Adverse male reproductive effects following subchronic exposure of rats to sodium dichloroacetate

FUNDAMENTAL

AND APPLIED

TOXICOLOGY

19, 57-63 (1992)

Adverse Male Reproductive Effects following Subchronic of Rats to Sodium Dichloroacetate

Exposure

G. P. TOTH,* K. C. KELTY,* E. L. GEORGE,* E. J. READ,? AND M. K. SMITH* *Reproductive and Developmental Biochemistry Branch, Developmental To.xicology Division, Health Efects Research Laboratory. U.S. Environmental Protection Agency: Cincinnati, Ohio 45268; and TComputer Sciences Corporation, Cincinnati, Ohio 45268 Received June 14, 1991; accepted January 7. 1992

acid (TCA), and chloral hydrate. Christman et al. (1983) reported that (in order of decreasing yield) TCA, chloroform, DCA, and dichlorosuccinic acid accounted for approximately 50% of the total organic halogen produced in the chlorination of humic acids. Formation of chlorinated reaction products (e.g., DCA) has also been demonstrated in vivo (Mink et al., 1983). DCA and TCA have been detected in tap water samples from two water treatment plants in the concentration range of 30 to 60 pg/liter (Uden and Miller, 1983). This concentration range was comparable to that of chloroform in drinking water. The present study examines the male reproductive toxicity of dichloroacetate. Katz et al. ( 198 1) previously reported, in addition to neurotoxic effects, degeneration of the seminiferous epithelium in rats and dogs dosed orally with 500 and 2000 mg/kg NaDCA for 3 months. However, Yount et al. ( 1982) reported that, in weanling rats which were orally dosed (300 to 500 mg DCA/kg/day in feed), dichloroacetate decreased growth rate and food consumption and caused neurotoxic effects. Testicular histology was normal. Stacpoole et al. (1990) reported that DCA in drinking water at doses of 50 mg and 1.1 g/kg/day for 7 weeks produced no testicular histopathology nor any reduction in testicular spermatid head counts in rats. On the basis of the results of pilot studies (IO-week oral gavage), we have studied the effects of a IO-week oral exposure to NaDCA at doses considerably lower than those previously reported to cause adverse male reproductive effects. The current study examined an extensive battery of male reproductive toxicity tests, including epididymal sperm motion analysis and histopathologic evaluation of the testis and epididymis.

Adverse Male Reproductive Effects following Subchronic Exposure of Rats to Sodium Dichloroacetate. TOTH, G. P., KELTY, K. C., GEORGE, E. L., READ, E. J., AND SMITH, M. K. (1992). Fundam. Appl. Toxicol. 19, 57-63. Dichloroacetate (DCA) activates the pyruvate dehydrogenase complex enhancing carbohydrate and lactate utilization in animals. As a result it is used clinically in the treatment of acute lactic acidosis and has therapeutic potential in the treatment of stroke. Adverse effects of chronic DCA treatment include polyneuropathy and testicular degeneration. Since DCA is a principal product of the aqueous chlorination of fulvic acids concern has arisen regarding the agent’s impact on environmental health. We treated male Long-Evans rats with 0, 31.25, 62.5, or 125 mg DCA/kg/day by oral gavage for 10 weeks. Compared to controls, preputial gland and epididymis weights were reduced at 3 1.25 mg/kg, body and liver weights at 62.5 mg/kg, and accessory organ weights at 125 mg/kg. Epididymal sperm counts were reduced and sperm morphology was impacted at the 62.5 and 125 mg/kg doses levels. Histologic examination of the testis and epididymis revealed inhibited spermiation in testes at the 125 mg/kg dose level. Computer-assisted sperm motion analysis revealed reductions in percentage motile sperm, curvilinear and straight-line velocity, linearity, and amplitude of lateral head displacement at both the 62.5 and the 125 mg/kg dose levels. In the assessment of fertility after an overnight mating, the number of viable implants on Day 14 of gestation was decreased only in the highest dose group. These studies demonstrate adverse effects of NaDCA treatment on the rat male reproductive system, primarily on the accessoryorgans and sperm within them at lower doses (31.25 and 62.5 mg/kg), and on the testis at the highest dose (125 mg/kg). Q 1992 Society of Toxicology.

