Reproductive toxicity of di-n-butylphthalate in a continuous breeding protocol in Sprague-Dawley rats.

The phthalate ester di-n-butylphthalate (DBP) is used extensively in the manufacture of plastics; its reproductive toxicity was tested in rats by the National Toxicology Program's Reproductive Assessment by Continuous Breeding protocol. Levels of 0.1, 0.5, and 1.0% DBP in the diet were selected, and this dosing design yielded average daily DBP intakes of 52, 256, and 509 mg/kg for males and 80, 385, and 794 mg/kg for females, respectively. DBP consumption by F0 rats reduced the total number of live pups per litter in all treated groups by 8-17% and live pup weights in the 0.5% and 1.0% dose groups by < 13%. In tests to determine the affected sex, the number of offspring was unchanged, but the weights of pups from treated females were significantly decreased and offspring from treated males were unchanged. At necropsy, high-dose F0 females had a 14% reduction in body weight, and both sexes had approximately 10-15% increased kidney and liver to body weight ratios compared to controls. Sperm parameters and estrous cyclicity were not affected. In the F1 mating trial, indices of mating, pregnancy, and fertility in the 1.0% dose group were all sharply decreased (one live litter was delivered out of 20 cohabited pairs), concomitant with a 13% decrease in dam body weight. Live F2 pup weights were 6-8% lower in all dose groups. F1 necropsy results revealed that epididymal sperm counts and testicular spermatid head counts were significantly decreased in the 1.0% dose group. Histopathologic investigation showed that 8 of 10 F1 males consuming 1.0% DBP had degenerated seminiferous tubules and 5 of 10 had underdeveloped or otherwise defective epididymides. No ovarian or uterine lesions were observed. In conclusion, this study showed that DBP is a reproductive/developmental toxicant in Sprague-Dawley rats exposed both as adults and during development; it also indicates that the adverse reproductive/developmental effects of DBP on the second generation were greater than on the first generation.

Evaluation of the binding patterns of eleven FITC-conjugated lectins in Fischer 344 rat testes.

The binding patterns of 11 recently commercially available fluorescein isothiocyanate-conjugated lectins that have been uncharacterized or undercharacterized in rat testes and/or have an unknown or complex carbohydrate specificity were evaluated in paraffin sections from Fischer 344 rat testes. Several of the lectins exhibited unique binding patterns that provide information about changes in carbohydrate domains, particularly during germ-cell maturation, that occur during spermatogenesis. Agaricus bisporus (ABA) lectin produced the most striking staining pattern in the cytoplasm of maturing germ cells, increasing in intensity until spermatid elongation, while the nuclei remained negative. In contrast, Cicer arietinum (CPA) strongly stained the nucleus of early leptotene/zygotene spermatocytes, decreasing to moderate intensity during maturation, until staining was irregular and scattered in elongated spermatids. This study describes new patterns of lectin staining during spermatogenesis and provides additional evidence of the complex carbohydrate modifications that occur as germ cells mature within the seminiferous tubule.

Adhesion and signaling proteins spatiotemporally associated with spermiation in the rat.

Spermiation, the process by which late spermatids separate from the Sertoli cell, is disrupted by a number of toxicants. In this study, we used immunohistochemistry (IHC) to identify some of the proteins associated with the spermatid-Sertoli junction. We confirmed the presence of tubulin, actin, and vinculin at the luminal edge of the seminiferous tubule, and we determined that paxillin is also present here. In other cell types, these proteins have been reported to colocalize with beta integrins. Numerous attempts to identify beta integrins by IHC and by use of Western blots were unsuccessful. Clear evidence was found for the presence of N-cadherin and its associated intracellular proteins: beta-catenin, pp120, desmoglein, pp60(src), and Csk. In addition, N-cadherin and desmoglein were found around spermatids retained by the epithelium. From these data and previous literature reports, we propose a hypothetical model for spermatid adhesion and the control of that adhesion, thus providing a framework for hypotheses on the steps involved in the complex process of spermiation in rat testes.

2-Methoxyacetic acid (MAA)-induced spermatocyte apoptosis in human and rat testes: an in vitro comparison.

