Ryan De Sotto, Sungpyo Kim, Carl Medriano, and Youngja Park High resolution metabolomics on zebrafish's response to low concentration of pharmaceuticals Ryan De Sotto, Sungpyo Kim, Carl Medriano, and Youngja Park

1 2 3 4 5 Background Objective Methodology Results Contents Conclusions and Implications Methodology Results

“Pharmaceuticals are compounds with different functionalities and physico- chemical properties.” (Kummerer, 2009. Journal of Environmental Management) Background Aquatic organisms are exposed to these pharmaceuticals which might have detrimental effects on them. “There is a need to obtain a more detailed understanding of the adverse environmental effects of pharmaceuticals and their toxicological effects on non-target organisms.” (Madden, 2009. Toxicology Letters) These past decades, pharmaceuticals have been found to present and persistent in various environmental matrices. Antibiotics are the most successful family of drugs for human and animal health, thus, their frequent detection in the environment. Antibiotics have adverse effect such as mutagenicity at genotoxicity to non target organisms (Isidori et al., 2005. Science of the Total Environment) How can we identify the effects of xenobiotic compounds to aquatic organisms?

Background It can be measured according to exposure time (acute or chronic), mode of effect (death, growth, reproduction), or effective response (lethal, sub lethal). (Kapanen and Itavarra, 2001, Ecotoxicology and Environmental Safety) But these tests are not designed for environmentally-relevant concentrations of antibiotics which ranges from ng/L-µg/L levels. Toxicity is the potential of a given substance to adversely affect living organisms. (Neumegen et al., 2005, Environmental Toxicology) Routine toxicity testing studies in aquatic organisms include EC50, IC50, and LC50. Therefore, we need a sensitive technology to determine the subtle effects of these antibiotics to non- target organisms at low concentrations.

What is metabolomics? Background Study of change of metabolites in a living life Metabolite changes in organisms due to environmental stress (Bundy et al., 2009) Quantitative and qualitative analysis of all metabolites (NMR, Mass Spectrometry) Untargeted metabolomics: Comprehensive analysis of all measurable analytes in a biological sample.

Zebrafish as model organism Small freshwater fish Background Zebrafish as model organism Small freshwater fish teleost ( bony fish with rayed fins and a swim bladder) easy to maintain in aquariums and its husbandry are well established (Briggs, 2002) Used in different scientific studies toxicology, integrative physiology, behavioral genetics (Hill, et al., 2005; Briggs, 2002; Norton and Bally-Cuif, 2010)

Objective To use high resolution metabolomics in understanding the effects of low concentration of clarithromycin, florfenicol, sulfamethazine, and their mixture to zebrafish.

Approach Material and Methods Exposure Sample Preparation Storage Measurement 72 hrs exposure to 100 ppb clarithromycin, florfenicol, sulfamethazine, and their mixture Ground and freeze-dried overnight Extraction of metabolites Bligh and Dyer Method Non-lipid layer Stored at -80˚C Analyzed using Agilent 6530 Q-TOF LC/MS 50-1000 m/z values

Metabolomics Material and Methods R program Metlin KEGG Pathways Metabolites Randomly chosen based on relevance with zebrafish One each antibiotic Significant metabolites FDR adjustment to eliminate false positives Identification from online database 10 ppm confidence limit Pathway mapping to check for affected compounds Top pathways affected in the metabolic pathway of zebrafish Identify compounds from each pathways

Results BEFORE AFTER Fishes did not show any physical defects nor changes in behavior and they are still alive after exposure to 100 ppb of antibiotics and their mixture No distinguishable impairment to the fish’s morphology after 72 hours. 100 ppb CLA 100 ppb FLF 100 ppb MXD 100 ppb SMZ

Results Manhattan plot of the significant metabolites after FDR adjustment at a significance threshold of q=0.01 Clarithromycin and florfenicol had the most number of significant metabolites Mixture of antibiotics yielded the least number

Nucleotide Metabolism Carbohydrate Metabolism Results Nucleotide Metabolism Carbohydrate Metabolism Lipid Metabolism Energy Metabolism Amino Acid Metabolism Metabolic Pathway Map of Zebrafish (KEGG Pathway)

Results Metabolomic pathway map after exposure to antibiotics CLARITHROMYCIN FLORFENICOL Some metabolites from lipid, energy, and amino acid metabolism SULFAMETHAZINE MIXED

Results CLARITHROMYCIN Down-regulation of phosphatidylcholine due to stimulation of phospholipase activity (PLD) Macrolides were observed to induce PLD eventually leading to degranulation of neutrophils (Abdelghaffar et al., 1997; Hoyt and Robbins, 2001) Might cause host tissue damage for misdirected activation (Soehnlein et al., 2008) As you can see from this figure, the signal of phosphatidylcholine in the fishes exposed to 100 ppb clarithromycin has decreased compared to control which is believed to be due to stimulation of phospholipase D. Neutrophils are white blood cells responsible for innate immune system (defense of host from infection) Degranulation: Is the release of cellular products to attack foreign bodies Phosphatidylcholine (Immune)

Results FLORFENICOL Increase in signal intensity of the metabolite Important in maturation and fertilization of adult fish (Meinelt et al., 1999) Florfenicol at 25 ug/mL was shown to inhibit production of prostaglandin E2 (Zhang et al., 2011) Arachidonic acid Prostaglandin E2 Essential in ovulation and spawning in zebrafish This figure shows the presence of a signal for a metabolite which is believed to be arachidonic acid in the presence of 100ppb florfenicol. Arachidonic acid is a precursor of Prostaglandin E2. The exposure of the fishes to florfenicol is believed to inhibit the use of this metabolite hence, inhibiting production of prostaglandin E2 as well. To support this claim, Zhang et al., in 2011 showed that florfenicol at 25 ug/ml inhibited prostaglandin E2 which is a sequential metabolite of arachidonic acid. Prostaglandin E2 is essential for the ovulation of the adult fish. Arachidonic acid (Reproductive)

Results SULFAMETHAZINE Important for production of glycogen Glycogen is important in otolith formation in zebrafish Otoliths (ear stones) play a key role in equilibrium and hearing of fish (Pisam et al., 2002) Similarly, the fishes exposed to sulfamethazine was seen to have an elevated UDP glucose signal when compared to control. This shows an impaired metabolism of the said metabolite which is an important compound for the formation of glycogen. Glycogen is essential in the formation of ear stones of the fish responsible for Hearing and equilibrium in its living environment. UDP-glucose (Equilibrium and Sensory)

Results ANTIBIOTIC MIXTURE Might be due to antagonism Of the individual antibiotics and the mixture, we have seen a consistent observation that the fishes exposed to the mixture of antibiotics had the least number of affected metabolite which is contrary to what is believed. This effect might be due to antagonism of the drugs in mixture. The figure on the right shows that while clarithromycin and florfenicol almost have the same number of affected metabolites followed by sulfamethazine, the mixture of these three in water affected zebrafish the least. Mixture of antibiotics have the least affected metabolites and pathways Might be due to antagonism Partial or complete inhibition of the effect of the other drug

Conclusions and Implications Individual antibiotics have more potent effect on adult zebrafish than their mixture. Although fishes did not die nor had morphological changes after exposure, changes in metabolites that might lead to detrimental effects to the fish have been seen. Metabolomics (untargeted) is a promising toxicity evaluation technique for preliminary identification of metabolic changes in organisms exposed to environmental stress.

This study was financially supported by the following: Acknowledgments This study was financially supported by the following: National Institute for Environmental Research, NIER-SP2014-188 Korea Institute of Science and Technology (KIST) Institutional Program (2E25312)

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