In the assessment of drinking water disinfection practices the toxicologic study of the chloroacetic acids has been a priority issue. Drinking water disinfectants (chlorine) and naturally occurring humic substances in the source water are the most probable precursors to the chloroacetic acids found in treated water (Fowle and Kopfler, 1986). The principal chlorinated products identified by Miller and Uden (1983) among chlorination by-products of reactions with humic acid were chloroform, dichloroacetic acid (DCA), trichloroacetic

MATERIALS

AND METHODS

Animal maintenance and dosing protocol. Long-Evans hooded rats (males, 100 days old: females, 70 days old) were received from Charles River Breeding Laboratories (Raleigh, NC). Upon arrival. the animals were fed Purina Lab Chow 5001 and filter-purified tap water ad libitum. Animal rooms were maintained at approximately 25°C and 55% humidity, with a 12: 12 light-dark cycle, light commencing at 0600 hours. Males were housed singly in plastic shoebox cagesfor at least 2 weeks before beginning treatment 57

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TOTH ET AL.

and females were housed two to a cage before and after the mating protocol of the study. Dichloroacetic acid (Sigma Chemical Co., St. Louis, MO) was neutralized with NaOH to pH 7.0, diluted with deionized, distilled water, and administered by gavage for 10 consecutive weeks (up to experimental Day 70) at doses of 3 1.25,62.5, and 125 mg sodium dichloroacetate (NaDCA)/kg/day. Control animals received 10 ml distilled water per kilogram body weight. Animals were killed by CO2 asphyxiation. Pilot studies had been previously conducted for 10 weeks at dosages of 31.25, 62.5, 125, 500, and 1000 mg NaDCA/kg. Male rats gavaged with 500 or 1000 mg NaDCA/kg had become paralyzed and died after 4 to 6 weeks of dosing. Analysis of male reproductive endpoints from the remaining dose groups in this pilot study was confounded by the discovery of a high percentage of bilateral testicular degeneration in the control group and in the 3 1.25, 62.5, and 125 mg NaDCA/kg dose groups. Since testicular weights in the highest viable dose group (125 mg NaDCA/kg) were so clearly dichotomized, with the higher weight group in the range of historical controls, inclusion of NaDCA-dosed animals for preliminary data analysis was based on the 80% confidence interval for testicular weights from “normal” control animals (1.36 to 1.7 1 g). Fifty-three percent of the controls and 54% of the treated animals were included in this interval (number of animals: 31.25 mg/kg, 10; 62.5 mg/kg, 12; 125 mg/kg, 12). Since technical problems precluded our evaluation of testicular spermatid head count in the present study, the following pilot study results are given: [in units of 106/g testis (*SD)] control, 100.4 (15.2); 31.25 mg/kg, 112.3 (14.4); 62.5 mg/kg, 113.7 (18.0); 125 mg/kg, 100.1 (39.2). Mule reproductive toxicology. On experimental Day 70, fertility was assessedby allowing each treated mate to mate overnight with one untreated, proestrus female. All mates received three overnight mating experiences with ovariectomized, hormonally primed females prior to mating trials (Weeks 6,7, and 9). Vaginal lavages were performed on the females the next morning to confirm the occurrence of mating. Mates that failed to mate were allowed another opportunity with another proestrus female the following night. Sperm positive females were allowed to advance to Day 14 of gestation (vaginal lavage = Day 0). Pregnant females were then examined for the number of live and dead implants, resorbed fetuses, and corpora lutea. Fertility was scored as “percentage implantation” [i.e., (implants/corpora lutea) X 100 = % implantation]. Males were terminated on experimental Day 75. At least a 2-day lapse following mating allowed replenishment of epididymal reserves that may have been depleted in the mating trial. At termination the following organs were excised and weighed: liver, kidney. spleen, testes, accessory organs (prostate and seminal vesicles), preputial glands, and epididymis. One testis and cauda epididymis were each homogenized in 0.9% NaCl, 0.0 1% Triton X-100 solution to obtain sperm head counts (Blazak et al., 1985). Sperm motion parameters were measured with the CellSoft (Cry0 Resources, Ltd., New York) computer-assisted sperm motion analysis system. For this analysis the contralateral cauda epididymis was nicked and subsequently incubated for 3 min at 37°C in 10 ml Dulbecco’s phosphate-buffered saline (+Ca*+ and Mg*+). pH 7.2, plus 10 mg/ml bovine serum albumin (Sigma, Fraction V). An aliquot of the mixture was then diluted IO- to 20-fold and 10 ~1 was placed on a Petroff-Hausser chamber (Hausser Scientific, Philadelphia, PA: 20 pm depth). Sperm motion, as viewed on an Olympus BH-2 microscope (12.5 power, pseudo dark field optics) equipped with a “Fryer” (Fryer Co. Inc., Carpentersville, IL) stage warmer (37°C) was videotaped and analyzed using the CellSoft instrument. The CellSoft system settings were as follows: frames analyzed = 15; framing rate = 30 frames/set; maximum velocity = 1100 pm/set; threshold velocity = 20 rm/sec; minimum sampling for motility = 2 frames; minimum sampling for velocity = 3 frames; minimum sampling for straight-line velocity, linearity, amplitude of lateral head displacement (ALH), and beat/cross frequency = 11 frames (determined with an auxiliary computer program); minimum linearity for ALH = 3.5; pixel scale = 3.40 pm/pixel; at least 10 fields and 200 cells analyzed per sample; maximum average number of cells/field = 40; cell size range = 20-200 pixels (Toth et al.. 1989).

