Potential adverse effects of ciprofloxacin and tetracycline on ARPE-19 cell lines

Abstract

BackgroundWe aim to determine the possible adverse effects of ciprofloxacin (CPFX) and tetracycline (TETRA), as examples of bactericidal and bacteriostatic agents, respectively, on cultured human retinal pigment epithelial cells (ARPE-19).MethodsCells were treated with 30, 60 and 120 µg/mL of CPFX and TETRA. Cell metabolism was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. JC-1 dye (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) assay was conducted to measure the mitochondrial membrane potential (MMP). The level of reactive oxygen species (ROS) was measured using the -2’,7’-dichlorodihydrofluorescein diacetate assay (H2DCFDA). Quantitative real-time PCR was performed to analyse the gene expression levels associated with apoptosis (BAX, BCL2-L13, BCL2, Caspase 3, Caspase 7 and Caspase 9), inflammatory (interleukin-1β (IL-1β), IL-6, IL-33, transforming growth factor-α (TGF-α), TGF-β1 and TGF-β2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4), along with the mitochondrial DNA (mtDNA) copy numbers.ResultsResults illustrated that while all three concentrations of CPFX decreased cellular viability of ARPE-19 during all incubation periods, the 120 µg/mL TETRA resulted in increased cellular viability. At 48 and 72 hours, levels of MMP and ROS decreased significantly with each antibiotic. BAX, BCL2-L13, CASP-7, CASP-9, SOD2 and GPX3 genes overexpressed by either antibiotics. There was higher expression of IL-6 and IL-1B with TETRA treatment. The level of mtDNA decreased using both treatments.ConclusionsClinically relevant concentrations of CPFX and TETRA have detrimental impacts on ARPE-19 cell lines in vitro, including upregulation of genes related to apoptosis, inflammation and antioxidant pathways. Additional studies are warranted to investigate if these harmful effects might be seen in retinal degeneration models in vivo.

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“…The transition of JC-1 from red fluorescence to green fluorescence can easily detect the drop of cell membrane points, and the transition from red fluorescence to green fluorescence of JC-1 can also be used as an early detection indicator of apoptosis (Salimiaghdam et al. 2020 ). The results from JC-1 staining and western blot were consistent with flow cytometry ( Figure 4(C–F) ).…”

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