Comparison of in vitro Toxicities of 8-Prenylnaringenin, Tartrazine and 17ß-Estradiol, Representatives of Natural and Synthetic Estrogens, in Rat and Human Hepatoma Cell Lines.

BACKGROUND: Phytoestrogens have been praised for their beneficial health effects, whereas synthetic xenoestrogens have been connected to ailments. AIMS: To ascertain whether the toxicities of natural and synthetic estrogens differ, we examined the potent phytoestrogen 8-prenylnaringenin (8-PN), the common synthetic xenoestrogen tartrazine, and the physiological estrogen 17ß-estradiol (E2). METHODS: These three compounds were tested for cytotoxicity, cell proliferation and genotoxicity in human HepG2 and rat H4IIE hepatoma cells. RESULTS: All three estrogens elicited cytotoxicity at high concentrations in both cell lines. They also inhibited cell proliferation, with E2 being the most effective. They all tended to increase micronuclei formation. CONCLUSION: Natural estrogens were no less toxic than a synthetic one.

Ginkgo biloba extract protects against tartrazine-induced testicular toxicity in rats: involvement of antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

The use of additives, especially colorants, in food and pharmaceutical industry is increasing dramatically. Currently, additives are classified as contaminants of emerging concern (CECs). Concerns have been raised about the potential hazards of food additives to reproductive organs and fertility. The present study investigates the reproductive toxicity of tartrazine (TRZ), a synthetic colorant, in male rats and aims to explore the curative effect of Ginkgo biloba extract (EGb) against TRZ-induced testicular toxicity. Twenty-four rats were divided into four groups: the control (0.5 ml distilled water), the EGb group (100 mg/kg EGb alone), the TRZ group (7.5 mg/kg TRZ alone), and the TRZ-EGb group (7.5 mg/kg TRZ plus 100 mg/kg EGb). The doses were administered orally in distilled water once daily for 28 days. Toxicity studies of TRZ investigated testicular redox state, serum gonadotropins, and testosterone levels, testicular 17 ß-hydroxysteroid dehydrogenase activity, sperm count and quality, levels of inflammatory cytokines, and caspase-3 expression as an apoptotic marker. Also, histopathological alterations of the testes were examined. TRZ significantly affected the testicular redox status as indicated by the increase in malondialdehyde and the decrease in reduced glutathione, superoxide dismutase, and catalase. It also disrupted serum gonadotropins (follicle stimulating hormone and luteinizing hormone) and testosterone levels and the activity of testicular 17ß-hydroxysteroid dehydrogenase. Additionally, TRZ adversely affected sperm count, motility, viability, and abnormality. Levels of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and expression of caspase-3 were increased in the testes. Histopathological examination of the testes supported the alterations mentioned above. Administration of EGb significantly ameliorated TRZ-induced testicular toxicity in rats. In conclusion, EGb protected against TRZ-induced testicular toxicity through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

The protective effect of aqueous extract of Stevia rebaudiana against tartrazine toxicity in male Wistar rat.

Tartrazine is a yellow colouring agent that is commonly used in foods; however, high dosages of Tartrazine affect fertility and create oxidative stress by generating free radicals. A plant species known as Stevia rebaudiana has natural antioxidants that show promise for protecting testicular tissue. Consequently, this study was intended to examine the ameliorative effect of the aqueous extract of S. rebaudiana (Stevia) on the fertility of male Wistar rats induced by the daily oral intake of Tartrazine. Utilizing gas chromatography-mass spectrometry, phytochemical identification was accomplished for Stevia extract. Study groups were separated into several groups: the first group (the control) got distilled water for up to 56 days; the Stevia group (1000 mg/kg), the Tartrazine group (300 mg/kg) and the Stevia and Tartrazine group (the group was given Tartrazine after 1 h of Stevia extract intake). Also, the oxidative damage in testicular tissues was assessed by measuring the levels of malondialdehyde (MDA) and antioxidants (catalase [CAT], superoxide dismutase [SOD] and glutathione reductase [GSH]). Further, histological alterations were examined. In addition, cyclic AMP-responsive element modulator (Crem) gene expression levels and their relative proteins were measured in the testicular tissues using quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assays, respectively. Sperm analysis and testosterone concentration were also performed. SPSS version 25 was used for the analysis of results while (p < .05) was regarded as significant. Compared with the control group, the results demonstrated that Tartrazine caused a significant reduction (p < .05) in the testosterone hormone level (0.70 ± 0.21) and the Crem protein quantity (1.21 ± 0.23) in the treated Tartrazine group. Also, it had a significant decrease (p < .05) in sperm motility, viability, count and antioxidant levels. Moreover, there was a significant increase (p < .05) in sperm abnormalities, MDA level (7.40 ± 1.10), kidney and liver function parameters, and DNA degradation in the treated Tartrazine group compared with the control group. On the contrary, the Stevia extract intake enhanced the testosterone (2.50 ± 0.60), antioxidants and Crem protein levels (2.33 ± 0.10) with an improvement in sperm quality in the Stevia and Tartrazine-treated group compared with the Tartrazine group. Stevia also caused a significant decrease (p < .05) in the MDA level (3.20 ± 0.20), and sperm abnormalities with an enhancement of the liver and kidney function parameters in the Stevia and Tartrazine-treated group compared to the Tartrazine group. Stevia administration has a protective effect on the testicular tissues and sperm quality against toxicity induced by Tartrazine exposure, so it will be a good antioxidant drug to be administered daily before daily administration of Tartrazine.

