Semen quality and reproductive health of young Czech men exposed to seasonal air pollution.

This study of male reproductive health in the Czech Republic resulted from community concern about potential adverse effects of air pollution. We compared young men (18 years of age) living in Teplice, a highly industrialized district with seasonally elevated levels of air pollution, to those from Prachatice, a rural district with relatively clean air. Surveys were scheduled for either late winter, after the season of higher air pollution, or at the end of summer, when pollution was low. Participation included a physical examination, donation of a semen sample, and completion of a questionnaire on health, personal habits, and exposure to solvents and metals through work or hobby. Analysis of data from 408 volunteers showed that the men from Teplice and Prachatice were similar in physical characteristics, personal habits, and work- or hobby-related exposures. Sixty-six percent (272) of these men donated a single semen sample for routine semen analysis, computer-aided sperm motion analysis, and sperm chromatin structure assay. The mean (median) sperm concentration and sperm count were 61. 2 (44.0) million/mL semen and 113.3 (81.5) million, respectively, and were not associated with district of residence or period of elevated air pollution. However, periods of elevated air pollution in Teplice were significantly associated with decrements in other semen measures including proportionately fewer motile sperm, proportionately fewer sperm with normal morphology or normal head shape, and proportionately more sperm with abnormal chromatin. These results suggest that young men may experience alterations in sperm quality after exposure to periods of elevated air pollution, without changes in sperm numbers.

Sperm motion predicts fertility in male hamsters treated with alpha-chlorohydrin.

An understanding of the relationship between altered sperm motion and sperm function (fertility) is important when interpreting the biological significance of toxicant-induced changes in sperm velocity in rodent test species. Previous studies showed that a brief (4-day) exposure of male hamsters to the model chemical alpha-chlorohydrin (ACH) results in significant deficits in epididymal and uterine sperm velocity, which are associated with both a delay and a failure of fertilization in vivo. To characterize this effect in terms of fertility, similarly treated male hamsters were bred to untreated females and pups were counted the day before parturition. ACH treatment resulted in a dose-dependent decline in the percentage of sperm-positive females that were pregnant at the end of gestation (100, 78, 67, 22, and 0 where males were treated with 0, 33, 49, 66, and 83 mg ACH/kg/day, respectively). Cauda epididymal sperm from the same males were assayed for motion characteristics using computer-assisted sperm analysis (CASA), and for fertilizing ability in vitro. While the percentage of motile sperm was unaffected by ACH treatment, sperm velocity declined in a dose-dependent manner at all ACH treatment levels. Furthermore, the velocity of sperm from infertile males was shifted downward consistently across the entire velocity distribution. Since treated males tended to either be infertile (no pups) or have near normal litter size, the correlation between sperm velocity and litter size was nonlinear. Therefore, logistic regression models using velocity cut-off values were the most useful models for predicting fertility. These results support the contention that fertility relies on there being a sufficient number of sperm that exceed a velocity threshold. Sperm from treated males were also less likely to support in vitro fertilization (IVF), providing further evidence of impaired sperm function associated with acute exposure to ACH.

Synchronous assessment of sperm motility and fertilizing ability in the hamster following treatment with alpha-chlorohydrin.

