A Simple Competitive Protein-Binding Assay for Adenosine-3′,5′-Monophosphate in Plasma and Urine

Sn, Madsen; Badawi, Ibrahim; Skovsted, L.

doi:10.1530/acta.0.0810208

Cited by 40 publications

(16 citation statements)

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“…cAMP and Tg were analyzed as previously described by a competitive protein binding method detected by a radioimmunoassay (RIA) and an enzyme-linked immunosorbent assay (ELISA), respectively [30;32]. To assess cAMP production, 3-isobutyl-1-methylxanthine was diluted in ethanol (final ethanol concentration 1%) and added to the cultures concurrently with DE-71 solutions.…”

Section: Methodsmentioning

confidence: 99%

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function in vitro

et al. 2017

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BackgroundNormal thyroid function is essential for general growth and metabolism, but can be affected by endocrine disrupting chemicals (EDCs). Polybrominated diphenyl ethers (PBDEs) have been used worldwide to reduce flammability in different materials and are suspected to be EDCs. The production of the commercial Penta- and OctaBDE mixtures is banned, but DecaBDEs and existing products may leak PBDEs into the environment. Our aim was to investigate the effect of the PentaBDE mixture DE-71 on human thyroid cells in vitro.Materials and methodsPrimary human thyroid cells were obtained as paraadenomatous tissue and cultured in monolayers. The influence of DE-71 on cyclic adenosine monophosphate (cAMP) and thyroglobulin (Tg) production was examined in the culture medium by competitive radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Real-time quantitative PCR analysis of thyroid-specific genes was performed on the exposed cell cultures. PBDE concentrations were determined in cellular and supernatant fractions of the cultures.ResultsDE-71 inhibited Tg-release from TSH-stimulated thyrocytes. At 50 mg/L DE-71, mean Tg production was reduced by 71.9% (range: 8.5–98.7%), and cAMP by 95.1% (range: 91.5–98.8%) compared to controls). Expression of mRNA encoding Tg, TPO and TSHr were significantly inhibited (p<0.0001, p = 0.0079, and p = 0.0002, respectively). The majority of DE-71 added was found in the cell fraction. No cytotoxicity was found.ConclusionsDE-71 inhibited differentiated thyroid cell functions in a two phase response manner and a concentration-dependent inhibition of Tg and cAMP production, respectively, as well as expression of mRNA encoding Tg, TPO and TSHr. Our findings suggest an inhibiting effect of PBDEs on thyroid cells.

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Section: Methodsmentioning

confidence: 99%

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function in vitro

et al. 2017

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“…Controls without cytokines were included in each culture plate. Cell func¬ tions were evaluated by the TSH-stimulated amount of cAMP (measured by a competitive protein binding assay (Madsen et al 1975)) and Tg in the medium after 72 h of cytokine exposure. 3-Isobutyl-L-methyl-xanthine (0-5 mmol/1), a phosphodiesterase inhibitor, was added during the last 72 h to inhibit the degradation of cAMP.…”

Section: Methodsmentioning

confidence: 99%

Influence of tumour necrosis factor-α, tumour necrosis factor-β and interferon-γ, separately and added together with interleukin-1 β, on the function of cultured human thyroid cells

Rasmussen

Kayser²,

Feldt‐Rasmussen³

et al. 1994

Journal of Endocrinology

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Interleukin (IL)-1, tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma have been demonstrated in thyroid tissue. We have previously shown that high concentrations of IL-1 inhibit and low concentrations stimulate human thyroid cell function in vitro. In the present study, TNF-alpha, TNF-beta and IFN-gamma all inhibited thyroglobulin (Tg) and cAMP production from cultured human thyroid cells. When TNF-alpha was added simultaneously with IL-1 beta, the highest concentration of TNF-alpha (10(6) U/l) enhanced the inhibition of Tg and cAMP induced by IL-1 beta (1-10(5) U/l). TNF-beta had no influence on IL-1 beta-induced inhibition. IFN-gamma (10(4) U/l) added together with IL-1 beta in lower concentrations (1-10(2) U/l) stimulated cAMP production, while at high concentrations of IL-1 beta (10(5) U/l), IFN-gamma enhanced the inhibitory influence of IL-1 beta on Tg production. The hormones of the immune system, IL-1, TNF and IFN-gamma, may thus contribute to the decreased thyroid function characteristic of some thyroid inflammatory diseases.

