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Robert E Chapin - One of the best experts on this subject based on the ideXlab platform.

  • protein kinase activity is central to rat germ cell apoptosis induced by Methoxyacetic Acid
    Toxicologic Pathology, 2001
    Co-Authors: Toshimasa Jindo, Robert N Wine, Robert E Chapin
    Abstract:

    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.

  • protection against Methoxyacetic Acid induced spermatocyte apoptosis with calcium channel blockers in cultured rat seminiferous tubules possible mechanisms
    Toxicology and Applied Pharmacology, 1997
    Co-Authors: Robert N Wine, David S Miller, Jeffrey M Reece, Marjo V Smith, Robert E Chapin
    Abstract:

    Abstract A calcium-mediated mechanism underlying spermatocyte apoptosis induced by 2-methoxyethanol (2-ME) has been previously proposed. This hypothesis was testedin vitroin 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 m m 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 μ m ) and nicardipine (20 μ m ), as well as verapamil (50 μ m ) and TMB-8 (50 μ m ), 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 IP3with manoalide, neomycin, and U73122 did not block the MAA-induced lesion. The protective effects of 50 μ m nifedipine and 50 μ m TMB-8 were also observed in ARSTs treated with 10 m m MAA for 5 hr. However, when rat testicular sections were immunohistochemically stained with monoclonal antibodies specific for the α1(the DHP receptor) or the α2subunits 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 MAA-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 μ m 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.

  • 2 Methoxyacetic Acid maa induced spermatocyte apoptosis in human and rat testes an in vitro comparison
    Journal of Andrology, 1996
    Co-Authors: Robert N Wine, Robert E Chapin
    Abstract:

    ABSTRACT: 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 ≥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 intemucleosomal 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.

Karen Smet - One of the best experts on this subject based on the ideXlab platform.

Kenneth S Korach - One of the best experts on this subject based on the ideXlab platform.

  • Research The Short-Chain Fatty Acid Methoxyacetic Acid Disrupts Endogenous Estrogen Receptor-α–Mediated Signaling
    2013
    Co-Authors: Derek V Henley, Stephanie Mueller, Kenneth S Korach
    Abstract:

    Bac k g r o u n d: Ethylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is Methoxyacetic Acid (MAA), a shortchain fatty Acid that inhibits histone deacetylase activity and alters gene expression. Objective: Because estrogen signaling is necessary for normal reproductive function and modulates gene expression, the estrogen-signaling pathway is a likely target for MAA; however, little is known about the effects of MAA in this regard. Me t h o d s: We evaluated the mechanistic effects of MAA on estrogen receptor (ER) expression and estrogen signaling using in vitro and in vivo model systems. Re s u l t s: MAA potentiates 17β-estradiol (E 2) stimulation of an estrogen-responsive reporter plasmid in HeLa cells transiently transfected with either a human ERα or ERβ expression vector containing a cytomegalovirus (CMV) promoter. This result is attributed to increased exogenous ER expression due to MAA-mediated activation of the CMV promoter. In contrast to its effects on exogenous ER, MAA decreases endogenous ERα expression and attenuates E 2-stimulated endogenous gene expression in both MCF-7 cells and the mouse uterus. Con c l u s i o n s: These results illustrate the importance of careful experimental design and analysi

  • the short chain fatty Acid Methoxyacetic Acid disrupts endogenous estrogen receptor α mediated signaling
    Environmental Health Perspectives, 2009
    Co-Authors: Derek V Henley, Stephanie Mueller, Kenneth S Korach
    Abstract:

    BackgroundEthylene glycol monomethyl ether (EGME) exposure is associated with impaired reproductive function. The primary metabolite of EGME is Methoxyacetic Acid (MAA), a short-chain fatty Acid th...