To investigate the relationship between sperm motion parameters and fertilizing ability, a model was developed to assess both of these endpoints synchronously using a toxicant that inhibits sperm motion. alpha-Chlorohydrin (ACH) was administered daily for 4 days to male hamsters at 0, 33, 49, 66, and 83 mg/kg body weight. These males were then allowed a 45-minute breeding period with untreated estrus females on the morning of day 5. One hour after breeding, sperm samples were surgically recovered from the uteri of the females for motility analysis. Six hours later, eggs were flushed from the oviducts and evaluated for fertilization. Cauda epididymal sperm were also collected from the males shortly after breeding. Proportions of motile and progressively motile sperm were manually quantified, and overall sperm velocity and the velocity of representative vigorously swimming sperm in both the uterine and epididymal samples were measured by computer-aided sperm analysis. Significant decreases in in vivo fertilization rates and epididymal sperm motion parameters were observed at 66 and 83 mg/kg ACH, whereas uterine sperm motion was adversely affected at all ACH dosages used. All sperm motion parameters except the percentage of motile sperm in the epididymis were significantly correlated with fertilization rates by both linear and logistic regression. Overall, uterine and epididymal sperm endpoints predicted fertilizing ability comparably well. Stepwise multiple linear regression gave a model containing epididymal sperm velocity (EVCL) and uterine sperm percent motility (UMOT) with an R2 value of 0.649. Stepwise multiple logistic regression gave models containing EVCL alone and EVCL and UMOT in binary (fertile/infertile) and quantal models, respectively.

Method of euthanasia does not affect sperm motility in the laboratory rat.

To determine if anesthetic agents used in laboratory animal euthanasia affected sperm motion parameters, rats (n = 10 per group) were euthanized by one of 5 different methods: decapitation alone, or decapitation following either ether, halothane, or Nembutal anesthesia, or CO2 asphyxiation. Sperm were collected from the distal cauda epididymis, diluted, and videotaped for computer-aided sperm analysis (CASA; HTM-2030, Hamilton-Thorn Research, Beverly, MA). The percentage of motile sperm (MOT), their straight-line velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linear index (LINX), and linearity (LIN) were measured on > or = 200 motile sperm per sample. No significant differences in any of these 6 motion parameters were found among the treatment groups. Thus, none of these 5 methods of euthanasia affect sperm motion as assessed by CASA methods, making them equally suitable for use in reproductive toxicology studies.

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.

Endpoints of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants.

Multiple endpoints of spermatotoxicity in short duration tests (1-5 days exposure; 2.5-week assay interval) were investigated in a number of chemicals reported to produce minimal to severe reproductive effects when administered subchronically. Six of these chemicals (boric acid, dinoseb, 2,5-hexanedione, methoxychlor, metronidazole, ornidazole) produced substantial spermatotoxicity after 1 to 5 doses. Spermatotoxic effects of chlordimeform were equivocal while p,p'-DDT, n-hexane, and sodium chlorite were judged negative. Four chemicals with known acute effects (benomyl, busulfan, ethylene glycol monomethyl ether, nitrobenzene) elicited expected histopathologic responses after a single dose. Testicular histology, testicular sperm head counts, cauda sperm counts, sperm morphology, and sperm velocity proved to be the most toxicologically sensitive endpoints in one or more of the studies, but histopathology of the testis and epididymis was the most consistent indicator of reproductive damage. The percentage of motile sperm and sperm concentration in the epididymal fluid were the least sensitive measurements. The data suggested that most chemicals with the potential to produce moderate to severe sperm damage are detectable with a short duration test. Complementary multiple endpoints enhanced the interpretation of results, often identified cellular targets, and provided insight on possible mechanisms. Specific responses were often similar to specific effects reported for subchronic exposures. A short duration test could be of value as a screen in structure-activity studies or to set priorities for chemicals requiring further evaluation. As a supplement to breeding studies, the data generated in the short test could also be used to enhance the design and interpretation of the longer tests.

Rat sperm motility analysis: methodologic considerations.

The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample chamber depth. In addition, sources of variation were identified and accuracy of the analysis was examined. All samples in this report were analyzed using a Hamilton Thorn Motility Analyzer (HTM-2000; Hamilton Thorn Research, Danvers, MA). We found that allowing the sperm to swim out from cuts made in the distal cauda epididymidis yielded samples with percentages of motile sperm 60% higher than samples collected using an aspiration method. Furthermore, sperm isolated from the distal cauda epididymidis exhibited slightly but significantly greater percentages of motile sperm and swimming speeds than sperm isolated from the proximal cauda epididymidis. Of the four motility media examined, all maintained a high percentage of motile sperm over an hour-long incubation period, but Medium 199 and modified Hanks' Balanced Salt supported substantially greater sperm velocity than Dulbecco's Phosphate Buffered Saline (with Ca++ and Mg++), with or without glucose. Motility and velocity endpoints were comparable in 200-, 100-, or 40-micron deep chambers, but significantly lower in 20-micron-deep chambers. Since these and presumably other variables in the preparation and analysis of rat sperm do influence the assessed motility endpoints, it is important to standardize these methods and to consider these issues when interpreting CASA data.