2-Methoxyethanol (2-ME) produces adverse reproductive effects in humans at an exposure level that is about 60-fold lower (2.6 mg/m3) than the concentration toxic to rat testes (167 mg/m3), suggesting that humans are much more sensitive to the testicular toxicity of 2-ME than rats. Previous studies found that 2-ME-induced germ cell death seen in vivo could be faithfully mimicked in vitro only in cultured seminiferous tubules, using the active metabolite methoxyacetic acid (MAA). To test whether human testis per se is more sensitive than rat testis to MAA, we compared the responses of cultured rat seminiferous tubules (RSTs) and human testicular tissues (HTT) in vitro. Degeneration in spermatocytes was observed in RSTs 19 hours after a 5-hour exposure to MAA at and above 1 mM. The dying germ cells showed necrotic-like morphology, as seen in vivo. Germ cells in HTT were also killed by doses > or = 1 mM, although the dying germ cells appeared apoptotic, rather than necrotic. For both species, doses lower than 1 mM were without visible effect. Interestingly, agarose gel electrophoresis of DNA from tubules of both species showed internucleosomal DNA fragmentation after MAA treatment, indicating that MAA induced apoptosis in both human and rat germ cells, though the dying cells showed different morphology in the two species. Furthermore, MAA-induced germ cell apoptosis in both species could be significantly attenuated by calcium channel blockers such as nifedipine or verapamil, which inhibit calcium movement through plasma membranes. In conclusion, the results suggest that: 1) human testis is equally sensitive to MAA compared to rat testis; and 2) MAA induces germ cell apoptosis both in human and rat, probably through similar, calcium-dependent mechanism(s). The precise steps in this germ cell apoptosis are under investigation.

Structure and control of a cell-cell adhesion complex associated with spermiation in rat seminiferous epithelium.

Spermiation, the release of late spermatids from the Sertoli cell, is disrupted by a number of toxicants. Control of the spermiation process, and the proteins that interact to adhere mature spermatids to Sertoli cells, is poorly understood. In these studies we used immunohistochemistry, coimmunoprecipitation/Western blotting, and mass spectrometry to refine an earlier model of sperm adhesion proposed by our laboratory. We have identified specific proteins linked together as part of a multiprotein complex, as well as several additional proteins (cortactin, ERK1/2, and 14-3-3 zeta) that may be functioning in both structural and signal transduction roles. The current and prior data suggest that protein phosphorylation is central to the control of spermiation. We also present and characterize an in vitro tubule culture system that allowed functional testing of the spermiation model by pharmacologic manipulation, and yielded data consistent with the importance of protein phosphorylation in spermiation.

Testicular toxicity of boric acid (BA): relationship of dose to lesion development and recovery in the F344 rat.

High-dose boric acid (BA) produces testicular lesions in adult rats, characterized by inhibited spermiation followed by atrophy. The present study addressed whether inhibited spermiation can be separated from atrophy based on dose, compared testis boron (B) dosimetry to lesion development, determined how inhibited spermiation was reflected by common reproductive endpoints, and examined reversibility of the testicular lesions. Rats were fed 3000, 4500, 6000, or 9000 ppm BA for up to 9 weeks and examined. Recovery was assessed for up to 32 weeks post treatment. Inhibited spermiation could be separated from atrophy based on dose (inhibited spermiation: 3000/4500 ppm; atrophy: 6000/9000 ppm), with each lesion aspect expressed at different threshold testis B concentrations (inhibited spermiation: 5.6 micrograms B/g and atrophy: 11.9 micrograms B/g) with no B accumulation during the 9-week exposure. These data suggest that separate mechanisms may be operating for these lesion aspects based on testis B concentration and that B dose rate was important for testicular toxicity. Inhibited spermiation was most reliably reflected by informed testicular histology, with the more severe cases decreasing epididymal sperm count to levels that could affect fertility. After treatment, serum and testis B levels in all dose groups rapidly fell to background levels at the earliest time points evaluated (7 days and 8 weeks posttreatment, respectively). The severely inhibited spermiation at 4500 ppm was resolved by 16 weeks posttreatment, but areas of focal atrophy were detected that did not recover posttreatment. Also, no signs of recovery from atrophy were observed (6000 and 9000 ppm). Atrophic tubules contained a normal complement of spermatogonia (2.6 to 2.9 germ cells/100 Sertoli cells), with occasional dividing and degenerating germ cells. Elevations in serum FSH and LH levels suggested an intact hormonal response to the atrophy. In summary, 1) the different aspects of the BA-induced testicular lesion can be separated using different doses, 2) inhibited spermiation does not necessarily proceed to atrophy, and 3) there is no recovery from the atrophy despite the absence of testis B after treatment. The ability to separate inhibited spermiation from atrophy based on dose and testis B dosimetry will be useful in evaluating possible mechanisms. Furthermore, the presence of dividing spermatogonia during long-term BA-induced atrophy suggests that this model should be useful for identifying critical components involved in the reinitiation of spermatogenesis.

