TOXICOLOGY AND APPLIED PHARMACOLOGY 107, I64- I72 ( 199 1)

Comparative Effects of Disulfiram and N-Methyltetrazolethiol on Spermatogenic Development in Young CD Rats

D. M. HOOVER,] J. A. HOYT, D. E. SEYLER, D. L. ABBOTT,

W. P. HOFFMAN, AND M. K. BUENING

Toxicology Division, Li1i.v Research Laboratories, A Division of Eli Lilly and Company, Greenfre[d, Indiana 46140

Received June I. 1990; accepted September 24, 1990

Comparative Effects of Disulfiram and N-Methyltetmzolethiol on Spermatogenic Development in Young CD Rats. HOOVER, D. M., HOYT, J. A., SEYLER, D. E., ABBOTT, D. L., HOFFMAN,

W. P., AND BUENING, M. K. (1991). Toxicol. Appl. Pharmacol. 107, 164-112. N-Methyltetra- zolethiol (NMTT) and NMTT-containing cephalosporin antibiotics cause characteristic testicular lesions in young but not adult rats. In addition, NMTT-containing cephalosporins inhibit aldehyde dehydrogenase and have been associated with a disulfiram-like reaction in humans and animals. Therefore, the potential testicular toxicity of disulfiram (IO, 30, or 100 mg/kg) was evaluated in 37-day-old rats given oral doses on Postpartum Days 6 through 36, and was compared to the toxicity induced by NMTT (100 mg/kg). NMTT and each dose of disulfiram caused a decrease in testes weight. By DNA flow cytometry. testicular cell suspensions from rats given 100 mg/kg of NMTT had a 40% reduction in spermatids while those from rats given 10. 30, or 100 mg/kg of disulfiram had reductions of 52, 61, or 89%, respectively. Microscopically, the testes of rats given either NMTT or disulfiram had qualitatively similar changes, characterized by delayed maturity of the leading waves of germinal cells which had reached early maturation phase in control animals. Moderate to severe reduction occurred in the total number of spermatids with complete absence of acrosome phase and maturation phase spermatids. There was also a prominent reduction in the number of spermatocytes. Reduction in number of spermatogonia was minimal. While the mechanism of toxicity is not known for either compound, it is possible that the toxicity was related to the enzyme-inhibitory effects which both compounds possess. By defining the mechanism of testicular toxicity for compounds which cause a NMTT-like testicular toxicity in rats, biological differences in the spermatogenic process between the young and adult rat may be further understood. Direct extrapolation of the testicular effects in neonatal rats to man is not possible because of the substantial differences in initiation of spermatogenesis between rodents and humans. 0 1991 Academic press. IK.

Both N-methyltetrazolethiol (NMTT; Fig. 1) and NMTT-containing cephalosporin anti- biotics cause testicular toxicity in neonatal rats (Comereski et al., 1987; Manson et al., 1987; Kurebe et al., 1986; Stahlmann and Chahoud, 1986; Hoover et al., 1989; Moe et al., 1989). Rats treated for 1 month begin- ning on Postpartum Day 6 have a charac- teristic delayed maturity of the germinal ep-

’ To whom correspondence should be addressed.

ithelium, with absence of the most mature waves of germinal cells. In contrast, NMTT alone and NMTT-containing cephalosporins have no effect on spermatogenesis in adult rats. The sensitivity of the young rat to these compounds has not been explained by pos- sible age-related differences in metabolism or compound distribution. Therefore, the mechanism of testicular toxicity may relate to the differences between spermatogenesis as it occurs in the adult and as it occurs dur-

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COMPARATIVE TESTICULAR TOXICITY OF DISULFIRAM AND NMTT 165

ing the initial phases of spermatogenesis in the neonate.

Spermatogenesis in the young rat begins with a burst of accelerated development; the first type A spermatogonia develop into lep- totene primary spermatocytes in less than half the time observed in the adult (Huckins, 1965). For the NMTT-containing cephalosporin, ce- famandole, it was reported that the testicular effects are related to phases of accelerated de- velopment in the first developing waves of germinal cells (Hoover et al., 1989). Perhaps this accelerated development increases the sensitivity of critical enzymatic processes to inhibition by NMTT. Alternatively, initiation of spermatogenesis could involve a unique process that is susceptible to inhibition by NMTT.