Acute inhalation exposure to epichlorohydrin transiently decreases rat sperm velocity.

The effect of inhaled epichlorohydrin on rat sperm motility characteristics was evaluated. Male F-344 rats were exposed to 100 ppm epichlorhydrin via inhalation for 4 hr in the morning of Day 0 and killed immediately and on Days 1, 2, 6, and 14 postexposure. Videotapes of cauda epididymal sperm were analyzed (300-350 sperm/sample) with a Hamilton Thorn motility analyzer (HTM-2000, Hamilton Thorn Research, Danvers, MA). Epichlorohydrin did not affect the percentage of motile sperm at any time. However, transient changes in sperm velocity were found. On Day 1 postexposure mean progressive (straight line) and mean path (smoothed curvilinear) velocity were significantly decreased to 80 and 85% of control, respectively. The progressive velocities of sperm from both control and treated rats were normally distributed, indicating a general effect of epichlorohydrin on all sperm as opposed to a more severe effect on a specific sperm subpopulation. Sperm velocity was not significantly affected at later times. Other endpoints (testis and epididymis weights, testicular spermatid counts, and cauda epididymal sperm reserves) were unaltered by epichlorohydrin. Thus, inhaled epichlorohydrin at 100 ppm produced specific, transient decreases in rat sperm velocity. Furthermore, computer-assisted sperm analysis was able to detect these relatively subtle, toxicant-induced changes in rat sperm velocity, demonstrating the utility of this technology in reproductive toxicology studies.

Unilateral depletion of testicular glutathione levels in the rat following intratesticular injections of diethylmaleate and buthionine sulfoximine.

A method was developed to selectively deplete glutathione (GSH) in a single rat testis. Using intratesticular injections of a mixture of two GSH-depleting agents, diethylmaleate and buthionine sulfoximine, testicular GSH levels were decreased to 33-54% of control 2 hr after injection and remained suppressed for 24 hr. GSH levels in the contralateral testis and liver were not affected by this treatment. Comparisons between GSH-depleted and vehicle-injected (contralateral) testes, evaluated 2 weeks later, showed that although testis and epididymal weights and cauda epididymal sperm reserves were slightly reduced (to greater than or equal to 90% of controls), no changes were seen in testicular spermatid counts or in the morphology or motility of cauda epididymal sperm. An increase in histologically abnormal tubules localized to the injection site occurred in some GSH-depleted testes; however, the proportion of normal tubules containing step 19 spermatids was not affected. Thus, intratesticular injections of GSH-depleting agents selectively lowered GSH levels in the treated testis, with minimal adverse effects. This protocol can now be applied to investigate specific roles of GSH in the testes, particularly with regard to the possible modulation of the effects of testicular toxicants.

Role of the 4-hydroxy intermediate in the in vitro embryotoxicity of cyclophosphamide and dechlorocyclophosphamide.