Rat testicular Src: normal distribution and involvement in ethylene glycol monomethyl ether-induced apoptosis.

Kinase activities were previously proposed to be central to germ cell apoptosis induced by ethylene glycol monomethyl ether (EGME) and its active metabolite methoxyacetic acid (MAA). We evaluated the role of tyrosine kinase pp60(c-src) in control and EGME-treated adult rat testis in vivo, as well as in vitro using cultured adult rat seminiferous tubules treated with MAA. In normal testicular tissue, immunoreactivity of Src was mostly detected in Sertoli cell cytoplasm and reached the maximum level around the lumen at spermiation. Src localization was confirmed by immunostaining of cocultures of Sertoli and germ cells and was further confirmed by electron microscopic observation that immunoreactivity was predominant in Sertoli cell cytoplasm as well as occasionally at the Sertoli/germ cell junctions. A single dose of 200 mg/kg EGME induced an increase of Src immunoexpression in both epithelium and interstitium in rat testis. Eight hours after treatment, an intensive immunostaining of Src began to be observed specifically in the cytoplasm of the dying spermatocytes. The apoptotic changes were replicated by exposure of 5 mM MAA in the adult rat seminiferous tubule culture model. Furthermore, spermatocyte degeneration was significantly prevented by cotreatment with 0.1 microM geldanamycin, 10 microM herbimycin A, or 10 microM PP2, which are inhibitors of Src activity. These data collectively suggest that pp60(c-src) mediates Sertoli-germ cell interaction in physiological events, and may play an important role in EGME/MAA-induced germ cell apoptosis.

Comparison of the testicular effects of 2-methoxyethanol (ME) in rats and guinea pigs.

Glycol ethers produce both hemato- and testicular toxicity in animals, which is dependent on both the alkyl chain length and animal species used. Ethylene glycol monobutyl ether (2-butoxyethanol, BE) causes hemolytic anemia in rats but not in guinea pigs, and red blood cells from both guinea pigs and humans are minimally affected in vitro by the active metabolite 2-butoxyacetic acid. This demonstrates the importance of animal species selection for assessing human risk to BE exposure. 2-Methoxyethanol (ME) produces testicular lesions in rats characterized primarily by the degeneration of spermatocytes undergoing meiotic division with minimal or no hemolytic changes. Because of the differential hemolytic response to BE between rats and guinea pigs, the present study addressed whether the testicular response to ME was similarly dichotomous. Adult rats or guinea pigs were given a single dose of either 200 or 300 mg ME/kg by gavage, and testicular and hemolytic changes were assessed 24 hr after treatment. Testis histology in rats showed dose-dependent degeneration of dividing spermatocytes in stage XIV tubules as expected, with only minimal hemolytic changes, also as expected. In contrast, no testicular or hemolytic effects were observed in guinea pigs 24 hr after either single ME dose. In a subsequent study, a single dose or multiple (3 daily) doses of 200 mg ME/kg were given, and animals were examined at 4 days after the start of treatment. Testes from rats given both single and multiple ME doses showed, as expected, tubules depleted of spermatocytes and early spermatids. In guinea pigs, spermatocyte degeneration was observed in stage III/IV tubules for both dosing schemes, but was much less severe and widespread and differed from rats in morphological characteristics, specifically in the appearance of nuclear chromatin degeneration. In the rat, degenerating spermatocytes showed uniformly condensed and dispersed chromatin, while in the guinea pig they showed marked chromatin condensation at the nuclear periphery. No hemolytic changes were observed in either species or dosing scheme. In summary, although ME-associated testicular lesions were observed in both species, they differed significantly in onset, characteristics, and severity. Both the nature of the differential testicular response to ME and a comparison to the in vitro human testicular response to the active metabolite 2-methoxyacetic acid are subjects of future study.

Protein kinase activity is central to rat germ cell apoptosis induced by methoxyacetic acid.

