Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Clin Infect Dis. May 25, 2017; 7(2): 21-31
Published online May 25, 2017. doi: 10.5495/wjcid.v7.i2.21
Antioxidant enzyme profile of two clinical isolates of Entamoeba histolytica varying in sensitivity to antiamoebic drugs
Lakshmi Rani Iyer, Neha Banyal, Shailendra Naik, Jaishree Paul
Lakshmi Rani Iyer, Neha Banyal, Shailendra Naik, Jaishree Paul, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Author contributions: Iyer LR and Paul J conceived and designed the experiments; Iyer LR, Banyal N and Naik S performed the experiments; Paul J contributed reagents, material, analysis tools; Iyer LR, Banyal N and Paul J analyzed the data; Iyer LR and Paul J prepared the manuscript; Iyer LR and Paul J participated in revising the manuscript.
Supported by WOS-A grant from Department of Science and Technology, New Delhi, Government of India to Iyer LR, No. SR/WOS-A/LS-98/2007; “Programme support on molecular parasitology”, Department of Biotechnology, New Delhi, India to Paul J, No. BT/01/CEIB/11/v/08.
Institutional review board statement: All stool samples from patients were taken after informed consent and ethical permission was obtained for participation in the study (Government of India, Institute Ethics committee, Safdarjung hospital and VMMC, letter no VMMC/SJH/PROJECT/JAN-14/21).
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
Data sharing statement: None.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Jaishree Paul, School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067, India. jpaul33@hotmail.com
Telephone: +91-11-26704156 Fax: +91-11-26742558
Received: September 23, 2016
Peer-review started: September 26, 2016
First decision: November 2, 2016
Revised: January 11, 2017
Accepted: February 10, 2017
Article in press: February 13, 2017
Published online: May 25, 2017
Processing time: 242 Days and 7.5 Hours
Abstract
AIM

To study the sensitivity and antioxidant enzyme response in two clinical isolates of Entamoeba histolytica (E. histolytica) during treatment with antiamoebic drugs, auranofin and metronidazole.

METHODS

E. histolytica were isolated from stool samples and maintained in Robinson’s biphasic culture medium. Clinial isolates were maintained in xenic culture medium, and harvested for determination of minimum inhibitory concentrations to the two antiamoebic drugs, Metronidazole and Auranofin using microtiter plate tests. The percent survival of the two isolates were determined using the trypan blue cell count. Isolate 980 was treated with 70 μmol/L and 2 μmol/L while isolate 989 was treated with 20 μmol/L and 0.5 μmol/L of metronidazole and auranofin respectively for 24 h. Fifty thousand cells of each isolate were harvested after 24 h of treatment for analysis of the mRNA expressions of the antioxidant enzymes, thioredoxin reductase, peroxiredoxin and FeSOD using the specific primers. Cell lysate was used for determination of enzyme activity of thioredoxin reductase by measuring DTNB reduction spectrophotometrically at 412 nm.

RESULTS

Minimum inhibitory concentration of the clinical isolates 980 and 989 for auranofin was 3 μmol/L and 1 μmol/L respectively while that for metronidazole was 80 μmol/L and 30 μmol/L respectively. Thioredoxin reductase, peroxiredoxin and FeSOD expression levels were significantly reduced in the isolate 980 when treated with Auranofin. Metronidazole treatment showed a down regulation of thioredoxin reductase. Though not significant both at the mRNA and the enzyme activity levels. Peroxiredoxin and FeSOD however remained unchanged. Auranofin treatment of isolate 989, showed an upregulation in expression of thioredoxin reductase while Peroxiredoxin and FeSOD did not show any change in expression. Upon treatment with metronidazole, isolate 989 showed an increase in thioredoxin reductase expression. Peroxiredoxin and FeSOD expressions however remain unchanged both at mRNA and enzyme activity level.

CONCLUSION

Clinical isolates from New Delhi NCR region show different sensitivities to antiamoebic drugs. Auranofin is effective against isolate showing higher tolerance to metronidazole as shown by its inhibition in thioredoxin reductase activity.

Keywords: Metronidazole; Entamoeba histolytica; Amoebiasis; Thioredoxin reductase; Minimum inhibitory concentrations; Auranofin

Core tip: Due to overuse of the mainstay drug against amoebiasis in an endemic country like India, there are concerns regarding the development of resistance towards metronidazole by the parasite. When Entamoeba histolytica (E. histolytica) from stool samples of diarrheal patients were cultivated in xenic medium, two clinical isolates of E. histolytica showed differential tolerance to the commonly used drug metronidazole. A new drug Auranofin was found to be effective on the isolate with higher tolerance to metronidazole. This was shown by inhibition of the antioxidant enzyme thioredoxin reductase as monitored by mRNA expression of TrxR gene and its enzyme activity.