NMTT-containing cephalosporins inhibit aldehyde dehydrogenase and have produced disulfiram-like reactions in humans and ani- mals (Buening and Wold, 1982; Lipsky, 1986). Symptoms typical of the disulfiram-like re- action are flushing, headache, nausea, vom- iting, tachycardia, and hypotension. Inhibition of aldehyde dehydrogenase results in increased concentrations of acetaldehyde, and the in- crease in acetaldehyde is generally believed to be the cause of the adverse reactions experi- enced by persons taking disulfiram (Fig. 2) followed by alcohol consumption (Per-man, 1962; Truitt and Walsh, 197 1). Aldehyde de- hydrogenase is present in the testes of young (20 days postpartum) and adult animals (Messiha, 1980) but its possible role in sper- matogenesis remains undefined.

HS

I CH,

FIG. 1. Chemical structure of N-methyltetrazolethiol.

FIG. 2. Chemical structure of disulftram.

Before this report, the characteristic delayed maturity of the testicular germinal epithelium of neonatal rats has been associated only with exposure to NMTT or NMTT-containing cephalosporins. The present study demon- strates a comparable testicular toxicity for di- sulfiram, a compound structurally distinct from NMTT but one with a similar inhibitory effect on at least one enzyme, aldehyde de- hydrogenase.

METHODS

Chemicals. NMTT (297% purity) was purchased from Sumitomo Corp. (Japan) and disulfiram was purchased from Sigma Chemical Co. (St. Louis, MO). Aqueous sus- pensions of disulfiram were prepared with 10% acacia. NMTT was dissolved in 0.9% sodium chloride solution, and sodium hydroxide was used to adjust the pH to ap- proximately 6. The concentration of each dose solution was adjusted to give each dose in a volume of 5 ml/kg body wt.

Animals. Adult CD rats were obtained from Charles River Breeding Laboratories (Portage, MI). After mating, the females were housed in polycarbonate shoebox cages suspended on a stainless-steel rack with an automatic wa- tering system. The cages contained aspen bedding chips and the animals were allowed free access to Purina Certified Rodent Chow No. 5002. The females were housed indi- vidually and maintained under conditions of constant temperature (22.2 f 1.7”C) and lighting (0600 to 1800 hr light cycle). Females were allowed to deliver naturally. On Postpartum Day 1, 10 litters were culled to six males and two females. and male progeny were individually identified by foot tattoo. Female pups were kept only to maintain mixed-sex litters with eight pups in each litter.

Treatment. Ten male pups (1 per litter) were assigned to each of six treatment groups: untreated controls, treated controls given 10% acacia, 100 mg/kg NMTT, and 10,30, or 100 mg/kg disulfiram. Doses were administered by gavage beginning on Postpartum Day 6 and continuing through Postpartum Day 36. Each male pup was weighed and the dose was adjusted daily on Postpartum Days 6 to 20 and every second day thereafter. The dam and female pups were killed on Postpartum Day 23, and the male

166 HOOVER ET AL

pups remained with littermates until caged individually on Postpartum Day 29. On Postpartum Day 37. the male pups were weighed and killed by asphyxiation with carbon dioxide. The testes were excised, trimmed, and weighed.

DNA/low cyfometr~~. The right testis from each animal was stripped of the tunica albuginea. placed in Hanks’ balanced salt solution. and minced with scissors until re- duced to small fragments, The cellular suspensions were sieved through 37-pm nylon mesh (Tetko, Inc., New York, NY), centrifuged (370g) for 10 min, and then fixed in 50% ethanol. Testicular cell suspensions with a cell concentra- tion of I-2 X I O6 cells/ml were prepared. Cells were treated with 0.1% (v/v) Triton X-100 (Technicon Instruments Corp., Tarrytown, NY) for 10 min and then washed and treated with RNase (I 74 F/ml, Sigma Chemical Co., St. Louis, MO) for 20 min at 37°C. Cells were washed and stained with propidium iodide (50 &ml, Calbiochem- Behring Co., La Jolla, CA) in the dark at 4°C for at least 1 hr. After staining, the cells were again filtered through 37-Grn nylon mesh and analyzed for relative DNA content using a Coulter Epics-C flow cytometer (Coulter. Hialeah. FL). The excitation wavelength was 488 nm. and the power of the laser beam was 550 mW. The number of fluorescent events collected per sample was dependent on cell yield, but a sufficient number for analysis was collected from all samples. At least 8500 fluorescent events per sample were analyzed and in most samples this number exceeded 25,000. The relative number of events within each flu- orescent peak was determined using the data analysis soft- ware of the flow cytometer. Rat diploid spleen and normal adult testis cells were used as ploidy reference markers.

