Rashid Kaveh*, Benoit Van AkenDepartment of Civil and Environmental Engineering, Temple University, Philadelphia, PA

*r.kaveh@temple.edu

Objectives

Conclusion

Background Results

Temple UniversityCollege of Engineering

The anti-influenza drugs, oseltamivir phosphate (OSP) and zanamivir (ZAN), are major medications currently used for the treatment of influenza. These drugs have been detected in municipal wastewater and water catchments. They are likely to contaminate agricultural plants through irrigation with reclamation water and/or land application of biosolids. However, little is known about the effects of antiviral drugs on plants at the molecular level.

Methods

Transcriptional analysis showed changes in genes expression that may reflect oxidative stress in exposed plants. The enzymatic functions and processes may lead to the drugs detoxification in the plant tissue this may help phytoremediation technologies to decrease the concentration of the drugs in the environment.Whole genome expression analysis may be useful for the detection of chronic toxicity of emerging contaminants on plants, even when short-term exposure does not result in observable physiological effects.

Control

OSP 5 m

g/L

OSP 20

mg/L

OSP 100

mg/L

ZAN 5 m

g/L

ZAN 20

mg/L

ZAN 100

mg/L

0

50

100

150

Bio

mas

s (m

g)

**

0 5 10 15 20

Immune systemMulti-organism

GrowthReproduction

SignalingRhythmic process

LocalizationCellular organizationBiological regulation

Multicellular organismalDevelopmental

CellularResponse to stimulus

Single-organismMetabolic

OSP Down-regulatedOSP Up-regulated

0 5 10 15 20

Immune systemRhythmic

ReproductionSignaling

Multi-organismGrowth

Cellular organizationLocalization

Multicellular organismalDevelopmental

Biological regulationResponse to stimulus

Metabolic processSingle-organism

Cellular

Genes per Category (%)

ZAN Down-regulatedZAN Up-regulated

A

B

0 20 40 60

Antioxidant

Receptor

Transcription factor

Molecular transducer

Transporter

Catalytic

Binding

OSP Down-regulated

OSP Up-regulated

0 20 40 60

Electron carrier

Receptor

Antioxidant

Enzyme regulator

Molecular transducer

Transporter

Structural molecule

Transcription factor

Catalytic

Genes per Category (%)

ZAN Down-regulatedZAN Up-regulated

A

B

To understand the potential physiological and transcriptional responses of the model plant Arabidopsis thaliana (A. thaliana) to different contamination levels of the antiviral drugs, OSP and ZAN using whole genome expression microarray.

Toxicity testing:A. thaliana was planted on gel medium in vented Magenta boxes under sterile conditions. The gel contained 0, 5, 20, and 100 mg/L on OSP and ZAN, separately. Exposure length was three weeks with incubation under 16 h/day fluorescent light.

Molecular techniques: A. thaliana plants exposed to 20 mg/L OSP and ZAN were chosen for transcriptional analysis. Plants were kept is RNAlater storage solution. RNA was extracted using TRIzol® Plus RNA Purification kit. RNA transcription to cDNA. RNA validity testing by RT-qPCR. Microarray analysis performed using Affimetrix Arabidopsis Gene 1.0 ST Array. Genomic data analysis:Data normalization by Affymetrix Gene Expression Console with Robust Multi-Array Average normalization algorithmBRB-ArrayTools package for statistical analysis and gene ontology analysis, BLAST2GO®online data bases were used. Fig. 4. Major gene ontology (GO) process categories

Acknowledgement: Dr. Yuesheng Li, Genomic facility, Fox Chase Cancer Center, Philadelphia.

ReferencesHruz, T. et al. (2008). Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Advances in Bioinformatics, 5 pages.Kaveh, R. et al. (2013). Changes in Arabidopsis thaliana Gene Expression in Response to Silver Nanoparticles and Silver Ions. Environmental Science and Technology, 10637-10644.

Genetic Response of Plants Exposed to Anti-Influenza Drugs

Fig. 1. Exposure to a) No drugs, b) OSP 20 mg/L, c) ZAN 20 mg/L.

Fig. 2. A. thaliana fresh weight after exposure to OSP and ZAN for three weeks. OSP samples are correlated with the applied concentrations.

Biomass and physiological effect:

Genetic response:

Fig. 3. Number of genes significantly up-and down-regulated by exposure to the antiviral drugs

Genes up-regulated (>2) Genes down-regulated (<0.5)

OSP OSPZAN ZAN

Fig. 5. Major gene ontology (GO) function categories

a) b) c)