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“…3-isobutyl-L-methyl-xanthine (0.5 mmol/1) was supple¬ mented for measurement of cAMP. The function of the cells was evaluated by release of extracellular cAMP mea¬ sured by a competitive protein binding assay (7). The amount of cAMP accumulated extracellularly after 3 days is corresponding to the intracellular amount of cAMP (8).…”

Section: Methodsmentioning

confidence: 99%

Differential effects of interleukin 1α and 1β on cultured human and rat thyroid epithelial cells

Rasmussen

Kayser²,

Bech³

et al. 1990

Acta Endocrinologica

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The effects of human recombinant interleukin 1\g=a\(20 pg/1-2 \g=m\g/l ) and 1\g=b\(200 pg/1-20 \g=m\g/l ) on two systems of thyroid cells have been compared. The thyroglobulin and cAMP secretion and the DNA content of human thyroid cells cultured in monolayer and of continuously grown rat thyroid cells, Fischer rat thyroid cell line have been studied. The growth of the rat thyroid cell line was inhibited by interleukin 1\ g=b\(20 ng/1-20 \g=m\g/l ), but not by interleukin 1\g=a\.None of the cytokines changed the cAMP production of the rat thyroid cells. In contrast, both cAMP production and thyroglobulin secretion were inhibited dose-dependently by the cytokines in human thyroid cells in secondary cultures. These results caution the interpretation and extrapolation of changes induced by interleukin 1 from one cell system to the other.Interleukin la (IL-la) and interleukin Iß (IL-Iß) belong to a group of bioactive polypeptides (mol wt 17 kD) produced by macrophages, but also by a broad spectrum of other cells (1). These cytokines are extremely potent and assumed to play a central role in immuno-inflammatory processes (1). IL-lß has also been recognised as a potent inhibitor of the function of endocrine tissues, such as rat pancreatic B-islets (2) and human thyroid cells in monolayer (3). In primary and secondary cultures of human thyroid cells, recombinant IL-lß (rIL-lß) (200 pg/1-20 p.g/1) inhibited both extracellular cAMP and thyroglobulin (Tg) secretion of the cells (3).The effects of IL-lß and IL-Ia appear to be me¬ diated through the same cell membrane receptor on lymphocytes, and probably, on other cells (1).Even so, IL-la has been shown to be more potent than IL-lß on a molar basis, particularly in the ac¬ tivation of T-and B-lymphocytes (1). The influ¬ ence and potency of IL-1 a on thyroid cells have not previously been reported.In the present study we compared the effect of rIL-la and rIL-lß on the function and growth of human secondary thyroid cultures and on the Fis¬ cher rat thyroid cell line (FRTL-5) (4) grown in monolayer and under similar culture conditions. FRTL-5 is a well characterized continuously grow¬ ing differentiated thyroid cell line convenient for repeptitive experiments (5). Material and Methods FRTL-5 cellsThe FRTL-5 cells (ATCC CRL 8305) were obtained from P. Ealey, University College Hospital, London, UK. The cells were grown as described by Ambesi-Impiombato et al. (4) in Ham's F12 medium modified by Coon (NordVacc, Stockholm, Sweden) supplemented with 5% new¬ born calf serum (NordVacc) penicillin (0.1 IU/1) strep¬ tomycin (100 mg/1) (NordVacc) and six growth factors (6H): bovine TSH 1 U/l (Armour, Kankakee, IL), human Parts of this study were presented in preliminary forms at

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A Simple Competitive Protein-Binding Assay for Adenosine-3′,5′-Monophosphate in Plasma and Urine

Cited by 40 publications

References 0 publications

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function in vitro

The flame retardant DE-71 (a mixture of polybrominated diphenyl ethers) inhibits human differentiated thyroid cell function in vitro

Influence of tumour necrosis factor-α, tumour necrosis factor-β and interferon-γ, separately and added together with interleukin-1 β, on the function of cultured human thyroid cells

Differential effects of interleukin 1α and 1β on cultured human and rat thyroid epithelial cells

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