TABLE 1 Body and Organ Weights following Treatment with NaDCA Dose(w/kg PO)

N

Final body weight’ (g) Weight gain (g) Right testis (g) /IO0 g BWb Right epididymis (g) /lOOgBW Right cauda epididymis (g) /lOO g BW Right preputial gland (g) /IO0 g BW Accessory organs (g) 1100 g BW Right kidney (g) /lOO g BW Liver (g) /lOO g BW Spleen (9) /lOOgBW

0

31.25

62.5

125

19

18

18

19

512.8 (35.8) t83.2 (28.4) 1.539 (0.189) 0.302 (0.048) 0.574 (0.060) 0.113 (0.017) 0.212 (0.026) 0.042 (0.007) 0.160 (0.028) 0.03 1 (0.005) 3.828 (0.572) 0.752 (0.132) 1.876 (0.137) 0.366 (0.020) 20.46 (1.26) 4.00 (0.33) 0.817 (0.175) 0.159 (0.030)

492.6 479.7” (26.8) (36.8) +64.4” +55.7” (19.4) (25.6) 1.550 I .475 (0.180) (0.248) 0.316 0.309 (0.040) (0.059) 0.529” 0.483” (0.044) (0.056) 0.108 0.101” (0.009) (0.016) 0.210 0.193” (0.024) (0.025) 0.043 0.041 (0.005) (0.007) 0.142O 0.120” (0.030) (0.023) 0.029 0.025” (0.006) (0.004) 3.829 3.769 (0.697) (0.452) 0.775 0.788 (0.119) (0.095) 1.883 1.897 (0.200) (0.205) 0.382 0.396” (0.029) (0.036) 21.44 22.86” (2.11) (2.80) 4.35” 4.76” (0.31) (0.40) 0.861 0.883 (0.114) (0.153) 0.175 0.184” (0.022) (0.027)

449.8” (28.9) +22.1a (23.2) 1.534 (0.131) 0.343” (0.042) 0.457” (0.045) 0.102” (0.015) 0.176” (0.024) 0.039 (0.007) 0.099” (0.020) 0.022” (0.004) 3.357” (0.376) 0.749 (0.098) 1.917 (0.127) 0.427” (0.035) 23.33” (2.22) 5.19” (0.43) 0.793 (0.095) 0.177” (0.024)

u Significantly different from control (p G 0.05). b BW, body weight. c Weights are expressed as mean + SD.

