Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

Tubaro, Aurelia; Florio, Chiara; Luxich, Elena; Vertua, R.; Loggia, R. Della; Yasumoto, Takeshi

doi:10.1016/0041-0101(96)00073-6

Cited by 80 publications

(39 citation statements)

References 17 publications

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“…Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay reported by Tubaro et al 32 , with some modification. The assay for each concentration of samples was performed in triplicate and the culture plates were kept at 37°C with 5% (v/v) CO 2 for 1 day.…”

Section: Assay For Cytotoxic Activitymentioning

confidence: 99%

Metabolite profile and in vitro activities of Phagnalon saxatile (L.) Cass. relevant to treatment of Alzheimer’s disease

Conforti

Rigano

Formisano

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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Section: Assay For Cytotoxic Activitymentioning

confidence: 99%

Metabolite profile and in vitro activities of Phagnalon saxatile (L.) Cass. relevant to treatment of Alzheimer’s disease

Conforti

Rigano

Formisano

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…This method is under increasing criticism not only from an ethical point of view but also because of its limited sensitivity and inability to distinguish between toxins of different groups (Quilliam and Wright, 1995;Hess et al, 2006). A number of alternative methods have been proposed ranging from cytotoxicity assays (Aune et al, 1991;Amzil et al, 1992.;Tubaro et al, 1996a), protein phosphatase assays (Luu et al, 1993;Honkanen et al, 1996;Simon and Vernoux, 1994;Tubaro et al, 1996b;Isobe et al, 1995;Vieytes et al, 1997), liquid chromatography (LC) coupled with fluorescence detection and LC coupled with mass spectrometry, capillary electrophoresis and numerous immunosorbent methods (James et al, 2000). Some of these methods lack the required sensitivity and others require long sample preparation.…”

Section: Introductionmentioning

confidence: 99%

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

Stewart

Elliott

Walker³

et al. 2009

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a b s t r a c tOkadaic acid (OA) and structurally related toxins dinophysistoxin-1 (DTX-1), and DTX-2, are lipophilic marine biotoxins. The current reference method for the analysis of these toxins is the mouse bioassay (MBA). This method is under increasing criticism both from an ethical point of view and because of its limited sensitivity and specificity. Alternative replacement methods must be rapid, robust, cost effective, specific and sensitive. Although published immuno-based detection techniques have good sensitivities, they are restricted in their use because of their inability to: (i) detect all of the OA toxins that contribute to contamination; and (ii) factor in the relative toxicities of each contaminant. Monoclonal antibodies (MAbs) were produced to OA and an automated biosensor screening assay developed and compared with ELISA techniques. The screening assay was designed to increase the probability of identifying a MAb capable of detecting all OA toxins. The result was the generation of a unique MAb which not only cross-reacted with both DTX-1 and DTX-2 but had a cross-reactivity profile in buffer that reflected exactly the intrinsic toxic potency of the OA group of toxins. Preliminary matrix studies reflected these results. This antibody is an excellent candidate for the development of a range of functional immunochemical-based detection assays for this group of toxins.

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“…Cytotoxicity was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. 13) …”

mentioning

confidence: 99%

“…As the total number of methoxyl and hydroxyl groups was five, the remaining oxygen should be ether. The positions of the three hydroxyls were determined by comparing the 13 C-NMR data of 2a (see Experimental) with those of 2. In the 13 C-NMR spectrum of 2a, the signals of C-4, C-7, and C-4Ј showed upfield shifts of 6.2, 5.7, and 6.7 ppm, respectively, and the signals of C-3, C4a, C-6, C-8, C-3Ј, and C-4aЈ showed downfield shifts of 7.1, 5.2, 7.1, 7.0, 7.0, and 5.2 ppm, respectively.…”

mentioning

confidence: 99%

“…The positions of the three hydroxyls were determined by comparing the 13 C-NMR data of 2a (see Experimental) with those of 2. In the 13 C-NMR spectrum of 2a, the signals of C-4, C-7, and C-4Ј showed upfield shifts of 6.2, 5.7, and 6.7 ppm, respectively, and the signals of C-3, C4a, C-6, C-8, C-3Ј, and C-4aЈ showed downfield shifts of 7.1, 5.2, 7.1, 7.0, 7.0, and 5.2 ppm, respectively. As the above evidence indicated, the locations of the three hydroxyls were at C-4, C-7, and C-4Ј, the two dihydrophenanthrene moieties were connected at C-1 and C-7Ј through an ether bond.…”

mentioning

confidence: 99%

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New Stilbenoids from Pholidota yunnanensis and Their Inhibitory Effects on Nitric Oxide Production