  • endocrine disrupting chemicals use distinct mechanisms of action to modulate endocrine system function
    Endocrinology, 2006
    Co-Authors: Derek V Henley, Kenneth S Korach
    Abstract:

    The term endocrine-disrupting chemicals is used to define a structurally diverse class of synthetic and natural compounds that possess the ability to alter various components of the endocrine system and potentially induce adverse health effects in exposed individuals and populations. Research on these compounds has revealed that they use a variety of both nuclear receptor-mediated and non-receptor-mediated mechanisms to modulate different components of the endocrine system. This review will describe in vitro and in vivo studies that highlight the spectrum of unique mechanisms of action and biological effects of four endocrine-disrupting chemicals–diethylstilbestrol, genistein, di(n-butyl)phthalate, and Methoxyacetic Acid–to illustrate the diverse and complex nature of this class of compounds.

Donald P. Mcdonnell - One of the best experts on this subject based on the ideXlab platform.

  • Copyright � American Society of Andrology Increased Expression of Estrogen Receptor � in Pachytene Spermatocytes After Short-Term Methoxyacetic Acid Administration
    2013
    Co-Authors: Oscar M. Tirado, David M. Selva, Núria Toràn, Carlos A. Suárez-quian, Donald P. Mcdonnell, Jaume Reventós, Francina Munell, Departament D’anatomia Patològica, Hospital Materno-infantil Vall
    Abstract:

    ABSTRACT: Degeneration of primary spermatocytes by apoptosis occurs during normal spermatogenesis, as well as in several pathological conditions, including exposure to specific testicular toxicants. The mechanisms that regulate the death and survival of primary spermatocytes, however, are still not well understood. The recent localization of estrogen receptor beta (ER�) and P450 aromatase in pachytene spermatocytes suggests a role for estrogens in this step of spermatogenesis. Using a well-known model of pachytene spermatocyte apoptosis in adult rats consisting of the administration of Methoxyacetic Acid (MAA), we investigated the participation of ER � during the initial phase of apoptosis, prior to germ cell loss. Adult rats were treated with a single intraperitoneal dose of MAA, and DNA laddering analysis confirmed apoptotic cell death in the testis. In enriched germ cell fractions and testis from MAA-treated animals, ER � mRNA increased significantly at 3 and 6 hours

  • short chain fatty Acids enhance nuclear receptor activity through mitogen activated protein kinase activation and histone deacetylase inhibition
    Proceedings of the National Academy of Sciences of the United States of America, 2004
    Co-Authors: Michelle S Jansen, Susan C Nagel, Phillippa J Miranda, Edward K Lobenhofer, Cynthia A Afshari, Donald P. Mcdonnell
    Abstract:

    In this study, we demonstrate that the pervasive xenobiotic Methoxyacetic Acid and the commonly prescribed anticonvulsant valproic Acid, both short-chain fatty Acids (SCFAs), dramatically increase cellular sensitivity to estrogens, progestins, and other nuclear hormone receptor ligands. These compounds do not mimic endogenous hormones but rather act to enhance the transcriptional efficacy of ligand activated nuclear hormone receptors by up to 8-fold in vitro and in vivo. Detailed characterization of their mode of action revealed that these SCFAs function as both activators of p42/p44 mitogen-activated protein kinase and as inhibitors of histone deacetylases at doses that parallel known exposure levels. Our results define a class of compounds that possess a dual mechanism of action and function as hormone sensitizers. These findings prompt an evaluation of previously unrecognized drug–drug interactions in women who are administered exogenous hormones while exposed to certain xenobiotic SCFAs. Furthermore, our study highlights the need to structure future screening programs to identify additional hormone sensitizers.

  • increased expression of estrogen receptor β in pachytene spermatocytes after short term Methoxyacetic Acid administration
    Journal of Andrology, 2004
    Co-Authors: Oscar M. Tirado, David M. Selva, Núria Toràn, Donald P. Mcdonnell, Jaume Reventós, Carlos A Suarezquian, Michelle S Jansen, Francina Munell
    Abstract:

