Lillian F. Strader

Spermatotoxicity of dichloroacetic acid

The testicular toxicity of dichloroacetic acid (DCA), a disinfection byproduct of drinking water, was evaluated in adult male rats given both single and multiple (up to 14 d) oral doses. Delayed spermiation and altered resorption of residual bodies were observed in rats given single doses of 1500 and 3000 mg/kg; these effects persisted to varying degrees on post-treatment days 2, 14, and 28. Delayed spermiation and formation of atypical residual bodies also were observed on days 2, 5, 9, and 14 in rats dosed daily with 1440, 480, 160, and 54 mg/kg. Distorted sperm heads and acrosomes were observed in step 15 spermatids after 14 doses of 480 and 1440 mg/kg. Decreases in the percentage of motile sperm occurred after 9 doses of 480 and 1440 mg/kg and 14 doses of 160 mg/kg. Increased numbers of fused epididymal sperm were observed on days 5, 9, and 14 in rats dosed with 1440, 480, and 160 mg/kg, respectively; other morphologic abnormalities occurred at 160 mg/kg and higher. On day 14, a significant decrease in epididymis weight was observed at 480 and 1440 mg/kg, and epididymal sperm count was decreased at 160 mg/kg and higher. These studies demonstrate that the testicular toxicity induced by DCA are similar to those produced by the analogue, dibromoacetic acid. However, the testicular toxicity of DCA is less severe at equal molar concentrations. Moreover, the DCA-induced testicular lesions occur with greater potency as the duration of dosing increases, indicating the importance of using low-dose subchronic exposures to assess the health risk of prevalent disinfection byproducts.

Spermatotoxicity of dibromoacetic acid in rats after 14 daily exposures

Halogenated acetic acids are major disinfection by-products of water chlorination and ozonation. Limited data in experimental animals indicate that repeated doses of dichloroacetic acid (DCA) or single doses of dibromoacetic acid (DBAA) cause testicular damage. In the present study, spermatotoxic effects were investigated in rats given oral doses of 0, 10, 30, 90, or 270 mg DBAA/kg/day for 14 days. In rats dosed with 270 mg/kg/day, there were marked effects on epididymal sperm motility and morphology including the flagellar fusion of 2 or more sperm. Testis weight, epididymis weight, and testicular sperm head counts were mildly reduced relative to control, whereas epididymal sperm counts were substantially decreased. Histologic changes in the testis included retention of Step 19 spermatids in Stages IX to XII, abnormal development of late spermatids, and the formation of atypical structures resembling residual bodies that were observed predominantly in Stages X to XIV and I of the cycle of the seminiferous epithelium. At the dosage of 90 mg/kg/day, effects on spermiation, spermatid development, epididymal sperm counts, sperm motility, and sperm morphology were less severe than at the higher dosage. Reduced caput sperm counts and mild effects on spermiation also occurred at 30 and 10 mg/kg/day. These studies indicate that subchronic exposure to DBAA has the potential to affect reproductive outcome in the rat. Compared to previous studies of DCA (12), DBAA, on a molar basis, appears to be a stronger testicular toxicant than the dichloro analogue.

Effect of acute exposure to boric acid on the male reproductive system of the rat

Adult male rats were dosed orally on d 0 with 0 or 2000 mg/kg of boric acid and killed on posttreatment d 2, 14, 28, and 57, or dosed with 0, 250, 500, 1000, or 2000 mg/kg of boric acid and killed on posttreatment d 14. At d 14, atypical structures that appeared to be enlarged irregular cytoplasmic lobes of Step 19 spermatids were observed in Stage VIII seminiferous tubules of rats dosed with 1000 and 2000 mg/kg. Abnormal retention of Step 19 spermatids and residual bodies was also observed in Stage IX-XIII tubules of these rats. The retained spermatids and residual bodies were seen in both the luminal and basal regions of the epithelium. A substantial increase in the testicular sperm head count occurred in animals dosed with 2000 mg/kg. Abnormal caput epididymal sperm morphology and reduced caput epididymal sperm reserves were observed at 1000 mg/kg and higher. Serum LH, FSH, TSH, and prolactin values were not affected at any dosage. At d 28, rats dosed with 2000 mg/kg exhibited continued retention of Step 19 spermatids into Stage X, abnormal caput and cauda sperm morphology, and decreased percentages of motile cauda spermatozoa with reduced straight-line swimming velocities. By d 57 substantial recovery was apparent; some retention of Step 19 spermatids into Stage X tubules was still present in two out of six rats but the sperm parameters were comparable to controls. The study indicated that acute oral exposure to boric acid adversely affected spermiation and sperm quality in the adult male rat. At the dosages used the effects appeared reversible. The no-effect level was 500 mg/kg.

