Testing of retinoids for teratogenicity in vitro: Use of micromass limb bud cell culture

Kistler, Andreas D.; Howard, W. Brian

doi:10.1016/0076-6879(90)90048-6

Cited by 10 publications

(6 citation statements)

References 5 publications

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“…No evidence based on chondrogenesis inhibition using CA and QN has been reported in the literature. This research validates the results of Flint and Orton [19] and Kistler and Howard [21], according to them RA and HC have teratogenic effects but CA and QN do not have teratogenic effects on fetal LB cells. RA-induced teratogenesis has been discovered in cultured periotic mesenchyme + otic epithelium from either E10.5 CBA C57/BL6 embryos or E13 Dlx5 mutant embryos [31].…”

supporting

confidence: 89%

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Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

et al. 2014

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CYP1A1, a P450 isoenzyme, is involved in the phase I xenobiotic metabolism including teratogen drugs. In the present study, the ability of teratogens to elevate the embryonic expression of CYP1A1 was examined. Micromass cell cultures prepared from day 13 rat embryo limb buds (LB). LB cells were cultivated and exposed for 5 days to retinoic acid (RA), hydrocortisone (HC), caffeine (CA) and quinine (QN). CYP1A1 protein expression and activity were measured using immunofluorescence staining and ethoxyresorufin O-deethylation (EROD) assay, respectively. The EROD activity increased significantly following LB cells exposure to RA and HC (p<0.05) but the expression of CYP1A1 protein was reduced by these drugs, whereas the expression of CYP1A1 protein and EROD activity decreased significantly following the addition of CA and QN (p<0.05, p<0.01). Our findings show that studied teratogens have potency to increase CYP1A1 activity.

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confidence: 89%

“…Differentiation of chondrocytes was determined according to the method of Kistler and Howard [21]. The absorbance at 620 nm is verified spectrophotometrically.…”

Section: Alcian Blue Staining For Assessment Of Differentiationmentioning

confidence: 99%

Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

et al. 2014

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“…Cultures were maintained in a humidified incubator at 37°C in an atmosphere of 95% O 2 per 5% CO 2 (v/v), allowing the cells to attach to the plate surface. These conditions are found to promote the maintenance and viability of LBCs (Kistler and Howard, 1987). Cells were cultured for 24–48 hr before transfection.…”

Section: Methodsmentioning

confidence: 99%

Misregulation of gene expression in the redox‐sensitive NF‐κb‐dependent limb outgrowth pathway by thalidomide

Hansen

Gong

Philbert

et al. 2002

Developmental Dynamics

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Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide-induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox-sensitive processes, such as transcription factor activation and/or binding. Nuclear factorkappa B (NF-B), a redox-sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide-induced redox shifts. Thalidomide-resistant Sprague-Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide-sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF-B and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf-10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF-B binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N-acetylcysteine and ␣-N-t-phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide-treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf-8, and Fgf-10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf-8, and Fgf-10 expression. These findings show that NF-B binding is diminished due to selective thalidomide-induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth.

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“…The micromass limb bud cell culture technique used was slightly modified from the original rat method as described by Flint and Orton (1984), Kistler and Howard (1987), and Kistler (1987). On GD 13 in rats and GD 12 in rabbits (similar developmental periods), animals were anesthetized with ether, and embryos were gathered and placed in warmed HBSS, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Thalidomide Modulates Nuclear Redox Status and Preferentially Depletes Glutathione in Rabbit Limb versus Rat Limb

Hansen¹,

Harris²,

Philbert³

et al. 2002

J Pharmacol Exp Ther

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Thalidomide produces numerous birth defects, the most notable being phocomelia. Mechanisms behind thalidomide-induced malformations have not been fully elucidated, although recent evidence suggests a role for reactive oxygen species. A thalidomide-resistant (rat) and -sensitive (rabbit) species were used to compare potential inherent differences related to oxidative stress that may provide a more definitive understanding of mechanisms of thalidomide embryopathy. Limb bud cells (LBCs) were removed from the rat and rabbit embryo, dissociated, and plated in culture for 24 h. A fluorescence (6-carboxy-2Ј,7Ј-dichlorofluorescin diacetate; DCF) assay for oxidative stress was used with varying concentrations of thalidomide (5-100 M). Thalidomide (100 M) showed a 6-fold greater production of oxidative stress in rabbit cultures than in rat. Lower concentrations (50 and 25 M) also showed a significant increase in reactive oxygen species. Confocal microscopy revealed DCF fluorescence preferentially in rabbit LBC nuclei compared with the uniform distribution of DCF fluorescence in rat LBC. Localization of glutathione (GSH) was determined using 5-chloromethylfluorescein diacetate fluorescent confocal microscopy. In rat cultures, significant thalidomide-induced GSH depletion was detected in the cytosol but the nuclei maintained its GSH content, but rabbit LBC showed significant GSH depletion in both compartments. GSH depletion was confirmed by high-performance liquid chromatography analysis. These observations provide evidence that thalidomide preferentially produces oxidative stress in the thalidomide-sensitive species but not the thalidomide-resistant species. Nuclear GSH content in the rabbit LBC is selectively modified and indicates a shift in the nuclear redox environment. Redox shifts in the nucleus may result in the misregulation of transcription factor/ DNA interactions and cause defective growth and development.

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Testing of retinoids for teratogenicity in vitro: Use of micromass limb bud cell culture

Cited by 10 publications

References 5 publications

Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

Effects of Teratogenic Drugs on CYP1A1 Activity in Differentiating Rat Embryo Cells

Misregulation of gene expression in the redox‐sensitive NF‐κb‐dependent limb outgrowth pathway by thalidomide

Thalidomide Modulates Nuclear Redox Status and Preferentially Depletes Glutathione in Rabbit Limb versus Rat Limb

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