    Degeneration of primary spermatocytes by apoptosis occurs during normal spermatogenesis, as well as in several pathological conditions, including exposure to specific testicular toxicants. The mechanisms that regulate the death and survival of primary spermatocytes, however, are still not well understood. The recent localization of estrogen receptor beta (ERbeta) and P450 aromatase in pachytene spermatocytes suggests a role for estrogens in this step of spermatogenesis. Using a well-known model of pachytene spermatocyte apoptosis in adult rats consisting of the administration of Methoxyacetic Acid (MAA), we investigated the participation of ERbeta during the initial phase of apoptosis, prior to germ cell loss. Adult rats were treated with a single intraperitoneal dose of MAA, and DNA laddering analysis confirmed apoptotic cell death in the testis. In enriched germ cell fractions and testis from MAA-treated animals, ERbeta mRNA increased significantly at 3 and 6 hours, respectively. Next, stage-specific induction of ERbeta mRNA was demonstrated by use of laser capture microdissection of seminiferous tubules in combination with semiquantitative reverse transcription-polymerase chain reaction. The ERbeta protein also increased significantly after 6 hours and was mainly immunolocalized in the cytoplasm of pachytene spermatocytes of afflicted tubules. The cytoplasmic localization was confirmed by Western blot analysis of isolated cytoplasmic and nuclear fractions of testicular extracts. Finally, the MAA activation of ERbeta was tested in vitro in HepG2 cells cotransfected with ERbeta and a reporter construct that contained a consensus estrogen responsive element. Addition of MAA at similar doses used in vivo elicited a similar estrogenic activation as did estradiol at 1 nmol/L concentration. The present results raise the possibility that cytoplasmic ERbeta participates in the apoptotic process of pachytene spermatocytes induced by MAA. Whether MAA interacts with ERbeta in the cytoplasm of primary spermatocytes, preventing the progression of the first meiotic division, however, remains to be determined.

Sri Sudarwati - One of the best experts on this subject based on the ideXlab platform.

  • Methoxyacetic Acid induced apoptosis on the forelimb bud of swiss webster mice
    Hayati Journal of Biosciences, 2011
    Co-Authors: Agus Haryono, Lien A. Sutasurya, Tien Wiati Surjono, Sri Sudarwati
    Abstract:

    Methoxyacetic Acid (MAA) causes digit malformations of mice when it was given orally on gestation day 11. Previous observation showed that malformation was caused by cell death. The aims of the research were to determine the types of cell death, first time of cell death and their distribution pattern on forelimb bud of Swiss Webster (SW) mice. Ten mM/kg of body weight (bw) of MAA were administered by gavage to SW mice on gestation day 11. Forelimb bud of mouse embryos of gestation day 11 + 0, 1, 2, 3, 4, 5 hours were processed with paraffin method and were made plantar section. Cell death at plantar section were colored with 4,6-diamino-2-phenylindole hydrochloride (DAPI) and hematoxylin. The result showed, that digit malformations initially by apoptosis mesenchymal cell at proximal of axial mesoderm in around of primary axial artery has done one hour after treatment. Apoptosis at the axial area, the site formation of digital ray III distributed to preaxial area where digits I and II are formed, and to the site formation of digits IV and V. The number of mesenchyme cell of digital rays II, III, and V was decrease by the increasing of gestation day, while digital ray was not formed and finally digits I, II, III, and V were missing. The reduction number of cell of digital ray IV were delayed time to be formed and its small size. Thereby it can be concluded, that MAA induced digit malformations of SW mice started by apoptosis which is occurrence has been increase in area of digital ray formation, so that digital ray can not be formed, but when formed it will not developed.

  • a teratoproteomics analysis heat shock protein 70 is upregulated in mouse forelimb bud by Methoxyacetic Acid treatment
    Birth Defects Research Part A-clinical and Molecular Teratology, 2005
    Co-Authors: Aceng Ruyani, Sri Sudarwati, Lien A. Sutasurya, Sony H. Sumarsono, Dong Jo Kim, Jun Ho Chung
    Abstract:

