Toxicity Assessment of Selected Antibiotic with Titanium Dioxide Nanoparticles and Evaluation of Quorum Quenching Potential Using Fish as Model Organism

Abstract

Growing trend of nano technology and excessive use of antibiotics in past few decades have given rise to global concerns regarding fate and toxicological impacts of these pollutants on ecosystem. Sulfamethoxazole (SMX) is a broad range bacteriostatic antibiotic widely used in animal and fish farming and also employed in human medicine. These antibiotics may ultimately end up in aquatic ecosystem and affect non-target organisms like fish. The overview of experimental work in this research followed three main phases linked to each other. In phase 1a, local fish Cyprinus carpio was used as bioindicator for toxicity assessment of SMX. Effects were determined by chronic exposure to environmentally relevant dosages of 25, 50,100 and 200 μg/L of SMX for 28 days.

Cytotoxicity assessed through hematological and biochemical profiling showed a dose- response relationship. Hemoglobin, platelets, and erythrocytes levels were significantly

reduced in exposed fish. Leukocytes count was considerably enhanced with values varying from 131-303 (x 103

/μL). Changes in biochemical indices showed biphasic trend with time and dosages tested in study. An inverse relation between concentrations applied and bioaccumulation in targeted fish muscles was discovered by HPLC analysis. The highest concentrations quantified in fish muscles exposed to 25, 50, 100 and 200 μg/L were 124, 202,104.5, and 123.2 ng/g, respectively at several sampling times. Moreover, exposure to SMX enabled ROS production and various histopathological lesions in various organs of SMX exposed fish. Organ pathological index showed that the intensity of tissue lesions increased as SMX dosage was increased. Upon completion of exposure time (28th day), quantitative analysis of gill morphology revealed that the severity of histopathology increased over time for all exposure groups, suggesting physio-metabolic turmoil brought on by the biological and molecular action of SMX. The current study also determined the exposure effects of a broad range of SMX concentrations to developing zebrafish, an ideal indexical organism for ecotoxicological studies. To discern the effect of SMX on developing zebrafish embryos and larvae, Phase 1 b of studies investigated a broad range of toxicity endpoints including survivability, hatchability, malformations, oxidative stress, behavioral changes, mitochondrial bioenergetics, apoptosis, and immune-related transcripts. Results showed that higher concentrations of SMX affect survivability, cause

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hatch delay and induce malformations including edema of yolk sac, pericardial effusion, bent tail and curved spine in developing embryos. Lower levels of SMX provoked an inflammatory response in larvae at 7 dpf as noted by up-regulation of ifn and IL-1β transcripts. SMX also increased transcript expression of genes related to apoptosis including bad and bax at 50 μg/L and decreased casp3 expression in a dose-responsive manner. SMX induced hyperactivity at 500 and 2500 μg/L based upon light/dark preference test. Titanium dioxide nanoparticles are most widely used nano materials employed in various industries. It has been reported that presence of nanoparticles alters the toxicity and bioavailability of organic toxicants. Still, combined toxic effects of nano titanium dioxide and SMX antibiotic that are used world-widely remains unclear. In Phase 2 of study, Cyprinus carpio was utilized as bioindicator for toxicity assessment of nano titanium and

SMX. Juvenile fish were exposed to selected dosages of 25-100 μg/L of SMX alone or co- exposed with 1.5 mg/L of nano titanium for 96 h period. Results revealed that nano titanium

bioaccumulates in fish and it may also adsorb SMX. Nitroblue-tetrazolium (NBT) reduction assay for determination of immunological changes provided clear evidence for increase in respiratory burst activity triggered by nanoparticles. Nano titanium accelerated the uptake of SMX, suggesting that it may increase the bioaccumulation of antibiotics in fish muscles. Fish biochemical characteristics including glucose, alanine transaminase, and total protein were also changed as a result of co-exposure to both contaminants. Current study demonstrated that nano titanium increased SMX bioaccumulation and enhanced SMX-induced toxicity in Cyprinus carpio. Phase 3 of thesis is focused on the in silico identification of promising phytochemical agent as drug candidate (and an alternative to conventional antibiotics) against vibriosis infections by considering LuxR protein as a primary target involved in quorum sensing (QS). Process of quorum sensing in Vibrio anguillarum (fish pathogen) depends on interaction of (AHL) autoinducer molecule with a receptor protein LuxR that is a positive transcriptional activator and results in vibriosis. In aquaculture vibriosis is responsible for severe economic losses worldwide. Phase 3 of current study was designed to investigate phytochemicals as an effective natural inhibitor (quorum quenching agent) of LuxR

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protein. The compounds with PubChem IDs; 99091, 443028, 44587196 and 42607999 showed successful binding with LuxR protein with minimum binding energy in the range of -10.1753 Kcal/mol to -8.79 Kcal/mol. Lipinski rule of five and ADMET analysis were further used to evaluate the drug like properties of selected compounds. Lead compound with best drug like properties was additionally evaluated by molecular dynamics simulations to evaluate the stability of the protein-ligand complex during the simulation period. The study's findings indicate that compound with PubChem ID_42607999 might serve as an effective quorum quenching agent for Vibrio anguillarum. Finally current study may possibly facilitate the development of cost-effective and natural drug against vibriosis in aquaculture.