In vitro estrogenic, cytotoxic, and genotoxic profiles of the xenoestrogens 8-prenylnaringenine, genistein and tartrazine.

Phytoestrogens have been widely praised for their health-promoting effects, whereas synthetic environmental estrogens are considered a toxicological risk to human health. The aim of this study was therefore to compare in vitro the estrogenic, cytotoxic, and genotoxic profiles of three common estrogen-like endocrine-disrupting chemicals: the phytoestrogens 8-prenylnaringenine (8-PN) and genistein and the synthetic xenoestrogen tartrazine. As assessed by a yeast bioreporter assay and estrogen-dependent proliferative response in human mammary gland adenocarcinoma cell line (MCF-7), 8-PN showed the highest estrogen-like activity of the three compounds, followed by tartrazine and genistein. After 24-h incubation on MCF-7 cells, all three compounds exhibited low cytotoxicity in the lactate dehydrogenase assay and no genotoxicity in the micronucleus assay. These results demonstrate that 8-PN, genistein and tartrazine possess variable estrogenic activity but display little cellular toxicity in short-term tests in vitro. No difference between phytoestrogens and a synthetic xenoestrogen could be established.

Evaluation of a magnetic coagulant based on Fe3O4 nanoparticles and Moringa oleifera extract on tartrazine removal: coagulation-adsorption and kinetics studies.

The lack of data regarding the mechanisms at work in the coagulation processes of different substances using magnetic coagulants makes it difficult to understand the phenomena involved and, consequently, makes it difficult to elucidate the mechanisms involved in the coagulation process. Thus, the present study aimed at evaluating the performance of a magnetic coagulant composed of iron oxide (Fe3O4) functionalised with Moringa oleifera (MO) salt extract in the treatment of a synthetic food industry wastewater simulated by the addition of dye to distilled water. From the data obtained in the coagulation/flocculation assays followed by magnetic sedimentation, the different mechanisms involved were evaluated for their fit to pseudo-first order, pseudo-second order, Langmuir and Freundlich theoretical models. The adjustments to the models were evaluated from the kinetic data and indicated that at pH 3 the best fit was to the pseudo-second order model, whereas for pH 6 and 9 the best fit was for the pseudo-first order model. The isothermal data were adjusted to the Langmuir model, suggesting adsorption of a monolayer, characterising chemical processes with selective adsorption. In relation to the mechanisms involved in the process, it is suggested that the neutralisation of charges was the predominant mechanism in the removal of tartrazine at pH 3, whereas at the other pH values evaluated the mechanism that prevailed was monolayer adsorption. Thus, the proposed magnetic coagulant was found to be an efficient alternative material for tartrazine removal, allowing easy separation in the sedimentation stage while also being compatible with environmental issues.

[Determination of common dyes in dyed safflower by near infrared spectroscopy].