Cyclophosphamide must be metabolically activated to produce malformations in cultured rat embryos. A 4-hydroxylated intermediate, 4-hydroxycyclophosphamide is initially formed during this activation. While 4-hydroxycyclophosphamide (and/or its open-ring tautomer, aldophosphamide) is believed to act as a transport form in mediating the antineoplastic activity of cyclophosphamide, its role in the teratogenicity of this drug is not known. In this study the effects of two "preactivated" cyclophosphamide analogs on cultured Day 10 rat embryos were determined. The first analog, 4-hydroperoxycyclophosphamide, is converted to 4-hydroxycyclophosphamide in aqueous solutions, releasing both acrolein and phosphoramide mustard, while the second, 4-hydroperoxydechlorocyclophosphamide, releases, in a similar manner, acrolein and the inactive metabolite, phosphoric acid diamide. Both cyclophosphamide analogs were teratogenic, embryolethal, and growth retarding in vitro, but the effective concentrations and the types of malformations produced were different. 4-Hydroperoxycyclophosphamide produced embryo deaths and malformations and decreases in embryonic growth and protein content at concentrations in the range of 5 to 25 microM. In contrast, 4-hydroperoxydechlorocyclophosphamide did not produce embryo deaths at concentrations below 100 microM and produced embryo malformations and growth retardation only at 125 microM. The concentration-response curve and the spectrum of malformations produced by 4-hydroperoxycyclophosphamide resembled those previously reported for phosphoramide mustard, while the concentration-response curve and types of malformations produced by 4-hydroperoxydechlorocyclophosphamide more closely resembled those observed with acrolein. Thus, the 4-hydroxy intermediates are similar as teratogens to the most potent of the metabolites which they produce; the 4-hydroxy compounds may serve as a transport form of cyclophosphamide but do not appear themselves to have a major role in teratogenicity.

Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes.

Sperm nuclear decondensing activity in mammalian oocytes is dependent upon the maturational state of the oocyte. It is maximal in mature, metaphase II oocytes and minimal or absent in immature germinal vesicle (GV) and fertilized pronuclear oocytes. Previous studies suggested that this difference may be due to the relative ability of an oocyte to reduce the protamine disulfide bonds in the sperm nucleus. The results of this study show that mature hamster oocytes contain significantly more glutathione (GSH), about 8 mM, and hence more disulfide reducing power, as compared with GV (4 mM) or pronuclear (6 mM) oocytes. Furthermore, the acquisition of sperm nuclear decondensing activity by maturing oocytes can be prevented or delayed by blocking GSH synthesis with L-buthionine-S,R-sulfoximine during the early stages of oocyte maturation. This is the first evidence that modulation of GSH levels during oocyte maturation and fertilization may be a mechanism by which sperm nuclear decondensing activity is regulated.

Protection of rat embryos in culture against the embryotoxicity of acrolein using exogenous glutathione.

The aldehyde acrolein is embryotoxic in vivo and in vitro. Since acrolein is reactive towards thiols, glutathione was evaluated for its protective effects against the in vitro embryotoxicity of acrolein. Day 10 rat embryos were cultured in the presence of acrolein and glutathione, either concurrently or sequentially, and evaluated for embryo deaths, malformations, growth retardation, and content of glutathione and protein. Acrolein, added alone at the initiation of the culture period, was embryolethal to 64 and 100% of the embryos at 120 and 160 microM respectively. At acrolein concentrations of 80 and 120 microM, 50 and 100%, respectively, of the surviving embryos were malformed. In addition, both of these concentrations of acrolein produced growth retardation manifested by significant decreases in the yolk sac diameter, crown-rump and head lengths, number of somites, and morphological score. Concurrent exposure to 100 or 500 microM glutathione markedly protected embryos against all of these effects. To study the mechanism of the protective effect of glutathione against the embryotoxicity of acrolein, the effects of sequential addition of acrolein and glutathione were determined. When rat embryos were cultured in the presence of acrolein for 2 hr prior to exposure to glutathione, even 500 microM glutathione could not offer any protection against the embryolethality, teratogenicity, and growth retardation induced by acrolein. However, a 6-hr preincubation with 500 microM glutathione, prior to exposure to acrolein (in the absence of exogenous glutathione), significantly decreased the incidence of embryo deaths at 160 microM acrolein and brought the number of deaths and malformations among embryos exposed to 120 microM acrolein down to a level not significantly different from control; unlike the embryos exposed concurrently to acrolein and glutathione, however, the sequential treatment with glutathione and acrolein did not protect against growth retardation. While there were some changes in the total glutathione and protein content of embryos and yolk sacs with acrolein exposure, none of the treatments had any overall effect on the glutathione concentration per mg protein. Thus, exogenous glutathione can protect against the in vitro embryotoxicity of acrolein. We propose that this protection is mediated in part by a direct interaction between glutathione and acrolein, added concurrently to the serum medium, and in part by an indirect effect on the embryo of glutathione added prior to acrolein.