Methoxyacetic acid (MAA) is a major metabolite of ethylene glycol monomethyl ether (EGME). Previous investigations of the testicular lesion induced by EGME have found that dividing meiotic cells are the most sensitive, although several stages of spermatocytes are also vulnerable. Preliminary data from this lab suggested the involvement of protein kinase activity in the development of this lesion, a hypothesis explored in the present studies. We used cultured seminiferous tubules (STs) from juvenile rats (25-day-old), exposed in vitro to MAA and several inhibitors of protein kinases. Nineteen h following a 5-h exposure to 5 mM MAA (the plasma level in vivo after a toxic dose of EGME), apoptotic spermatocytes were seen in early- and late-stage STs. Cell death was prevented by cotreatment with broad-spectrum inhibitors of protein kinases such as H-7, H-8, K-252a, W-7, and genistein. In corroboration, immunocytochemistry with antibodies to various kinases (PKCmu, zeta, and gamma, AKAP220, CaMKII, MLCK, and Src) showed increased staining around dying spermatocytes following EGME treatment in vivo. 2D-PAGE, autoradiography, and nanospray mass spectrometry was used to separate and identify proteins whose phosphorylation status was most greatly changed following exposure to MAA. One protein was identified by sequence analysis as being glucose-regulated protein 94 (grp94). Westem blotting and immunocytochemistry confirmed this finding. The data we present implicate kinase activities in the pathogenesis of this lesion and suggest the involvement of Sertoli cells.

Cyclophilin A is present in rat germ cells and is associated with spermatocyte apoptosis. Reproductive Toxicology Group.

Recent investigations in our laboratory revealed divalent cation-dependent endonuclease activity in testes from 2-methoxyethanol-treated rats, which was able to cleave substrate DNA into a pattern of DNA fragmentation consisting of approximately 180-200 base pairs. Further studies were undertaken to characterize the active nuclease. F344 rats were treated with 2-methoxyethanol, a glycol ether that causes the death of pachytene spermatocytes in juvenile and adult rats. The active nuclease was found in nuclear extract from treated animals, but not controls. A radioactive gel nuclease assay, which detects degradation and loss of 32P-labeled DNA from a DNA-containing polyacrylamide gel, localized the nuclease activity to a band of approximately 18 kDa. This activity was dependent on calcium and was inhibited by both zinc and aurintricarboxylic acid. Amino acid sequence data showed that this protein was identical to cyclophilin A. Immunohistochemistry using antibodies against cyclophilin A found specific staining in pachytene spermatocytes, spermatids, interstitial cells, and Sertoli cell nuclei. Cyclophilin A staining was present in both control and 2-methoxyethanol-treated rat testes in a stage-dependent manner, with pachytene spermatocytes in stage-VIII-XIV seminiferous tubules most heavily stained. These data demonstrate that rat testis germ cells contain relatively high levels of cyclophilin A whose nuclease activity is associated with spermatocyte apoptosis induced by 2-methoxyethanol.

Spermatocyte toxicity of 2-methoxyethanol (ME) in rats and guinea pigs: evidence for the induction of apoptosis.

2-Methoxyethanol (ME) produces testicular lesions characterized by pachytene spermatocyte degeneration in rats and guinea pigs which differ in onset, severity, and morphological characteristics. In the rat, degenerating spermatocytes appear necrotic at 24 hr, while in the guinea pig they appear apoptotic 96 hr after the start of three daily doses. To further examine if the spermatocyte degeneration in both species represented necrosis or apoptosis, the extent and nature of nuclear DNA fragmentation after ME exposure were assessed both visually using an in situ nucleotide 3' end-labeling (ISEL) procedure and by DNA gel electrophoresis. Testes from rats given a single oral dose of ME (200 mg/kg) showed the expected pachytene spermatocyte degeneration 24 hr after dosing, with the nuclear chromatin degradation typical of necrosis. In contrast, testes from guinea pigs given daily oral doses of ME (200 mg/kg) showed spermatocyte degeneration at only 96 hr after the start of dosing, with marked peripheral nuclear chromatin condensation characteristic of apoptosis. Coincident with the appearance of morphologic changes, degenerating spermatocytes in both species contained fragmented DNA as revealed by the ISEL procedure. The pattern of DNA fragmentation on agarose gels in both species consisted of ordered multiples or "ladders" of approximately 200 base pairs, a hallmark of apoptosis, with their appearance coincident with the time course of morphologic spermatocyte degeneration and ISEL staining. Preliminary data reveal the appearance of divalent metal cation-dependent endonuclease activity at pH 7.0 in ME-treated immature (24-day-old) rat testis that produces a similar pattern of DNA fragmentation and which appears to be distinct from activity associated with the spontaneous germ cell degeneration observed in testes of this age. In summary, in vivo ME exposure induces spermatocyte apoptosis in both the rat and guinea pig despite differing morphological classifications and time of onset of cell death. Future studies will focus on further characterization of the testicular endonuclease in the rat and the potential role of increased intracellular Ca2+ as a "triggering" stimulus in ME-induced spermatocyte apoptosis.