Because the red and bright color of corolla is the main indicator for the quality assessment of good safflower,the dyed safflower is sometimes found at the herbal market,what is influence on this herb quality and efficacy. A total of 127 safflower samples was therefore collected from different cultivating areas and herbal markets in China to develop a rapid method to identify the dyed safflower. Near-infrared spectroscopy(NIRS) combined with characteristic identification,high performance liquid chromatography(HPLC),principal component analysis(PCA) and partial least squares regression analysis(PLS) were employed to differentiate safflower from dyed safflower samples,and further quantify the levels of the 6 dyes,i.e. tartrazine,carmine,sunset yellow,azorubine,acid red 73 and orange Ⅱ in the dyed safflower. The results indicated that the 50 safflower samples and 77 dyed safflower samples were located at different regions in PCA cluster diagram by NIR spectra. Tartrazine,carmineand and sunset yellow were found in the 77 dyed safflower samples with the amounts of 0. 60-3. 66,0. 11-1. 37,0. 10-0. 71 mg·g-1,respectively. It indicated that the three dyes were the common and main dyes in the dyed safflower. However,azorubine,acid red 73 and orange Ⅱ were not detected in all herb samples. A total of 62 dyed safflower samples were chosen as calibration samples to develop the model for estimating the amount of dyes in dyed safflower. The estimating accuracy was verified by another 15 dyed safflower samples. The values of tartrazine,carmine and sunset yellow in dyed safflower samples were compared between the NIRS and HPLC methods. Each value of mean absolute difference(MAD) was less than 5%. The correlation coefficients of tartrazine,carmineand and sunset yellow were 0. 970,0. 975,0. 971,respectively. It indicated the data quantified by NIRS and HPLC were consistence. It is concluded that NIRS can not only differentiate safflower from dyed safflower,but also quantify the amount of the dyes. NIRS is suitable for rapidly identify the quality of safflower.

High-Dose Aspirin Reverses Tartrazine-Induced Cell Growth Dysregulation Independent of p53 Signaling and Antioxidant Mechanisms in Rat Brain.

Tartrazine, an azo dye used in food, cosmetics, and pharmaceuticals with the effects on cell cycle, is not well understood. Therefore, we investigated the toxicity of tartrazine in rat brain with high-dose aspirin. Male Wistar rats (n = 24) were divided into (C) control, (T) tartrazine (700 mg/kg body weight [BW] at weeks 1 and 2), (A) aspirin (150 mg/kg [BW] at weeks 1, 2, and 3), and (TA) aspirin + tartrazine (150 mg/kg [BW] aspirin at weeks 1, 2, and 3 and 700 mg/kg [BW] tartrazine at weeks 1 and 2) groups. The expression of p53, B cell lymphoma-2 extra-large (Bcl-xL), cyclin-dependent kinase 2 (CDK2), p27, and Ki67 was evaluated by quantitative reverse-transcription PCR. A histopathological analysis of brain tissue and oxidative stress level was assessed based on reduced glutathione (GSH), ascorbic acid (AA), and malondialdehyde levels. We found that Bcl-xL, Ki67, CDK2, and p27 were upregulated and p53 was downregulated in the tartrazine-treated group as compared to the control group. Aspirin administration reversed these changes except P53 expression. Tartrazine had no effect on lipid peroxidation but altered AA and GSH levels with no reversal by aspirin treatment. Histopathological analysis revealed that aspirin prevented tartrazine-induced damage including increased perivascular space and hemorrhage. These results indicate that aspirin protects the brain from tartrazine-induced toxicity independent of p53 signaling and antioxidant mechanisms.

Food Yellow4 reprotoxicity in relation to localization of DMC1 and apoptosis in rat testes: Roles of royal jelly and cod liver oil.

Food Yellow 4 (FY4) is a lemon-yellow-colored synthetic organic azo dye, which is used widely for imparting pleasant and attractive appearance to foods and cosmetics. The present study aimed at evaluating the possible mechanism underlying the FY4-induced reprotoxicity in rats, and the potential supportive role of royal jelly (RJ) or cod liver oil (CLO), which is a natural remedy with several pharmacological benefits, against induced toxicity. Forty-eight male rats were divided into different groups-the control group, the CLO group (0.4 mL/kg), the RJ group (300 mg/kg), the FY4 group (500 mg/kg b.w.), and the co-treated groups (FY4 + CLO or FY4 + RJ). Semen analysis, serum hormones, and enzyme activities were estimated. Immunohistochemical staining was performed using anti-PCNA, anti-Sox 9, anti-STRA8, anti-DMC1, and anti-ssDNA antibody. The FY4 group exhibited a significant decrease in sperm concentration and motility percentage (%) and a substantial reduction in the TES and LH levels. Testicular LDH, ACP, and SDH were observed to be inhibited. Furthermore, co-localization of DMC1 and ssDNA, which reflected apoptotic induction in the leptotene and zygotene spermatocytes, respectively, was observed to have markedly elevated in the FY4 treated rats, with fewer PCNA-positive and SOX9-positive cells and higher ssDNA-positive cells in the seminiferous epithelium in comparison to the control groups. Interestingly, co-treatment with CLO or RJ exhibited healthy sperms and restored their features, activated the enzyme production, and raised the levels of sexual hormones. In addition, both RJ and CLO restored the features of the testicular tissue as observed under a light microscope, and limited the apoptosis as observed through antibody staining. Collectively, the results of the present study revealed that the co-administration of RJ or CLO with FY4 improved the biochemical, hormonal, and structural aspects of the testicular tissue in rats. Therefore, CLO and RJ may be considered promising agents that would be able to improve the testicular structure and function in the FY4-exposed individuals.

