids were overtly degenerating as evidenced by condensed nuclei being present near the basement membrane. Also on day 3 1, large intraepithelial vacuoles appeared in the Sertoli cell cytoplasm in Stages XIV and I-VII (predominantly in Stage I). These vacuoles are considered early evidence of Sertoli cell damage (27,28). Day 31 testes examined by EM revealed acrosomal vesiculation beginning in early spermatids. Similar acrosomal defects have been observed in rabbits after exposure to a mixture of drinking water contaminants (29).

After 42 doses of 250 mg/kg, extensive atrophy of seminiferous tubules was apparent in the animals examined after the 6-month posttreatment recovery period. Tubular disorganization, spermatid degeneration, and symplast formation were observed in the few tubules that were producing sperm suggesting permanent alterations in Sertoli cell function. It is unclear if the systemic toxicity associated with this test group contributed to testicular atrophy; however, less severe effects on spermiation and sperm morphology were also observed in lower dosage groups that did not exhibit signs of toxicity (13). In general, the reproductive effects of the 250 mg/kg dosage appear to be specific to the reproductive system.

Several effects described in rats dosed with 250 mg/kg were also recognized in animals dosed with 50

mg/kg, although the response was much less pronounced than at the higher dosage. ARB and retention of mature

spermatids were observed on day 31. Unlike the tran- siency at the higher dosage, the incidence of ARB did not diminish within the time frame of the study. The abnormal retention of Step 19 spermatids increased between days 3 1 and 79, possibly indicating a level of change that had stabilized between days 31 and 79, or was slowly intensifying. No unequivocal evidence of histopathologic changes was observed in rats dosed with 10 mg/kg although moderately increased retention of Step 19 spermatids was observed in three rats on each of days 3 1 and

79, whereas, only 1 of 40 control rats were judged to have a similar degree of retention of Step 19 spermatids. In an earlier study, semiquantitative counts of retained spermatids suggested also that some animals may be mildly affected at a dosage of 10 mg/kg (12). No histopathologic changes were noted in rats dosed with 2

mghg. In the absence of evidence of direct killing of sper-

matogenic cells, the various responses described in these studies suggest that DBAA adversely affects the struc- ture and/or function of the Sertoli cell. Indeed, most or all of the testicular changes described could be effected by alterations in the Sertoli cell cytoskeleton. Actin, in- termediate filaments, microtubules, and various associated proteins provide numerous structural, positioning, and transport functions attributed to the Sertoli cell (30,31). The cytoskeletal elements participate in sperm shaping, maintenance of intercellular bridges, germ cell- Sertoli cell attachments, spermiation, and sperm release, transport of residual bodies, and endocytosis, all of which appear to be targets of DBAA.

Our own unpublished observations and studies reported by Toth and colleagues (10) suggest that the DBAA analogue, dichloroacetic acid, attacks the same testicular targets as DBAA. These analogues as well as several other haloacids probably occur simultaneously in water supplies (4,8). Although the dosages of DBAA used in the present study are considerably higher than those likely to occur in humans, the possible intermixture of several disinfection by-products suggests that male reproductive toxicity of these chemical could be additive. Studies of the mixed halogen acetates and the various disubstituted brominated and chlorinated propanoates and butanoates may be particularly useful. Possible cel- lular markers of common effects include ARB and fused sperm. The current study suggests that experiments di- rected at elucidating cytoskeletal and biochemical targets in the Sertoli cells are critical to understanding the un- derlying mechanisms responsible for the testicular toxicity of the halogenated acids. Considering that a number of related by-products may coexist in drinking water supplies, the identification of subcellular markers indicative

56 Reproductive Toxicology

of similar mechanisms of action would expedite the char- acterization of the toxic potential of these chemicals.

~i.~~~~~~ze~ - This article has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or rec- ommendation for use.

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Histopathologic changes in the testes of rats exposed to dibromoacetic acid

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