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Barbara F Hales

Publications and source records attributed to Barbara F Hales.

27 records · Page 2Linked to original sources
PubMed2003

Caspase-3 mediates retinoid-induced apoptosis in the organogenesis-stage mouse limb.

BACKGROUND: Caspases are key mediators in the regulation and execution of apoptosis, a crucial part of the morphogenetic process during limb development. Caspase-8 and -9 are upstream caspases. Caspase-8 mediates the extrinsic pathway of apoptosis triggered by signaling through TNF-R1 family receptors. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria. Caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Vitamin A is a potent teratogen that causes limb reduction defects in embryos exposed during organogenesis. Previous in vitro studies have shown that exposure of the organogenesis-stage murine limb to vitamin A results in excessive levels of apoptosis. The goal of this work was to characterize the involvement of caspase-3, -8, and -9, as well as cytochrome-c release from the mitochondria, in the apoptotic cascade induced by vitamin A. METHODS: Limb buds from gestational day 12 CD-1 mice were cultured in a chemically defined medium in the absence or presence of vitamin A. Cultures were terminated after 6 days to examine the effect of the drug on gross morphology. Apoptosis was detected by TUNEL staining after culture for 24 hr. Caspase activation was determined by Western blotting and localized by immunohistochemistry of control and treated limbs. The release of cytochrome-c into the cytoplasm was assessed by Western blotting after cell-fractionation. RESULTS: Limbs cultured in the presence of vitamin A showed a dose-dependent growth reduction and dysmorphogenesis of the cartilaginous anlagen. Apoptosis was increased in the interdigital, anterior, and posterior marginal zones and in the apical ectodermal ridge. Western-blotting confirmed the presence of activated caspase-3 that increased with time in culture and vitamin A concentration. Cleaved caspase-3 immunoreactivity colocalized with TUNEL stained limb regions and increased dramatically with increasing drug concentrations. In contrast, procaspase-8 and -9 were not activated. Exposure to high concentrations of vitamin A did, however, increase cytoplasmic cytochrome-c, suggesting mitochondrial involvement. CONCLUSIONS: Caspase-3 is a key effector caspase in the apoptotic pathway induced by Vitamin A. While caspases-8 and -9 are not responsible for the activation of caspase-3 in response to the drug, cytochrome-c release from mitochondria may play an upstream role.

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PubMed2002

DNA repair during organogenesis.

DNA damage caused by genotoxic agents can impact on virtually any cellular process due to its ability to affect gene expression and subsequent gene products. The importance of repairing damaged DNA is evidenced by the variety of DNA repair pathways that have evolved in all living organisms, and the human syndromes caused by a lack of this repair ability. This review focuses on the expression and activity of DNA repair pathways during mammalian organogenesis, and the role of these pathways in ensuring the stability of the conceptal genome. DNA repair capacity may play a role also in the response of the conceptus to genotoxic agents that may induce malformations; the consequences of exposure to a genotoxic agent during organogenesis depend on the extent of the damage and on the ability of the embryo to respond by repairing DNA or arresting cell division. The four main repair pathways (nucleotide excision repair, base excision repair, mismatch repair, and recombination repair) are expressed to various degrees during organogenesis, as are members of the genotoxic stress-activated cell cycle checkpoint pathways. Developmental-stage-specific alterations in transcript levels, protein levels, as well as activity, indicate that the regulation of DNA repair pathways during development is complex. The importance of DNA repair pathways in endogenous damage control is illustrated by the sensitivity of development to their disruption if some of these genes are mutated. Furthermore, the conceptus has a limited capacity to alter DNA repair responses following exposure to genotoxic agents.

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PubMed2002

Expression and activity of the DNA repair enzyme uracil DNA glycosylase during organogenesis in the rat conceptus and following methotrexate exposure in vitro.