Using vitamin E to prevent the impairment in behavioral test, cell loss and dendrite changes in medial prefrontal cortex induced by tartrazine in rats.

Tartrazine is a food color that may adversely affect the nervous system. Vitamin E is a neuro-protective agent. This study aimed to evaluate the effects of tartrazine and vitamin E on the performance of rats in memory and learning tests as well as the structure of medial Prefrontal Cortex (mPFC). The rats were first divided into seven groups which received the followings for a period of seven weeks: distilled water, corn oil, vitamin E (100mg/kg/day), a low dose (50mg/kg/day) and a high dose (50mg/kg/day) of tartrazine with and without vitamin E. Behavioral tests were conducted and the brain was extracted for stereological methods The high dose of tartrazine decreased the exploration time of novel objects (P<0.01). The low and high doses of tartrazine led into an increase in working and reference memory errors in acquisition and retention phases (eight-arm radial maze) compared to distilled water group (P<0.01). Additionally, the high dose of tartrazine induced a reduction in the volume of mPFC (∼13%) and its subdivision. Not only that, but the number of neurons and glial cells (∼14%) as well as the mushroom and thin spines per dendrite length declined. The length of dendrites per neuron also reduced in comparison to the distilled water group (P<0.01). Nonetheless, concomitant treatment of the rats with vitamin E plus tartrazine prevented the above-mentioned changes. An acceptable daily dose of tartrazine could induce impairment in spatial memory and dendrite structure. Moreover, a high dose of tartrazine may defect the visual memory, mPFC structure, the spatial memory and also cause dendrite changes. Vitamin E could prevent the behavioral and structural changes.

Determination of green, blue and yellow artificial food colorants and their abuse in herb-coloured green Easter beers on tap.

Beer is one of the most popular alcoholic beverages worldwide. For consumer acceptance, significant factors are its taste, flavour and colour. This study determines selected synthetic green, blue and yellow food colorants in popular Easter herb-coloured green beers on tap produced in breweries on Holy Thursday. The abuse of beer colouring with Tartrazine (E 102), Quinoline yellow (E 104), Sunset yellow (E 110), Patent blue (E 131), Indigo carmine (E 132), Brilliant blue FCF (E 133), Green S (E 142) and Fast green FCF (E 143) was assessed in 11 green beer samples purchased in local restaurants. HPLC was used for the separation and detection of artificial colorants with diode-array detection and a Chromolith Performance CN 100 × 4.6 mm column with guard pre-column Chromolith CN 5 × 4.6 mm. Separation was performed in gradient elution with mobile phase containing methanol-aqueous 2% ammonium acetate at pH 7.0. The study showed that eight beers (70%) marketed in the Czech Republic contained artificial colorants (Tartrazine and Brilliant blue FCF). The concentration of colorants found in analysed green herb-coloured beers ranged from 1.58 to 3.49 mg l(-)(1) for Tartrazine, 0.45-2.18 mg l(-)(1) for Brilliant blue, while Indigo carmine was detected only once at concentration 2.36 mg l(-)(1). Only three beers showed no addition of the synthetic colorants. However, the levels of artificial colorants found in beers marketed in the Czech region were very low and did not show a serious risk for consumers' health.

Food additives: Sodium benzoate, potassium sorbate, azorubine, and tartrazine modify the expression of NFκB, GADD45α, and MAPK8 genes.