Protection against methoxyacetic-acid-induced spermatocyte apoptosis with calcium channel blockers in cultured rat seminiferous tubules: possible mechanisms.

A calcium-mediated mechanism underlying spermatocyte apoptosis induced by 2-methoxyethanol (2-ME) has been previously proposed. This hypothesis was tested in vitro in the present study using cultured juvenile (25 days old) and adult rat seminiferous tubules (JRST and ARST, respectively) with methoxyacetic acid (MAA, the active metabolite of 2-ME). In JRST, spermatocyte degeneration was morphologically obvious 19 hr after a 5-hr exposure to 5 mM MAA. The lesion was unaffected by the presence or absence of extratubular Ca2+. However, MAA-induced cell death was significantly prevented by cotreatment with the dihydropyridines (DHP) nifedipine (50 microM) and nicardipine (20 microM), as well as verapamil (50 microM) and TMB-8 (50 microM), all of which are able to inhibit calcium movement through plasma membranes. However, neither ryanodine, dantrolene, nor cyclosporin A and ruthenium red, which inhibit Ca2+ mobilization from intracellular stores (endoplasmic reticulum and mitochondria), affected the MAA-induced cell death. Inhibition of calcium mobilization through IP3-sensitive pathways by blocking the product of IP3 with manoalide, neomycin, and U73122 did not block the MAA-induced lesion. The protective effects of 50 microM nifedipine and 50 microM TMB-8 were also observed in ARSTs treated with 10 mM MAA for 5 hr. However, when rat testicular sections were immunohistochemically stained with monoclonal antibodies specific for the alpha 1 (the DHP receptor) or the alpha 2 subunits of DHP-sensitive calcium channels, no positive staining was found. Finally, in an attempt to see whether the intracellular free calcium concentrations ([Ca2+]i) in germ cells were increased after the MAA treatment, intact seminiferous tubules were loaded with indo-1 and were measured using laser-scanning confocal microscopy. No detectable increase in the signal in MA A-sensitive spermatocytes was observed, while a 34-54% increase in the signal could be detected in the same cell types when tubules were exposed to 10 microM of the calcium ionophore 4-bromo-A23187 for 5 min. Collectively, these data suggest that the protective effect of calcium channel blockers against the MAA-induced spermatocyte apoptosis is probably not through their blocking effect on DHP-sensitive calcium channels. We postulate alternate mechanisms based on stabilization of cells membranes, or interactions with calmodulin or protein kinase C.

The effects of dietary boron on bone strength in rats.

Previous studies from our laboratory found that when boric acid (BA) was administered in the diet to rats, boron levels in bone were approximately fourfold greater than serum levels. The current studies were undertaken to determine if these elevations produced adverse effects on several bone-related measures, including serum electrolyte levels, bone structure, and bone strength. Data from two studies are presented: in the first study, young adult male rats consumed a powdered diet containing 0, 3000, 4500, 6000, or 9000 ppm BA for 9 weeks. Endpoints were serum calcium, phosphorous, potassium, and chloride, as well as blood and bone boron concentrations ([B]) measured weekly during the 9-week exposure period, and at 8, 16, 24, and 32 weeks after the end of exposure. In the second study, the male and female young adult rats diet contained 0, 200, 1000, 3000, or 9000 ppm BA for 12 weeks; endpoints measured weekly were serum levels of calcium, phosphorous, and magnesium, bone [B], and bone structure (humerus) and strength (tibia, femur, and lumbar vertebrae). In treated rats, calcium was reduced in the first study but not the second. Serum phosphorous was reduced in both studies; potassium was unchanged, chloride was increased by 1%, and magnesium was reduced in all BA-exposed groups in the second study, to a maximal 19% reduction. Bone [B] was consistently increased in all treated groups, to concentrations approximately fourfold those of serum. After cessation of exposure, serum and urinary boron concentrations dropped to within control values within a week. However, even 32 weeks after the end of exposure, bone [B] remained threefold greater than controls. Male tibia and femur resistance to bending was unchanged. However, vertebral strength in compression was significantly increased by 5-10% in all dose groups (200 to 9000 ppm). The pattern was substantially similar in females. Only the humerus was examined by light microscopy and was found to be unchanged at any level of BA consumption. These data show that, despite a reduction in some serum electrolyte levels, BA consumption increased vertebral resistance to crush force, without detectably altering the microscopic structure of the humerus or the resistance of femur and tibia to a bending load. This increase in compression resistance occurred at exposure levels substantially below those that were previously reported to be reproductively toxic.