One testis was prepared for histological examination by immersion in 10% neutral-buffered formalin for 24 hr. Following preparation of testis cross sections, fixation was done in 5% glutaraldehyde. Sections were stained with hematoxylin and periodic acid/SchitTs reagent and subsequently embedded in glycol methacrylate (Chapin et al., 1985). Epididymides from additional study males (four control and four high dose animals) were also prepared for histological examination by perfusion fixation, staining with toluidine blue, and embedding in epoxy (Russell, 1983). For the evaluation of sperm morphology, air-dried slides of sperm from the vas deferens were prepared and stained with the triple stain of Bryan (1970). Statistical analysis. Animal weights, organ weight, sperm motion endpoints, testicular spermatid head counts, and cauda epididymal sperm head counts were analyzed by analysis of variance with pairwise contrasts (two-

MALE

REPRODUCTIVE

EFFECTS TO SODIUM

TABLE 3 Sperm Morphology following NADCA Treatment

TABLE 2 Epididymal Sperm Counts following NADCA Treatment Dose (w/kg

11: Cauda Epididymal Sperm Countsb ( I 06/g cauda)

59

DICHLOROACETATE

PO)

Dose Gw/k

1

31.25

62.5

125

19

18

18

19

630.3 (204.8)

582.5 (137.0)

502.6” (163.5)

367.8” (91.6)

Percentage abnormal intact sperm ’ Significantly different from control (p G 0.05). h Counts are expressed as means + SD.

0

31.25

62.5

125

20

19

17

19

85.1 (19.2) 7.6

86.7 (16.9) 5.8

80.4b (14.1) 6.1 (5.1) 12.5” (10.9) 1.0 (1.1)

58.9” (16.2) 8.4 (5.0) 28.7” (16.4) 4.1” (3.4)

N Percentage normal intact sperm’

Percentage abnormal loose heads

RESULTS

(6.4)

(8.6) Percentage normal loose heads

sided) to compare the individual dose groups to the control group (Winer, 197 1). Percentages of motile sperm and percentages of circularly swimming sperm were transformed with the arcsine transformation before analysis. Percentages of individual sperm morphology groups were analyzed by the rank-based Wilcoxon test for pairwise differences (Lehmann. 1975). The Wilcoxon test was also used for analysis of pregnancy success(implantation rate) and the number of implants per pregnant female.

PO)

5.3

6.2

(6.9)

(6.9)

2.0 (5.6)

1.3 (4.1)

a Significantly different from control (p G 0.05). b Significantly different from control (0.05 i p G 0.10). ’ Percentages are means + SD.

sperm heads rather than an increase in intact abnormal sperm forms.

Animal and Organ Weights

Sperm Motion Analysis

Reduced final animal weights relative to the controls were observed after treatment with 62.5 and 125 mg NaDCA/kg and slower weight gain was seen for all NaDCA-treated males (Table 1). The dose groups started the study at comparable weights (data not shown). At 3 1.25 mg NaDCA and higher, relative liver weights were increased, while relative kidney and spleen weights and absolute liver weight were increased at 62.5 and 125 mg NaDCA. Of the male reproductive organs measured, only the testis did not show reductions in absolute weight (Table 1). Reductions in absolute weights of the epididymis and preputial glands were observed at all NaDCA dose levels while the accessory organs (prostate and seminal vesicles) were reduced at 125 mg NaDCA. Reductions in the relative weights of the epididymis and preputial glands resulted from NaDCA treatment at 62.5 mg NaDCA and higher, while relative cauda epididymis and accessory organ weights were unchanged. Relative testis weights were increased at 125 mg NaDCA.

The percentage of motile sperm as well as four descriptive sperm motion endpoints (curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement) were reduced at 62.5 and 125 mg NaDCA (Table 4). The percentage of circularly swimming sperm and the beat/ cross frequency were unchanged.