It has been reported that some of the food additives may cause sensitization, inflammation of tissues, and potentially risk factors in the development of several chronic diseases. Thus, we hypothesized that expressions of common inflammatory molecules - known to be involved in the development of various inflammatory conditions and cancers - are affected by these food additives. We investigated the effects of commonly used food preservatives and artificial food colorants based on the expressions of NFκB, GADD45α, and MAPK8 (JNK1) from the tissues of liver. RNA was isolated based on Trizol protocol and the activation levels were compared between the treated and the control groups. Tartrazine alone could elicit effects on the expressions of NFκB (p = 0.013) and MAPK8 (p = 0.022). Azorubine also resulted in apoptosis according to MAPK8 expression (p = 0.009). Preservatives were anti-apoptotic in high dose. Sodium benzoate (from low to high doses) dose-dependently silenced MAPK8 expression (p = 0.004 to p = 0.002). Addition of the two preservatives together elicited significantly greater expression of MAPK8 at half-fold dose (p = 0.002) and at fivefold dose (p = 0.008). This study suggests that some of the food preservatives and colorants can contribute to the activation of inflammatory pathways.

Comparative protective effects of royal jelly and cod liver oil against neurotoxic impact of tartrazine on male rat pups brain.

This study is aimed to evaluate the possible neurotoxic effect of tartrazine (T), an extensively used synthetic azo dye, as well as to determine the potential modulatory role of cod liver oil (CLO) or royal jelly (RJ) against such effects. For this purpose, thirty-six male rat pups were allocated into six groups. The 1st group received distilled water (control group), the 2nd group was given 300 mg RJ/kg bw (RJ group), the 3rd group was given 0.4 ml CLO/kg bw (CLO group), the 4th was given 500 mg T/kg bw (T group). The 5th group was given T concurrently with RJ (TRJ group) and the 6th group was given T concurrently with CLO (TCLO group), at the same doses as the former groups. All treatments were given orally for 30 consecutive days. The concentrations of different brain neurotransmitters, gamma amino butyric acid (GABA), dopamine (DA) and serotonin (5HT) as well as the antioxidant and oxidative stress biomarkers were measured in the brain homogenates. An immunohistochemical staining of the cerebral cortex was applied with the anti-ssDNA antibody (an apoptotic cell marker) to reveal the changes in brain structure. The T group revealed a significant decrease in the concentration of the brain neurotransmitters, a sharp shortage in the level of antioxidant biomarkers (super oxide dismutase, catalase and the reduced glutathione), a marked increase in malondialdehyde levels, and numerous apoptotic cells in the brain cortex compared with the other groups. Interestingly, all the previously mentioned parameters were almost retrieved in both the TRJ and TCLO groups compared to the T group. These results conclusively demonstrate that RJ and CLO administration provides sufficient protection against the ruinous effects of T on rat pups brain tissue function and structure.

Immune reactivity to food coloring.

Artificial food dyes are made from petroleum and have been approved by the US Food and Drug Administration (FDA) for the enhancement of the color of processed foods. They are widely used in the food and pharmaceutical industries to increase the appeal and acceptability of their products. Synthetic food colorants can achieve hues not possible for natural colorants and are cheaper, more easily available, and last longer. However, since the use of artificial food coloring has become widespread, many allergic and other immune reactive disorders have increasingly been reported. During the past 50 y, the amount of synthetic dye used in foods has increased by 500%. Simultaneously, an alarming rise has occurred in behavioral problems in children, such as aggression, attention deficit disorder (ADD), and attention-deficit/hyperactivity disorder (ADHD). The ingestion of food delivers the greatest foreign antigenic load that challenges the immune system. Artificial colors can also be absorbed via the skin through cosmetic and pharmaceutical products. The molecules of synthetic colorants are small, and the immune system finds it difficult to defend the body against them. They can also bond to food or body proteins and, thus, are able to act in stealth mode to circumvent and disrupt the immune system. The consumption of synthetic food colors, and their ability to bind with body proteins, can have significant immunological consequences. This consumption can activate the inflammatory cascade, can result in the induction of intestinal permeability to large antigenic molecules, and could lead to cross-reactivities, autoimmunities, and even neurobehavioral disorders. The Centers for Disease Control (CDC) recently found a 41% increase in diagnoses of ADHD in boys of high-school age during the past decade. More shocking is the legal amount of artificial colorants allowed by the FDA in the foods, drugs, and cosmetics that we consume and use every day. The consuming public is largely unaware of the perilous truth behind the deceptive allure of artificial color.

Highly-sensitive and rapid detection of ponceau 4R and tartrazine in drinks using alumina microfibers-based electrochemical sensor.