Interpretation of the fluorescence peaks was according to Toppari et al. (I 985).

Hi.stopntho/ogy. The left testis from each animal was preserved in neutral buffered formalin. The fixed tissues were sectioned, processed, and embedded in glycol meth- acrylate. Histologic preparations of the testes were stained using a periodic acid-Schiff method with hematoxylin, eosin, and phloxine as counterstains. Terminology specific to spermatogenic kinetics was according to Leblond and Clermont ( 1952).

Statistics. A two-way analysis of variance was used to evaluate the effects of treatment and litter on final body weight and body weight gain. Absolute and relative weights of testes and fluorescent events were analysed using two- way analyses of variance. The acacia control along with three dose levels of disulfiram were tested for linear trend in final body weight, body weight gain, absolute and relative weight of testes, and fluorescent events. The two control groups were compared to ensure that no effect was asso- ciated with dosage of the acacia vehicle. The untreated control group was used for evaluations of the NMTT group while the acacia control group was compared to the di- sulliram groups. All tests withp values < 0.05 were reported statistically significant.

RESULTS

One and four males of the 30 and 100 mg/ kg disulfiram groups, respectively, had dried

TABLE I

EFFECT OF NMTT AND DISULFIRAM ON BODY WEIGHT AND TESTES WEIGHT AFTER ORAL ADMINISTRATION TO YOUNG RATS

Treatment group

Mean” (&SE)* Mean” (*SE) b Mean” (?SE)b Mean” (&SE)* testes/body

terminal body weight body weight gain testes weight weight N k) k) k) (g/100 I4

Untreated control IO 158 (2.8) 144 (2.8)

Acacia control 10 159 (2.8) 144 (2.8) NMTT (mg/kg)

100 9 I46 (3.0)t 132 (2.9)t Disulfiram (mg/kg)

10 10 143 (2.8)* 129 (2.8)* 30 9 134 (3.0)* 120 (2.9)*

100 9 119 (3.0)* 105 (2.9)*

* Statistically significant different from acacia control, p value < 0.05. j’ Statistically significant different from untreated control, p value < 0.05. ’ Least-squares mean. b Standard error of least-squares mean.

I .4 1 (0.034) 0.90 (0.023) 1.38 (0.034) 0.87 (0.023)

0.99 (0.036)t 0.68 (0.025)T

0.92 (0.034)* 0.65 (0.023)* 0.90 (0.036)* 0.67 (0.025)* 0.72 (0.036)* 0.60 (0.025)*

COMPARATIVE TESTICULAR TOXICITY OF DISULFIRAM AND NMTT 167

DNA Content

FIG. 3. DNA content distribution histograms of rat tes- ticular cells from an adult (A and D), a 37day-old control (B and E), and a 37-day-old treated with 100 mg/kg di- sulfiram (C and F). Relative fluorescence intensity mea- sured on 256-channel resolution is plotted against relative number of cells per channel (A-C). Peak 1, maturation phase spermatids; peak II, round and elongating sper- matids; peak III, diploid cells: peak IV, tetraploid cells. (D, E, and F) three-dimensional plots of DNA content X cell size X relative cell count. The modal value of the diploid peak (peak III) was normalized between 115 and 120 in all measurements.

blood and scabbing around the anus during the first week of treatment. Blood was noted in the urine of one male of the 100 mg/kg disulfiram group. Three animals died and were not evaluated further. The death of a male treated with NMTT was attributed to a gav- aging error. The causes of death of two males treated with 30 or 100 mg/kg disulfiram were not evident.

A statistically significant decreasing trend was observed in final body weight, body weight gain, and absolute and relative weights of testes for each dose of disulfiram (Table 1). The per- centages of reduction, respectively, for doses of 10,30, and 100 mg/kg disulfiram compared to acacia control group were 10, 15, and 25% for final body weight; 10, 17, and 27% for body weight gain; 33, 35, and 48% for absolute testis weight; and 25, 23, and 3 1% for relative testis weight. In comparison with the untreated control group, rats given 100 mg/kg NMTT

had an 8% reduction in final body weight, 7% reduction in body weight gain, 30% reduction in absolute testis weight, and 24% reduction in relative testis weight. The untreated control and the acacia control did not differ signifi- cantly.