Cauda Epididymal Sperm Head Counts; Sperm Morphology Table 2 shows that counts of cauda epididymal sperm heads were reduced at 62.5 and 125 mg NaDCA/kg. In the evaluation of sperm morphology (Table 3), NaDCA treatment resulted in a reduction of the percentage of normal, intact sperm [borderline significantly different at 62.5 mg NaDCA (a = 0.06)]. However, this reduction reflected an increase in the percentage of normally shaped detached

TABLE 4 Sperm Motion Analysis following NADCA Treatment Dose (w/kg

N Percentage motile spermb Curvilinear velocity (pm/set)

0

31.25

62.5

125

15

14

17

19

54.6 (10.2) 139.5

54.1 (I 1.2) 143.2 (7.9) 56.3

39.5” (12.0) 124.9” (13.4) 42.8” (9.7) 3.55” (0.45) 5.62” (0.78) 10.09 (0.98) 19.2 (6.3)

27.1” (9.8) 110.7* (10.0) 32.0“ (7.2) 3.04” (0.47) 4.68” (0.77) 10.38 (1.08) 17.8 (5.8)

(6.3 Straight-line velocity (qm/sec) Linearity Amplitude of lateral head displacement (pm) Beat/cross frequency (Hz) Percentage circular cells

PO)

57.2 (5.4) 4.15 (0.32) 6.69 (0.48) 10.64 (0.47) 21.1 (3.3)

(6.2) 3.94 (0.42) 6.96 (0.55) 10.10 (0.66) 23.3 (5.5)

’ Significantly different from control (p < 0.05). b Motion endpoints are means + SD.

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TOTH ET AL.

f. 1. Photomicrographs of stage 10 seminiferous tubules from (a) testis from control animal and (b. c) testis from an animal treated with I NaDCA/kg. (Hematoxyl~n; periodic acid-Schiq 100X).

Histological

Evaluation

No gross lesions (degenerated or atrophic seminiferous tubules, Leydig cell hyperplasia or hypoplasia) were evident in the cross sections of testes excised from males treated with 125 mg NaDCA/kg. However, as represented in Fig.-1 b, there was evidence of retention of the late-step elongated spermatids beyond stage 8. Of the 9 high dose animals whose testes were examined histologically, all showed a predominance of retention of step 19 spermatids into stage 10 seminiferous tubules. At 62.5 mg NaDCA, 4 of 10 rats showed retention into stage 9 seminiferous tubules. Retention of latestep spermatids was not evident in the controls or in the 31.25-mg NaDCA dose group. Evidence of mature sperm heads drawn down toward the basal compartment in stage 10 tubules can be seen in Fig. lc. Examination of the epididymal epithelium revealed normal cellular structures. Fertility Assessment NaDCA treatment did not result in a statistically significant reduction in the pregnancy rate (Table 5). A reduction in the number of live implants per dam was observed at 125

mg

mg NaDCA. However, none of the three NaDCA groups was found to be significantly different from the controls for implantation rate. DISCUSSION The results provided from the organ weight measurements alone showed evidence of an apparent adverse effect of sodium dichloroacetate treatment on the male reproductive accessory organs, including the epididymis. Sperm isolated from the cauda epididymis of treated males showed reductions in percentages of motile sperm, indicators of sperm vigor and pattern (velocity, linearity, ALH), and sperm head counts. Sperm isolated from the vas deferens of treated males had increased numbers of detached sperm heads. From the basic indicators of testicular integrity, organ weight and testicular late-step spermatid head count, this organ appeared to have been spared any adverse effects at the doses used in this study. It seemed unusual that epididymal sperm counts were reduced without the concurrent reduction in testicular late-step spermatid head counts. However, examination of the seminiferous tubule cross sections from the histological

MALE

REPRODUCTIVE

EFFECTS TO SODIUM

DICHLOROACETATE

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TOTH ET AL.

