title: Tetrabromobisphenol A (TBBPA)-stimulated reactive oxygen species (ROS) production in cell-free model using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay-limitations of method.
abstract: Tetrabromobisphenol A (TBBPA) is a widely used brominated flame retardant, applied in a variety of commercial and household products, mainly electronic ones. Since the production of reactive oxygen species (ROS) is considered one of the principal cytotoxicity mechanisms, numerous studies undertake that aspect of TBBPA's mechanism of action. The present study verifies if the fluorogenic substrate 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) should be used to detect ROS production induced by TBBPA. To determine the ability of TBBPA alone to stimulate the conversion of H2DCFDA to its fluorescent product 2',7'-dichlorofluorescein (DCF), we used a cell-free model. In the experiments we check different cultured media also in combination with free radical scavenger N-acetyl-l-cysteine (NAC). Additionally, experiments with stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH·) have been made. The presented data showed that TBBPA in all tested concentrations interacts with H2DCFDA in phosphate-buffered saline (PBS) buffer while in micromolar concentrations in the DMEM/F12 medium with and without serum. The addition of NAC inhibited the interaction of TBBPA with H2DCFDA. Experiments with DPPH· showed that, in the presence of NAC, TBBPA acts like a free radical. TBBPA has similar properties to free radical and is susceptible to free radical scavenging properties of NAC. Our results indicated that H2DCFDA assay cannot be used to evaluate cellular ROS production in TBBPA studies. The study connected with TBBPA-stimulated ROS production in cell culture models using the H2DCFDA assay should be revised using a different method. However, due to the free radical-like nature of TBBPA, it can be very difficult. Therefore, further investigation of the nature of TBBPA as a compound with similar properties to free radical is required.
authors.affiliation: Department of Public Health, Dietetics and Lifestyle Disorders, University of Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225, Rzeszow, Poland. konrad.szychowski@gmail.com.
authors.forename: Konrad A
authors.initials: KA
authors.lastname: Szychowski
authors.affiliation: Department of Environmental Microbiology, Laboratory of Mycology, University of Life Sciences, Leszczyńskiego 7, 20-069, Lublin, Poland.
authors.forename: Kamila
authors.initials: K
authors.lastname: Rybczyńska-Tkaczyk
authors.affiliation: Department of Public Health, Dietetics and Lifestyle Disorders, University of Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225, Rzeszow, Poland.
authors.forename: Marcin L
authors.initials: ML
authors.lastname: Leja
authors.affiliation: Department of Animal Biotechnology, Animal Sciences Faculty, University of Agriculture, Redzina 1B, 30-248, Krakow, Poland.
authors.forename: Anna K
authors.initials: AK
authors.lastname: Wójtowicz
authors.affiliation: Department of Public Health, Dietetics and Lifestyle Disorders, University of Information Technology and Management in Rzeszow, Sucharskiego 2, 35-225, Rzeszow, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Gmiński
mesh_terms: D002474:Cell-Free System; D005452:Fluoresceins / Q000737:chemistry*; D016166:Free Radical Scavengers / Q000737:chemistry*; D011075:Polybrominated Biphenyls / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000737:chemistry*
chemical_list: C110400:2',7'-dichlorodihydrofluorescein diacetate; D005452:Fluoresceins; D016166:Free Radical Scavengers; D011075:Polybrominated Biphenyls; D017382:Reactive Oxygen Species; C020806:tetrabromobisphenol A
keywords: DPPH; Free radical; H2DCFDA; ROS; TBBPA