Alumina microfibers were prepared and used to construct an electrochemical sensor for simultaneous detection of ponceau 4R and tartrazine. In pH 3.6 acetate buffer, two oxidation waves at 0.67 and 1.01 V were observed. Due to porous structures and large surface area, alumina microfibers exhibited high accumulation efficiency to ponceau 4R and tartrazine, and increased their oxidation signals remarkably. The oxidation mechanisms were studied, and their oxidation reaction involved one electron and one proton. The influences of pH value, amount of alumina microfibers and accumulation time were examined. As a result, a highly-sensitive, rapid and simple electrochemical method was newly developed for simultaneous detection of ponceau 4R and tartrazine. The detection limits were 0.8 and 2.0 nM for ponceau 4R and tartrazine. This new sensor was used in different drink samples, and the results consisted with the values that obtained by high-performance liquid chromatography.

Decolourization of azo dyes using magnesium-palladium system.

Magnesium-palladium system was found to efficiently decolourize reactive black 5, sunset yellow FCF and tartrazine dyes. There is complete loss of visible range absorption peaks and extent of colour removal exceeded 95% within 24 h of reaction. There is appearance of new peak(s) in the UV region and/or gradual and significant shift of the lambda(max) in the UV range during 1-24 h of reaction of dyes with Mg/Pd system. LC-MS analyses following the reaction of dyes with magnesium palladium system suggest reductive cleavage of azo bonds and formation of amines as the end products. Kinetic analyses of dye decolourization indicate that the reaction follows first order kinetics. Agreement between the experimental and predicted Michaelis-Menten plots for the decolourization of reactive black 5, sunset yellow FCF and tartrazine dyes by Mg(0)/Pd(4+) system, suggests the correctness of Michaelis-Menten model for the prediction of dye decolourization rates by Mg(0)/Pd(4+) system. Our investigations reveal that Mg(0)/K(2)PdCl(6) system is more effective in decolourizing dyes as compared to Mg(0)/Pd(0)-alumina or Mg(0) alone. Results obtained from reuse experiments suggest that Pd(0) pellets have the potential for recycling which will make the treatment process cost effective. Mg(0)/Pd(4+) system was found to be efficient in decolourizing mixture of drimarene, remazol and procion dyes as well as raw effluent generated by textile dye manufacturing company.

Food Additives and Asthma / 소아알레르기및호흡기학회지

PURPOSE: To review the role of food additives in asthma and provide a practical approach for evaluation, diagnosis, and management of additive-induced asthma. METHODS: Information was gathered from original articles, selected reviews and abstracts published in peer-reviewed journals and from selected textbook chapters, supplemented by the clinical experience of the authors. RESULTS: In some patients, food additive ingestion can induce bronchospasm or exacerbation of symptoms in patients with chronic asthma. The most implicated agents are sulfites, followed by tartrazine, monosodium glutamate and others. However, geographic variations exist depending on the dietary habits. CONCLUSION: Food additives are worth considering as possible causes of bronchospasm or worsening of asthma. The medical history may be suggestive, particularly when symptoms occur to commercially prepared foods or to multiple unrelated foods. Physicians should also think of food additives in patients whose asthma is poorly controlled in spite of appropriate routine allergy evaluation, environmental control, and optimal pharmacologic therapy. Except for a few natural additives, allergy skin test and in-vitro tests are unreliable. A titrated oral challenge testing, preferably in a blind fashion would be the definitive diagnostic procedure.

A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists.

In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.

Impact of a red-shifted dye label for high throughput fluorescence polarization assays of G protein-coupled receptors.

High throughput screening fluorescence polarization assays using G protein-coupled receptors (GPCRs) as targets have been compared using fluorescein and BODIPY TMR-labeled peptides. The red-shifted BODIPY TMR dye exhibits improved assay performance relative to fluorescein due to improvement in both ligand affinity to the GPCRs and assay precision brought about by the higher intensity probe. Furthermore, the red-shifted dye demonstrates an insensitivity to the effects of the highly colored compound tartrazine, which can produce false-negative results for assays conducted with fluorescein as a label.

Precipitation of asthma attacks in Melanesian adults by sodium metabisulphite.

Seven Melanesian asthmatic patients were challenged with substances that have been shown to precipitate asthma attacks in asthma patients in developed countries. Patients were challenged in a double-blind fashion using placebo and active substances. The active substances were tartrazine, sodium metabisulphite, aspirin and betel nut. All 7 patients were challenged with tartrazine and sodium metabisulphite; 5 were challenged with aspirin also, but only 2 were challenged with betel nut. Asthma attacks were precipitated by sodium metabisulphite in 3 patients. No other substances precipitated asthma. As sodium metabisulphite is a common food additive, these results suggest that processed foods introduced into developing countries may have an important role in precipitating asthma attacks in susceptible persons.