TABLE 5 Fertility Assessment following NADCA Treatment

causes rapid and substantial atrophy of the seminal vesicles and the prostate and causes inhibition of sebaceous gland function [e.g., preputial glands (Neumann et al., 1980)]. AnDose (w/kg PO) tiandrogens of the cyproterone acetate type lead to androgen deprivation for dependent tissues (Neumann and Toper, 0 31.25 62.5 125 1986). They also have antigonadotrophic activities which 15 14 17 18 Iv” lead to inhibition of testicular testosterone biosynthesis and testicular degeneration. Nonsteroidal pure antiandrogens, Number pregnant 1s/20 17120 18/19 14/19 13.9 13.4 14.3 8.9* Implants per damc.d such as flutamide, have been shown to interfere with andro(4.1) (4.2) (1.8) (6.6) genie negative feedback in neural centers, leading to a Leydig Implantation rateC 85.3 81.3 92.0 61.9 cell hyperplasia. No effects from either type of antiandrogen (26.8) (26. I) (13.0) (43.0) were evident in our histological sections. An assay of hora Number of samples for which both motion and fertility data existed. mone levels following NaDCA treatment would be infor‘Significantly different from control (p G 0.05). mative in this context. ’ Figures are based on animals for which both motion and fertility existed. No clear link is evident between dichloroacetate’s multiple d Endpoints are means + SD. effects on pathways of intermediary metabolism and the reported male reproductive effects. The activation of the pyruvate dehydrogenase complex by DCA (Stacpoole, 1989) preparations revealed that elongated spermatids were being has been proposed to play a role in the agent’s pharmacologic retained beyond the stage (8) at which spermiation is known effects. DCA has been suggested to enhance regional lactate to occur (retention evident in stages 9 and 10). The appear- removal in experimental states of cerebral ischemia (Katayance of spermatid heads located toward the basal compartama and Welsh, 1989). Considering the dependence of the ment in stage 10 tubules suggested phagocytosis of these re- developing sperm cells in the testis on lactate (Grootegoed tained cells. The evidence of impaired spermiation and pos- et al., 1984) removal of this substance could lead to an imsible phagocytosis of late-step spermatids allows the balance in the energy metabolism of the testis. cr-Chlorohypossibility that late-step spermatids were retained and sub- drin (Ford and Harrison, 1987), 2,5-hexanedione (Chapin sequently lost without being transferred to the epididymis. et al., 1983) and ethoxyethanol (Oudiz and Zenick, 1986) Technical problems precluded our measurement of tes- are male reproductive toxicants which act via interference ticular late-step spermatid head counts in the primary portion with the energy metabolism of sperm cells or testicular Sertoli of this study and necessitated our inclusion of results from cells. Clear similarities between adverse effects of these agents the pilot study (see Materials and Methods). Interpretation and DCA were not evident. Given the increases in relative of these results in conjunction with those of the primary kidney, spleen, and liver weights in the same dose range as study could be questioned. However, as shown by Stacpoole the reproductive effects, we were not able to eliminate syset al. ( 1990), at higher doses of NaDCA ( 1.1 g/kg/day) no reductions in testicular late-step spermatid head count were temic toxicity as a confounding influence in these studies. 2-Chloropropionate and trichloroacetic acid are two other evident. The retention of spermatids discussed above could halogenated acetic acids that have been shown to activate have masked a reduction of spermatids in the testis. Interpretation of the fertility results was made difficult due pyruvate dehydrogenase (Crabb et al., 198 1). 2-Chloroproto the insensitivity of the overnight mating trials which allow pionate caused testicular abnormalities manifested by testicular maturation arrest and degeneration of germ cells multiple mating opportunities. Restricted mating protocols (Clegg et al.. 1989) may allow for clearer relationships to be (Yount et al., 1982). This agent is not metabolized to glyoxylate and oxalate, as is DCA. Since both DCA and 2-chlodrawn between the myriad NaDCA-induced effects reported ropropionate activate pyruvate dehydrogenase and produce here and fertility. Taken together, a polytypic male reproductive toxicity has male reproductive lesions, it could be suggested that glyoxresulted from sodium dichloroacetate exposure. Since an- ylate and oxalate do not underly DCA’s reproductive toxicity. drogens are required for the development and function of Clearly, research into the structure/activity relationships of the male reproductive tract and the accessory sex organs the chloroacetic acids relative to their male reproductive (Eddy, 1988) and since NaDCA treatment resulted in sig- toxicities and their effects on intermediary metabolism is in nificant adverse effects on the accessory sex organs, a hor- order. A pilot study in our laboratory with monochloroacetic monal effect could underly the toxicity of NaDCA. The re- acid (10 weeks, 60 mg/kg/day) revealed no significant reduction in sperm motility following NaDCA treatment is ductions in reproductive organ weights, cauda epididymal consistent with this hypothesis since maturation of sperm in sperm counts, sperm motility, or sperm motion parameters. the epididymis (i.e., acquisition of motility and fertility) is We are currently studying the effects of trichloroacetic acid dependent upon testosterone. Treatment with antiandrogens ( 10 weeks, 200 and 1000 mg TCA/kg/day, oral gavage).