Guo

Wang

et al. 2006

CHEMICAL & PHARMACEUTICAL BULLETIN

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Pholidota yunnanensis ROLFE (Orchidaceae) is a perennial herb distributed in Yunnan, Guangxi, Sichuan, and Guizhou provinces in China. The whole plant or pseudobulb is used as a folk medicine for the treatment of cough, rheumatism, stomachache, and trauma. 1,2) In previous investigations on this plant, five triterpenes together with n-nonacosane, n-dotriacontanoic acid, n-octacostyl ferulate, and b-sitosterol were reported. [3][4][5] The 60% ethanol extract of the air-dried whole plant of P. yunnanensis showed inhibitory effects on nitric oxide (NO) production in murine macrophages (RAW 264.7) activated by lipopolysaccharide (LPS) and interferon (IFN)-g in our preliminary screening (inhibition %ϭ92.1%, 30 mg/ml). Therefore the study of its chemical constituents was undertaken to identify the NO production inhibitors.The 60% ethanol extract of P. yunnanensis was suspended in H 2 O and partitioned with CHCl 3 , EtOAc, and n-BuOH successively. The CHCl 3 layer (inhibition %ϭ90.3%, 30 mg/ml) and EtOAc layer (inhibition %ϭ97.1%, 30 mg/ml) that showed NO production inhibitory activity were isolated by repeated column chromatography on silica gel, ODS, Sephadex LH-20, and preparative HPLC (ODS), respectively, to afford four new stilbenes (3-6) and two new stilbenoids (1, 2). In this report, we describe the isolation, structural elucidation, and inhibitory effects on NO production by activated murine macrophages of these new compounds. Results and DiscussionPhoyunnanin D (1), an amorphous powder, was subjected to high-resolution electron impact ionization (HR-EI)-MS and exhibited [M] ϩ at m/z 484.1874 to give the molecular formula C 30 H 28 O 6 . The UV spectrum showed the presence of aromatic rings (205, 280 nm H-detected heteronuclear multiple-quantum coherence (HMQC) spectra exhibited the signals for four methylenes, two methoxyls, and 24 aromatic carbons that consisted of 11 protonated carbons, seven quaternary carbons, and six oxygenated carbons. The presence of a dihydrophenanthrene moiety and a bibenzyl moiety was confirmed by the 1 H-detected heteronuclear multiple-bond correlation (HMBC) correlations (Table 1). This was further supported by the nuclear Overhauser effect spectroscopy (NOESY) correlations between the methylene protons at d 2.69 and H-1, H-8, and between the methylene protons at d 2.73 and H-3Ј, H-2Љ, and H-6Љ. In the NOESY experiment, the correlations between the methoxyl protons at d 3.80 and H-1, H-3, and between the methoxyl protons at d 3.70 and H-5Ј further identified the positions of the two methoxyls located at C-2 and C-6Ј, respectively. Acetylation of 1 with acetic anhydride and pyridine (1 : 1, v/v) afforded a triacetate product (1a), indicating the presence of three hydroxyls. As the total number of methoxyl and hydroxyl groups was five, the remaining oxygen should be ether. The positions of these hydroxyls were deduced from NMR data (see Experimental) of 1a in which the signals of H-3, H-3Ј, H-5Ј, H-2Љ, and H-4Љ were shifted downfield of 0.18, 0.34, 0.33, 0.21, and 0.21 ppm, respec...

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Suitability of the MTT-based cytotoxicity assay to detect okadaic acid contamination of mussels

Cited by 80 publications

References 17 publications

Metabolite profile and in vitro activities of Phagnalon saxatile (L.) Cass. relevant to treatment of Alzheimer’s disease

Metabolite profile and in vitro activities of Phagnalon saxatile (L.) Cass. relevant to treatment of Alzheimer’s disease

Development of a monoclonal antibody binding okadaic acid and dinophysistoxins-1, -2 in proportion to their toxicity equivalence factors

New Stilbenoids from Pholidota yunnanensis and Their Inhibitory Effects on Nitric Oxide Production

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