    Methoxyacetic Acid (MAA) causes fetal limb abnormalities when the substance is administrated on gestation day (GD) 11 in mice. Limb abnormalities are caused mainly by extensive cell death in the mesoderm of the limb plate. This investigation focused on identifying a protein that is linked with mouse limb teratogenicity.A single dose of MAA at 10 mmol/kg body weight was administered by gavage on GD 11; controls were administered vehicle only. Dams were killed by cervical dislocation 4 hr after treatment and forelimb buds were isolated from both the control and treated embryos. Proteins in forelimb buds GD 11 + 4 hr were precipitated out using 40-60% ammonium sulfate and were then analyzed by 2D SDS-PAGE. Excised protein spots were identified by mass spectrometry and amino Acid internal sequence analysis. Identified protein was further confirmed by Western blotting.Two-dimensional gel analysis indicated that 1 protein spot of 81.7 kDa/pI 7.3 was overexpressed, and the protein matched heat shock protein 70 (HSP70; accession no. P08109, SwissProt).The results suggest that MAA, when administered to pregnant mice, upregulates HSP70 in the forelimb buds.

  • maternal contribution in revealing the effects of Methoxyacetic Acid maa administered before implantation on the embryonic development of swiss webster mice mus musculus
    ANNALES BOGORIENSES, 2004
    Co-Authors: Ekayanti Mulyawati Kaiin, Tien Wiati Surjono, Sony H. Sumarsono, Sri Sudarwati
    Abstract:

    Maternal contribution to and direct action of Methoxyacetic Acid (MAA) on the embryonic development bad been examined by conducting embryo tTansfer. To reveal the maternal contribut ion, compacted morulae and early blastocysts, which were collected from untreated Swiss Webster donor mice on day 3 of gestation, were transferred to day 2 pseudopregnant recipients, after having been treated with 2 .0 mmollkg body weight (b.w.) MAA by gavage on day 1 of pseudopregnancy. Direct effect of MAA on the embryonic development were observed by transferring compacted morulae and early bla tocysts, simi larly recovered from day 3 pregnant donor mice, after MAA treatment on day 2 of gestation with the same method and dosing, to untreated day 2 pseudopregnant recipients. Control donor mice and recipient were given distilled water only as the MAA olvent. Observations on fetuses resulting fTom embryo transfer wert: carried out on day 16 of gestatlOn . Administration of MAA to the donors tended to decrease the unplantatlOn rate and the survival rate of the implanted embryos. W11en MAA was given to the recipients the implantation rate and survival rate of embryos transferred decreased significantly (p<0 .05) but the survival rate of implanted embryos were significantly higher (p<0.05) Lf compared to those of MAA treated donors. The intrauterine death tended to inc rease either in the treated donors or recipients. Th re was no et1ect of MAA on the fetal body weight and in producing fetal malformations . It is concluded that at the beginning of implantation, maternal contribution in revealing the effects of MAA on the embryonic development of Swiss Webster mice is predominant, whereas after Implantation took place, the quality of the embryos become ';lore important for their survival.

  • The laminin binding protein p40 is involved in inducing limb abnormality of mouse fetuses as the effects of Methoxyacetic Acid treatment
    Toxicological Sciences, 2003
    Co-Authors: Aceng Ruyani, Sri Sudarwati, Lien A. Sutasurya, Sony H. Sumarsono, Torsten Gloe
    Abstract:

    This study is intended to characterize a protein that is linked with mouse limb teratogenicity as the effects of Methoxyacetic Acid (MAA) treatment. A single dose of MAA (10 mmol/kg body weight) was given by gavage on gestation day (GD) 11, whereas the control group were administered vehicle only. The pregnant mice were killed at 4 h after MAA treatment, and forelimb buds were isolated from both the control and treated group embryos. Proteins from forelimb buds GD 11 + 4 h, which were precipitated out using 40-60% ammonium sulfate, then were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) technique. The 2-D gels reveal one protein with 41.6 kDa and pI 6.4, which expression was downregulated after MAA treatment. Tentative protein identification via peptide mass database search and definitive protein identification via a primary sequence database search indicate that the protein matches exactly to 34/67 kDa laminin binding protein (LBP; P14206, SwissProt), which is encoded by p40 gene (MGI:105381). The identity was further verified by Western blotting with an antibody against the 67 kDa LBP. The results suggest that MAA treatment to pregnant mice downregulates the LBP-p40 in the forelimb buds.