MALE

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EFFECTS TO SODIUM

The polyneuropathy brought about by DCA administration has been shown to be associated with signs typical of thiamine deficiency, increased oxalate excretion and decreased red cell transketolase activity (Stacpoole et al., 1990). Thiamine replacement ameliorated these changes and provided a beneficial action on the peripheral neuropathies. While thiamine deficiencies have historically been known to lead to neurological changes, detailed male reproductive studies are lacking. Before thiamine replacement can be considered as “a nossible safe and effective means of minimizing toxicity while preserving the efficacy of DCA in the chronic treatment of various metabolic disorders in man” (Stacpoole et al., 1990), considerable research needs to be conducted on the mechanism of DCA’s male reproductive effects and the maximum tolerated dose before onset of adverse reproductive effects. Previously, Stacpoole et al. (1990) reported no adverse effects of NaDCA treatment at the 50 mg/kg/day level for a subchronic (7 week) exposure. The present work found a “Lowest Observed Adverse Effect Level (LOAEL)” at 3 1.25 mg/kg. Although the reductions in preputial gland and epididymis weights were the only adverse effects observed at this dose level, the discussion above on the accessory organs as a targets for antiandrogens supports the inclusion of this observation. A “No Observed Effect Level” was not found in this study. ACKNOWLEDGMENTS

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Crabb, D. W., Yount, E. A., and Harris, R. A. (198 1). The metabolic effects of dichloroacetate. Metabolism 30, 1024- 1039. Eddy, E. M. (1988). Duct system and accessory glands of the male reproductive tract. In Physiology and Toxicology of Male Reproduction (J. C. Lamb and P. M. D. Foster, Eds.), pp. 35-69. Academic Press, San Diego. Ford, W. C. L., and Harrison, A. (1987). Futile substrate cycles in the glycolytic pathway of boar and rat spermatozoa and the effect of cr-chlorohydrin. J. Reprod. Fertil. 79, 2 1-32. Fowle, J. R., and Kopfler, F. C. (1986). Water disinfection: Microbes versus molecules-An introduction of issues.Environ. Health Perspect. 69, 3-6. Grootegoed, J. A., Jansen, R., and van der Molen, H. J. (1984). The role of glucose, pyruvate, and lactate in ATP production by rat spermatocytes and spermatids. Biochem. Biophys. Acta 767,248-256. Katayama, Y., and Welsh, F. A. (1989). Effect of dichloroacetate on regional energy metabolites and pyruvate dehydrogenase activity during ischemia and reperfusion in gerbil brain. J. Neurochem. 52, I8 17- 1822. Katz, R., Tai, C. N., Diener, R. M., McConnell, R. F.. and Semonick, D. E. (198 1). Dichloroacetate, sodium: 3-Month oral toxicity studies in rats and dogs. Toxicol. Appl. Pharmacol. 57, 273-287. Miller, J. W., and Uden, P. C. (1983). Characterization of nonvolatile aqueous chlorination products of humic substances. Environ. Sci. Technol. 17, 150-157. Mink, F. L., Coleman, W. E., Munch, J. W., Kaylor, W. H., and Ringhand, H. P. ( 1983). In vivo formation of halogenated reaction products following peroral sodium hypochlorite. Bull. Environ. Contam. Toxicol. 30, 394-399. Neumann, F., Schleusener, A., and Albring, M. (1980). Pharmacology of Antiandrogens. In Androgenization in Women-Acne, Seborrhoe, Androgenetic Alopecia and Hirsutism. (J. Hammerstein, U. Lachnit-Fixon, F. Neumann, and G. Plewig, Eds.), pp. 147-192. Excerpta Medica, Amsterdam. Neumann, F., and Toper, M. (1986). Pharmacology of antiandrogens. J. Steroid

The authors thank Jeffrey Rennecker, and Patricia Horton for their technical assistance. This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Approval does not signify that the contents necessarily reflect the view or policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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