Uracil incorporation into DNA occurs under conditions that limit thymidine biosynthesis; uracil is removed by two isoforms of uracil DNA glycosylase (UNG; EC 3.2.2.3), UNG1 and UNG2. We hypothesize that UNG is important in protecting the mid-organogenesis stage [gestational day (GD) 10-12] rat conceptus against conditions that limit thymidine biosynthesis. Transcripts for both UNG isoforms were expressed highly in the yolk sac and embryo, increasing over 400% in the embryo between GD11 and 12. GD10 and 11 yolk sacs showed the highest levels of putatively active UNG2 protein, with little UNG1 protein. Moderate levels of UNG2 and UNG1 proteins were found in the embryo on GDs 10 through 12; no significant increase in either protein occurred on GD12. UNG activity was higher in yolk sac than embryo on GDs 10 and 11, mirroring protein levels. Exposure to the teratogen methotrexate (MTX) leads to nucleotide pool imbalance, uracil incorporation into DNA, and genotoxic stress-induced cell death. Concentration-dependent decreases in developmental growth parameters, decreased yolk sac vasculature, and malformations such as kinked tail and retarded limb development were observed in embryos exposed to MTX (0.5, 2.5, or 5 microM). UNG transcripts were elevated 30-40% in both yolk sac and embryo after a 6-hr culture with 0.5 microM MTX; however, protein expression and activity were unaffected. Thus, MTX exposure caused malformations but did not modify UNG protein expression or activity, indicating an inability to increase the removal of MTX-induced genotoxic damage. Furthermore, UNG expression was developmental stage- and tissue-specific; the discrepancy between transcript and protein levels suggests post-transcriptional regulation.

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PubMed2002

Buthionine sulfoximine embryotoxicity is associated with prolonged AP-1 activation.

BACKGROUND: Many teratogens induce oxidative stress, altering redox status and redox signaling; this has led to the suggestion that developmental toxicants act by disturbing redox status. The goal of these studies was to determine the consequences of altering glutathione homeostasis during organogenesis on embryo development, total DNA methylation, and activator protein-1 (AP-1) DNA binding activity and gene expression. METHODS: Gestational day 10.5 rat embryos were cultured in vitro for up to 44 hour in the presence of L-buthionine-S,R-sulfoximine (BSO), an irreversible inhibitor of gamma-glutamyl-cysteine synthetase, the rate limiting step in glutathione biosynthesis. Effects of BSO on total, oxidized and reduced glutathione, embryo development, DNA methylation, AP-1 DNA binding activity and gene expression were investigated. RESULTS: Significant depletion of glutathione by BSO was first noted at 6 hr in the embryo and at 3 hr in the yolk sac; total glutathione in the conceptus was depleted to the same extent after treatment with either 0.1 or 1.0 mM BSO. Exposure to 0.1 mM BSO did not cause a significant increase in embryotoxicity, although some impairment of growth and development was observed. In contrast, exposure to 1.0 mM BSO severely inhibited growth and development, significantly increasing the incidence of swollen hindbrains and of blebs in the forebrain, limb and maxillary regions. No significant treatment-related differences in total DNA methylation were observed. Interestingly, AP-1 DNA binding activity was similar in control and 0.1 mM BSO-treated conceptuses; however, exposure to 1.0 mM BSO increased AP-1 DNA binding at 6, 24, and 44 hr. The expression of several AP-1 family genes and of gamma-glutamylcysteine synthetase was induced in embryos cultured with 1.0 mM BSO. CONCLUSION: Exposure of embryos in vitro to BSO at a concentration that was embryotoxic induced prolonged AP-1 DNA binding activity and altered gene expression. These data suggest that AP-1 induction may serve as a biomarker of embryo stress.

Animals↗
PubMed2002

Role of caspases in murine limb bud cell death induced by 4-hydroperoxycyclophosphamide, an activated analog of cyclophosphamide.

