Cyto-genotoxicity studies on green carbon dots developed from biomass for biosensing applications.

INTRODUCTION: The production of an increasing amount of biomass/biowaste is an environmental challenge that our society urgently needs to address. Many residues from industrial processes contain valuable biocompounds, either as such or that can be transformed into such by simple and sustainable transformation processes. Biomasses can be converted into graphite-based carbon (carbon dots, CD) by a hydrothermal process, using only water as a green solvent, i.e. without toxic solvents and with minimal impact on the environment. OBJECTIVE: The purpose of this study is to assess the safety of two green CDs compounds made from waste biomaterials, i.e. Cork powder and Fish scales, on biotechnologists who produce disposable biosensing devices. Cork powder is a residue of the cork stopper production and Fish scales are a residue of the fish processing industry. We performed both a cytotoxicity and a genotoxicity test (the Trypan Blue Staining Assay and the Alkaline Comet Assay, respectively) to investigate the effects of these bio-nanomaterials in Jurkat cells, a human tumorderived lymphoblastoid cell line defective for P53 activity, exposed to the compounds. The two assays complement each other since the former informs about induction of cell death, while the latter provides a quantitative estimate of DNA damage induction in (surviving) cells. Scaling concentrations (up to 100 ug/ml) were selected for both compounds and prolonged exposure times, up to 72h, were studied. MATERIALS AND METHODS: Green carbon dots from Fish Scale and Gill (FSG-CDs) and Cork (C-CDs) were prepared from the Nile tilapia (Oreochromis niloticus) and cork powder, respectively, by the hydrothermal treatment (described in poster by Dawood et al., ‘Green carbon dots developed from biomass for sensing Marine Biotoxins’). Jurkat cells were cultured in RPMI supplemented with RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml and maintained in a humidified incubator at 37 °C and 5% CO2/air. FSG-CDs and C-CDs water solutions (2mg/ml) were sterilized (0,22um filters) and diluted in complete culture medium (1mg/ml) before cell administration. Appropriate volumes were then added to the cell suspensions to obtain the final concentrations of 0, 5, 25, 50, 100 ug/ml, also analysing three times of prolonged exposure (24h, 48h, 72h). In addition to negative control (no treatment), hydrogen dioxide (H2O2) was used as positive control (150uM, 30 mins at 37°C). Trypan Blue Staining Assay was carried out before and immediately after the end of treatments by counting viable and nonviable cells in a hemocytometer. Slide preparation for alkaline Comet Assay was performed at the end of exposure, at +4°C, in the dark. One single electrophoretic run was carried out per each test compound, where all concentrations and incubation times were inserted in the basin and run together. The software “Comet IV” (Instem) was used for comet image analysis, and a minimum of 50 comets / experimental point were measured. RESULTS: No significant induction of cell death was induced by FSG-CDs and C-CDs at the different concentrations and times of exposure. No genotoxic effect was detected for FSG-CDs at all concentrations and exposure times, while a slight but significant increase of DNA damage was induced by C-CDs. An extremely significant increase of DNA damage was recorded in the positive control (H2O2), as expected.