For flow cytometry, frequency histograms of testicular cell DNA fluorescent events were characterized in the adult rat by four main peaks representing step 17- 19 spermatids (hy- pofluorescent haploid) and step 1-14 (hap- loid), diploid, and tetraploid cells (Fig. 3). The DNA synthesizing cells are located in the re- gion between the diploid and the tetraploid peaks. Histograms of testicular cells from 37- day-old control rats had three main peaks, in- dicating that the majority of spermatids had not matured beyond step 14. Both NM’IT and disulfiram caused significant changes in the ratios of testicular cell types present (Fig. 4). Rats treated with 100 mg/kg NMTT had a statistically significant reduction in spermatids of 40% compared to the untreated control rats. The acacia control group and the 10, 30, or 100 mg/kg disulfiram-treated groups had a statistically significant linear decreasing trend in spermatids. The reductions of spermatids for the 10,30, or 100 mg/kg disulfiram-treated rats compared to the acacia control group were 52,6 1, or 89%, respectively. A statistically sig-

Ploidy

FIG. 4. Relative proportions of cells with IN, 2N, and 4N states of DNA ploidy in testicular suspensions from 37-day-old rats. Least-squares means ? SE.

168 HOOVER ET AL.

FIG. 5. Testes of 37-day-old rats (a) of the control group given acacia. (b) of the group given 100 mg/kg NMTT, and (c) of the group given 10 mg/kg disulfiram. The testis of the rats given NMTT or disuliiram had a reduced number of spermatocytes and spermatids and no maturation phase or Golgi phase spermatids. Modified PAS. X 160

nificant increasing trend was observed in the percentages of diploid cells of the acacia con- trol and the three disulfiram groups. The in- creases in the percentage of diploid cells com- pared to the acacia control group were 146, 17 1, and 270% for the three disulfiram groups. The increases in the tetraploid cells of the 10 and 30 mg/kg disulfiram-treated groups from the acacia control group were statistically sig- nificant. However, the 100 mg/kg disulfiram group did not differ significantly from the aca- cia control group in tetraploid cell ratios.

Rats given either NMTT or disulfiram had qualitatively similar histologic changes. The most prominent feature was a reduction in the number of spermatids associated with a delay in the maturity of the leading waves of the germinal cells. In control rats, the leading

waves of spermatids were early maturation phase, usually step 15. In comparison, the most advanced waves of spermatids in rats given either 100 mg/kg of NMTT or 10 mg/ kg of disulfiram were in Golgi phase or early cap phase, steps 1 to 6 (Fig. 5). The average decrease in the number of spermatids for these groups ranged from moderate, with approxi- mately half the normal number of sperrnatids present, to severe with total or almost total absence of spermatids (Table 2). Both the 100 mg/kg NMTT group and the 10 mg/kg disul- firam group had minimal or moderate reduc- tion in number of spermatocytes. In a few an- imals, a minimal reduction in number of spermatogonia occurred. With disulfiram, the changes were dose-related with progressively greater effects in the groups given 30 and 100

COMPARATIVE TESTICULAR TOXICITY OF DISULFIRAM AND NMTT 169

FIG. 5-Continued

170 HOOVER ET AL.

TABLE 2

AVERAGE GRADE“ FOR HISTOL~CX CHANGES IN THE TESTIS OF RATS GIVEN NMTT AND DISULFIRAM

Treatment group

Decreased Decreased Decreased Degeneration of spermatids spermatocytes spermatogonia spermatocytes

N mean (&SE) mean (*SE) mean (*SE) mean (*SE)

Untreated control Acacia control NMTT (mg/kg)

100 Disulliram (mg/kg)

10 30

100

10 0 0 0 0 10 0 0 0 0

9 3.8 (0.22) 2.7 (0.29) 0.6 (0.18) 1.0 (0.17)

10 4.0 (0.15) 2.2 (0.25) 0.2 (0.25) 0.5 (0.22) 9 4.3 (0.17) 2.7 (0.17) 2.7 (0.17) 0.6 (0.18) 9 5.0 (0.00) 3.4 (0.18) 1 .o (0.00) 1 .O (0.24)

a 0, Normal; 1, minimal (< 10% affected); 2, slight; 3. moderate; 4, marked; 5, severe (>90% affected).

mg/kg. In the 100 mg/kg disulfiram group, the decrease in number of spermatids was uni- formly severe with complete or almost com- plete absence of spermatids. Where spermatids were present, they were limited to early Golgi phase, usually step 1. The number of sper- matocytes in the rats given 100 mg/kg of di- sulfiram was reduced by approximately half. A few tubules had an absence of spermato- gonia but the overall change in spermatogonia was minimal. A few animals treated with NMTT or disulfiram had degeneration of ger- minal cells. However, the degeneration was minimal and involved only a small number of individual spermatocytes.