BACKGROUND: Caspases play a pivotal role in the regulation and execution of apoptosis, an essential process during limb development. Caspase 8 activation is usually downstream of the Fas/FasL death receptors, whereas caspase 9 mediates the mitochondrial signaling pathway of apoptosis. Caspase 3 is an effector caspase. Previous studies have shown that the exposure of embryonic murine limbs in vitro to 4-hydroperoxycyclophosphamide (4-OOHCPA), an activated analog of the anticancer alkylating agent, cyclophosphamide, induced limb malformations and apoptosis. The goal of this study was to determine the role of caspases in mediating apoptosis in this model system. METHODS: Limb buds from gestational day 12 CD-1 mice were excised and cultured in roller bottles in a chemically defined medium for up to 6 days in the absence or presence of 4-OOHCPA. Apoptosis was indicated by internucleosomal DNA fragmentation, as detected by TUNEL staining. The profile of caspase activation was characterized by Western blot analysis and immunohistochemistry of control and treated limbs. To determine the consequences to limb morphology of inhibiting caspase activation, DEVD-CHO, a caspase-3 inhibitor, was added to the cultures. RESULTS: Limbs cultured in the presence of 4-OOHCPA were growth retarded and malformed; apoptosis was increased in the apical ectodermal ridge and interdigital areas. Western blot analysis showed that 4-OOHCPA exposure did not activate procaspases 8 or 9 in limbs. In contrast, procaspase-3 cleavage was increased in a concentration and time-dependent manner after exposure of limbs to 4-OOHCPA. Immunoreactive activated caspase-3 was localized in the interdigital areas and the apical ectodermal ridge region in control limbs; staining in these areas and in the interdigital areas was increased dramatically in limbs exposed to 4-OOHCPA. Inhibition of caspase 3 activation with DEVD-CHO partially protected limbs from insult with 4-OOHCPA. CONCLUSION: Caspase-dependent and caspase-independent pathways of cell death are both important is mediating the abnormal limb development triggered by insult with 4-OOHCPA.

Animals↗
PubMed2002

Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells.

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.

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PubMedDate not supplied

Spermiogenic germ cell phase-specific DNA damage following cyclophosphamide exposure.

The production of genetically competent spermatozoa is essential for normal embryo development. The chemotherapeutic drug cyclophosphamide creates cross-links and DNA strand breaks in many cell types, including germ cells. This study assessed the phase specificity of the susceptibility of spermiogenic germ cells to genetic damage induced by cyclophosphamide. Adult male rats were given cyclophosphamide using one of four schedules: 1) high dose/acute- day 1, 100 mg/kg; 2) low dose/subchronic, 4 days-days 1-4, 6.0 mg/kg/d; 3) high dose/subchronic, 4 days-day 1, 100 mg/kg, and days 2-4, 50 mg/kg/d; and 4) low dose/chronic-daily, 6.0 mg/kg/d for 14-28 days. To capture cauda epididymal spermatozoa exposed to cyclophosphamide during late, mid-, and early spermiogenesis, animals were sacrificed on days 14, 21, and 28, respectively. Spermatozoa were analyzed for DNA strand breaks using the comet assay. No dramatic increases in damage were seen after high-dose/acute exposure to cyclophosphamide. Subchronic exposure showed a dose-related increase in DNA damage; maximal damage, as demonstrated by comet tail parameters, was seen after 21 days, reflecting an increased susceptibility of step 9-14 spermatids. Low-dose chronic exposure to cyclophosphamide induced DNA damage, which reached a plateau by day 21. The magnitude of damage at all time points after low-dose chronic exposure was much greater than that following low-dose exposure for 4 days, indicating an accumulation of damage over time. Thus, the DNA damage induced by cyclophosphamide is germ cell phase-specific. The most damaging effects of cyclophosphamide occurred during a key point of sperm chromatin remodeling (histone hyperacetylation and transition protein deposition). We speculate that strand breaks disrupt chromatin remodeling, hence affecting chromatin structure and embryo development.

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