Enhancement of the embryotoxicity of acrolein, but not phosphoramide mustard, by glutathione depletion in rat embryos in vitro.

The intracellular thiol glutathione is known to protect cells against the toxicity of certain drugs and reactive intermediates. In this study, the role of glutathione in protecting the embryo against two embryolethal and teratogenic metabolites of cyclophosphamide, and anticancer drug, was assessed in vitro using the rat whole embryo culture system. Day 10.5 rat embryos were cultured in rat serum medium containing phosphoramide mustard (1, 10, or 25 microM) or acrolein (10, 25, 50 or 100 microM), with and without buthionine sulfoximine (10 or 100 microM), a compound which depletes glutathione by inhibiting its synthesis. After 45 hr, embryos were assessed for viability, malformations, growth and development, and the glutathione content of embryos exposed to buthionine sulfoximine alone was assayed. The glutathione levels of the embryos and their yolk sacs were decreased significantly by 100 microM buthionine sulfoximine, whereas 10 microM buthionine sulfoximine decreased glutathione levels in the yolk sacs only. Phosphoramide mustard alone, at concentrations of 10 and 25 microM, did not produce embryo deaths but did cause malformations and growth retardation in 100% of the exposed embryos. The addition of buthionine sulfoximine (100 microM) had no effect on the teratogenicity or growth-retarding effects of phosphoramide mustard. Acrolein alone produced a 25 and 48% incidence of embryo deaths at 50 and 100 microM, respectively, and a 46% incidence of embryo malformations, as well as significant growth retardation, among the surviving embryos at 100 microM. Buthionine sulfoximine (10 or 100 microM) significantly enhanced the embryotoxic effects of acrolein. The addition of 10 microM buthionine sulfoximine resulted in 100% embryolethality at 100 microM acrolein; this buthionine sulfoximine concentration decreased the EC50 values for embryo deaths and malformations to 50% of those for acrolein alone. The addition of 100 microM butionine sulfoximine significantly potentiated the embryolethality of acrolein at 25, 50 and 100 microM; the combination of 100 microM acrolein plus 100 microM buthionine sulfoximine was 100% embryolethal. The incidence of embryo malformations was enhanced significantly at 10 and 25 microM acrolein by 100 microM buthionine sulfoximine. The EC50 values for embryo deaths and malformations were decreased to 50 and 20%, respectively, of those values for acrolein alone. Both butionine sulfoximine concentrations produced significant growth retardation at all acrolein concentrations compared to either acrolein or buthionine sulfoximine alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of glutathione depletion by buthionine sulfoximine on rat embryonic development in vitro.