The Effect of Ambient Air Pollution on Sperm Quality

Background Research has suggested an association with ambient air pollution and sperm quality. Objectives We investigated the effect of exposure to ozone (O3) and particulate matter < 2.5 μm in aerodynamic diameter (PM2.5) on sperm quality. Methods We reexamined a previous cohort study of water disinfection by-products to evaluate sperm quality in 228 presumed fertile men with different air pollution profiles. Outcomes included sperm concentration, total sperm per ejaculate (count), and morphology, as well as DNA integrity and chromatin maturity. Exposures to O3 and PM2.5 were evaluated for the 90–day period before sampling. We used multivariable linear regression, which included different levels of adjustment (i.e., without and with season and temperature) to assess the relationship between exposure to air pollutants during key periods of sperm development and adverse sperm outcomes. Results Sperm concentration and count were not associated with exposure to PM2.5, but there was evidence of an association (but not statistically significant) with O3 concentration and decreased sperm concentration and count. Additionally, a significant increase in the percentage of sperm cells with cytoplasmic drop [β = 2.64; 95% confidence interval (CI), 0.21–5.06] and abnormal head (β = 0.47; 95% CI, 0.03–0.92) was associated with PM2.5 concentration in the base model. However, these associations, along with all other sperm outcomes, were not significantly associated with either pollutant after controlling for season and temperature. Overall, although we found both protective and adverse effects, there was generally no consistent pattern of increased abnormal sperm quality with elevated exposure to O3 or PM2.5. Conclusions Exposures to O3 or PM2.5 at levels below the current National Ambient Air Quality Standards were not associated with statistically significant decrements in sperm outcomes in this cohort of fertile men. However, some results suggested effects on sperm concentration, count, and morphology.

Optimization of the Hamilton-Thorn Computerized Sperm Motility Analysis System for Use with Rat Spermatozoa in Toxicological Studies

To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.

Histopathologic changes in the testes of rats exposed to dibromoacetic acid

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-XII 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.

The healthy men study: An evaluation of exposure to disinfection by-products in tap water and sperm quality

Background Chlorination of drinking water generates disinfection by-products (DBPs), which have been shown to disrupt spermatogenesis in rodents at high doses, suggesting that DBPs could pose a reproductive risk to men. In this study we assessed DBP exposure and testicular toxicity, as evidenced by altered semen quality. Methods We conducted a cohort study to evaluate semen quality in men with well-characterized exposures to DBPs. Participants were 228 presumed fertile men with different DBP profiles. They completed a telephone interview about demographics, health history, water consumption, and other exposures and provided a semen sample. Semen outcomes included sperm concentration and morphology, as well as DNA integrity and chromatin maturity. Exposures to DBPs were evaluated by incorporating data on water consumption and bathing and showering with concentrations measured in tap water. We used multivariable linear regression to assess the relationship between exposure to DBPs and adverse sperm outcomes. Results The mean (median) sperm concentration and sperm count were 114.2 (90.5) million/mL and 362 (265) million, respectively. The mean (median) of the four trihalomethane species (THM4) exposure was 45.7 (65.3) μg/L, and the mean (median) of the nine haloacetic acid species (HAA9) exposure was 30.7 (44.2) μg/L. These sperm parameters were not associated with exposure to these classes of DBPs. For other sperm outcomes, we found no consistent pattern of increased abnormal semen quality with elevated exposure to trihalomethanes (THMs) or haloacetic acids (HAAs). The use of alternate methods for assessing exposure to DBPs and site-specific analyses did not change these results. Conclusions The results of this study do not support an association between exposure to levels of DBPs near or below regulatory limits and adverse sperm outcomes in humans.

Testicular toxicity and infertility in male rats treated with 1,3‐dinitrobenzene

Weanling male Sprague-Dawley rats were gavaged 5 d/wk with 1,3-dinitrobenzene (m-DNB) at dosages of 0, 0.75, 1.5, 3.0, and 6.0 mg/kg X d. Males were bred to untreated females during treatment wk 10 and were killed during treatment wk 12. Although males dosed with 3 mg/kg X d inseminated the females and evidence of mating was observed in males dosed with 6 mg/kg X d, none of the males in these groups sired litters. Diminished sperm production (reduced testicular sperm head counts), decreased cauda epididymal sperm reserves, nonmotile spermatozoa, atypical sperm morphology, decreased weights of the testes and epididymides, seminiferous tubular atrophy, and incomplete spermatogenesis were also observed in these groups. Sperm production was also decreased in males dosed with 1.5 mg/kg X d. Changes in the spleen included increased weight at dosages of 1.5 mg/kg X d or higher and splenic hemosiderosis, which ranged from slight in rats treated with 0.75 mg/kg X d to moderately severe in those dosed with 6 mg/kg X d. The data indicate that m-DNB is a potent testicular toxicant in the male rat, capable of producing extensive damage to reproductive tissues and reproductive failure. Limited data on four rats that received 6 mg/kg X d and were allowed a 5-mo posttreatment recovery period suggested that the testicular effects are at least partially reversible.

Evaluation of reproductive parameters in adult male Wistar rats after subchronic exposure (gavage) to benomyl.

Proven-breeder 102-d-old male Wistar rats were gavaged daily with 0, 1, 5, 15, or 45 mg/kg.d benomyl. The animals were bred to untreated females after 62 d and killed after 76-79 d for evaluation of selected male reproductive end points. Minimal to moderate changes were observed in rats dosed with 45 mg/kg.d; these included decreased testis and epididymis weight, reduced cauda sperm reserves, decreased sperm production, increased numbers of decapitated spermatozoa, and increased numbers of seminiferous tubules containing multinucleated giant cells. Reproductive performance, seminal vesicle and prostate weight, sperm motility, serum luteinizing hormone, follicle-stimulating hormone, prolactin, and androgen binding protein were not affected by any of the dosages tested. Based on these end points, the no-effect level was 15 mg/kg.d.

Reproductive toxicity of a single dose of 1,3-dinitrobenzene in two ages of young adult male rats☆☆☆

These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5-6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.