DISCUSSION

The present study demonstrated a disulfi- ram-induced testicular toxicity in young rats similar to that previously described only for NMTT and NMTT-containing cephalosporin antibiotics. Previous descriptions and our findings for NMTT are consistent, allowing for differences in use of terminology and du- ration of treatment. DNA flow cytometric analysis of testicular cells identified a decrease in the ratio of spermatids (haploid cells) for both NMTT and disulfiram. Microscopically,

NMTT treatment resulted in an absence of the leading waves of germinal cells in the ma- turing testes. Interestingly, while a decrease in the number of spermatocytes and spermato- gonia in the young CD rats given 100 mg/kg daily of NMTT for 4 weeks was observed in this study, another study found no effect on these cells in Sprague-Dawley rats given 300 mg/kg of NMTT beginning on Postpartum Day 6 and continuing for 5 weeks (Moe et al., 1989). However, this difference could be ex- plained in part if the effect is limited to the first maturing generations of germinal cells and recovery occurs while treatment continues. It has been shown for one NMTT-containing cephalosporin, cefamandole, that the testicular effect occurs early in the treatment period and is associated with degeneration of the first waves of germinal cells, the subsequent waves appearing to develop normally (Hoover et al.,

1989). Disulfiram is used extensively in the rubber

industry (Del Gatto, 1968) and in the treat- ment of alcoholism (Hald et al., 1948; Ritchie, 1985). There are no reports of testicular tox- icity caused by disulfiram alone. In one study, adult Sprague-Dawley rats fed disulfiram at a dietary concentration of 0.15% for 40 days (approximately 68 mg/kg/day) had no testic- ular changes, although rats given a combina-

COMPARATIVE TESTICULAR TOXICITY OF DISULFIRAM AND NMTT 171

tion of disulfiram and 1,2-dichloroethane had testicular atrophy (Igwe et al., 1986). The 10 mg/kg dose of disulfiram used in the present study is the maximum recommended for therapy (Ritchie, 1985). This dosage caused a degree of testicular toxicity roughly equivalent to that induced by 100 mg/kg of NMTT. Doses of 30 or 100 mg/kg of disulfiram caused an increased effect but did not prevent devel- opment of the germinal epithelium.

Disulfiram was selected for the present study because it, like NMTT, is an inhibitor of al- dehyde dehydrogenase (Ritchie, 1985). How- ever, most enzymes with crucial sulfhydryl groups can be inhibited by disulfiram, and di- sulfiram could have its effect through other enzymatic pathways. Besides aldehyde dehy- drogenase, NMTT also inhibits y-carboxyla- tion of glutamic acid (Kitson, 1984; Lipsky, 1984) but it is not known whether NMTT has broader enzyme inhibitory effects, similar to disulfiram. It remains to be determined if other compounds which induce disulfiram-like re- actions in patients following alcohol con- sumption can also induce an NMTT-type tes- ticular toxicity.

Direct extrapolation of the observation made in the young rat to humans is not pos- sible because differences in the initiation of spermatogenesis and differences in spermato- genie cycles between rodents and humans are substantial (Steinberger and Steinberger, 1972). The implications to humans of the findings in the young rat have been discussed previously (Hoover et al., 1989). Briefly, the human has no direct counterpart to the rapid phase of spermatogenic development as it oc- curs initially in the young rat. To expose hu- mans during germinal cell development com- parable to the sensitive period in rats would require therapy over a period of years and only a small proportion of the germinal cells would be expected to be in a sensitive phase during a usual course of antibiotic therapy. The char- acteristics of the young rat appear to make it unsuitable as a model for direct extrapolation to humans. Defining the mechanism of testic-

ular toxicity for compounds which cause an NMTT-like testicular toxicity in rats may help define the biological differences in the sper- matogenic process between the neonatal and the adult rat.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the assistance of K. S. Howell in preparation of the manuscript and the technical assistance of R. M. Tague.

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