The intracellular thiol glutathione has many functions within cells including protection against xenobiotic and oxidative damage, and a role in protein and DNA synthesis and amino acid transport. Consequently, glutathione might be an important substance for normal growth and development. In this study the extent of glutathione depletion by buthionine sulfoximine, an agent which depletes glutathione by inhibiting its synthesis, and the subsequent effects of the depletion on rat embryonic growth and development were assessed. Day 10.5 rat embryos were cultured in rat serum medium in the presence of L-buthionine-S,R-sulfoximine (0.01 to 2.0 mM) and examined for viability, malformations, growth and development 45 hr later. The glutathione concentrations of the cultured embryos and their yolk sacs were also determined. Exposure to buthionine sulfoximine produced marked and significant (P less than or equal to 0.05) depletion of glutathione at a buthionine sulfoximine concentration of 0.10 mM in the embryos and 0.05 mM in the yolk sacs. Exposure to 1 mM buthionine sulfoximine depleted glutathione to less than 7% of control in both of these tissues. None of the concentrations of buthionine sulfoximine tested had a significant effect on embryo viability; however, buthionine sulfoximine caused a significant (P less than or equal to 0.05) incidence of malformed embryos at concentrations of 0.25, 0.5, 1.0 and 2.0 mM. The types of defects induced by buthionine sulfoximine were blebs of the maxillary or nasal processes, prosencephalon or forelimb buds, small or misshapen heads, small prosencephalons and swollen hind brains, and tail defects. Embryonic growth was the most sensitive, of the variables assessed, to the effects of buthionine sulfoximine. Significant (P less than or equal to 0.05) growth retardation was observed at buthionine sulfoximine concentrations as low as 0.01 mM. At 2.0 mM buthionine sulfoximine, the yolk sac diameter, embryo crown-rump length, head length, number of somites and morphological score were reduced to 65, 72, 77, 90 and 80% of control levels respectively. We propose that the embryotoxic effects of buthionine sulfoximine are due to glutathione depletion and, consequently, that a certain basal level of endogenous glutathione is essential to allow for normal development.

The embryolethality and teratogenicity of acrolein in cultured rat embryos.

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.

Sodium 2-mercaptoethane sulfonate protection against cyclophosphamide-induced teratogenicity in rats.

Certain deleterious effects of cyclophosphamide, for example urotoxicity, can be prevented by the administration of thiol compounds such as 2-mercaptoethane sulfonate (MESNA) without altering the therapeutic efficacy of cyclophosphamide. To evaluate the effect of MESNA on the teratogenicity of cyclophosphamide, pregnant Sprague-Dawley rats were divided into nine treatment groups. Individual groups were administered 0.9% NaCl or cyclophosphamide (10 or 15 mg/kg) alone or in combination with MESNA at one of two doses (5 or 30 mg/kg) on Day 13 of gestation. The fetuses were examined for malformations on Day 20 of gestation. Cyclophosphamide alone produced malformations in 50% (10 mg/kg) or 100% (15 mg/kg) of the fetuses. The abnormalities observed were hydrocephaly, hind- and forelimb defects, open eyes, cleft palate, edema, micrognathia, omphalocele, and various skeletal defects. MESNA alone did not induce a significant number of fetal malformations compared to control. The low dose of MESNA had no significant effect on the total incidence of external malformations produced by either dose of cyclophosphamide. The high dose of MESNA significantly reduced the total number of externally abnormal fetuses and fetuses with skeletal defects produced by both 10 and 15 mg/kg of cyclophosphamide. This protection, although statistically significant (p less than or equal to 0.05), is probably not extensive enough for MESNA to be considered effective in protecting pregnant women from the teratogenic effects of cyclophosphamide chemotherapy.

Teratogenicity and embryolethality of acrolein and structurally related compounds in rats.

Acrolein, a metabolite of the anticancer agent cyclophosphamide, is teratogenic to rats after intraamniotic administration. It is not known whether acrolein or a metabolite of acrolein is responsible for the teratogenicity of this compound. We assessed the teratogenicity and embryolethality of acrolein and five structurally related compounds: acrylic acid, allyl alcohol, glycidol, glyceraldehyde, and propionaldehyde by intraamniotic injections in Sprague-Dawley rats on day 13 of gestation. All compounds tested were significantly embryolethal with at least one concentration of the drug. Acrolein was the most embryolethal of the drugs, causing a significant increase in resorptions with as little as 0.1 micrograms/fetus; the other drugs were embryolethal at doses 100-10,000 times that of acrolein. Acrolein was also the most teratogenic of the drugs tested; a dose as low as 5 micrograms/fetus caused a significant increase in the incidence of fetal malformations. Of the other compounds tested, only glycidol at a dose of 1,000 micrograms/fetus induced a significant number of malformed fetuses compared to control. These results suggest that it is acrolein itself that is responsible for its teratogenicity.