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Evelyn Assadian*1,2, Jalal Pourahmad1, Mohammad hadi Zarei1

Hamid Degampanah2, Ali Ghanadzadeh gilani2

1Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Chemistry, Faculty of Science ,Guilan University,Gilan, Iran 

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SUBJECT

Toxicity of Copper oxide (CuO) nanoparticles on human blood

lymphocytes

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CUO METAL OXIDE OF NANO PARTICLES

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ABSTRACTCopper oxide (CuO) nanoparticles have different application in various part of human life such as medicine, engineering and technology. In this study we investigated the cytotoxicity of CuO nanoparticle on human lymphocytes and then the effect of CuO nanoparticles on different cellular and subcellular factor was assessed. Our results showed CuO Nanoparticles induced cytotoxicity on human blood lymphocytes was associated with significant increase at intracellular ROS level and loss of mitochondrial membrane potential and lysosomal membrane leakage.

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INTRODUCTIONCopper oxide (CuO) nanoparticles have a lot of use in different human life sectors such as medicine, engineering and technology. These nanoparticles were used as catalysts, semiconductors, sensors, gaseous and solid ceramic pigments, magnets rotatable devices, electrode materials, hydrophilic materials and super conductors.Furthermore, CuO nanoparticles were used in the pharmaceutical industry and in the production of anti-microbial fabric treatment or prevention of infections caused by Escherichia coli and methicillin-resistant Staphylococcus aureus (Ren, Hu et al. 2009).

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Possible routes of exposure to Cuo nanoparticles are through inhalation and skin exposure.Toxicity of this nanoparticles have been reported in various studies, but no study so far investigated complete cellular mechanisms involved in CuO nanoparticles toxicity on human cells (Chang, Zhang et al. 2012).In this study we aimed to assess the cytotoxicity of Cuo nanoparticles on human blood lymphocytes.

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NECESSITY OF THIS RESEARCH

There is evidence that these substances affect the immune system, but no comprehensive mechanistic research has been done about them.

Extensive contact with nanoparticles of copper in its applications in industry, medicine and even textiles

The critical role of lymphocytes in the immune system

Increase in diseases related to the immune system

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METHODS Blood lymphocytes were isolated from healthy male volunteers’ blood, using Ficoll polysaccharide followed by gradient centrifugation.Cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, glutathione levels, and damage to mitochondria and lysosome were determined in blood lymphocytes after 6-h incubation with different concentrations of CuO Nanoparticle (Salimi, Vaghar -Moussavi et al. 2016).

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THE RESEARCH PROTOCOL

Böyum A, 1996

2 h 4 h 6 h 12 Hour

EC50

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Nontoxic Toxicitythreshold Toxic

Before 12 hour 12 hour After 12 hourMechanistic

Study EC50 Cytotoxic Study

June 29, 2015 10

RPTC RP

TC

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WAYS OF RESEARCH ON THE IMMUNOSUPPRESSION

  

Measurement of cytotoxicity using trypan blue and determination EC50

•Pourahmad J et al, 2012

Measuring reactive oxygen radicals with the reagent dichlorofluorescein diacetate• Chignell CF et al, 2003

Measurement GSH,GSSG with Hissin- Hilf method

Hissin PJ et al, 1976

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WAYS OF RESEARCH ON THE IMMUNOSUPPRESSION

Evaluation of lipid peroxidation by measuring(MDA)

Ohkawa H et al, 1979

Measurement of mitochondrial membrane potential with Cations Rhodamine 123

Andersson B et al, 1987

Measurement of membrane damage of lysosomes with using acridine orange reagentPourahmad J et al, 2001

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STATISTIC ANALYSIS

ط آزمونBج توسBز نتايBآناليANOVA ک طرفه وBي و TukeyدوطرفBه و بBه ترتیBب بBا پسBت تست

Bonferroni بررسی جهBBت گیرد. مBBی انجام تست از گروه، يBک در واريانBس هموژنيسBيته

Leven ورت ميانگينBج بصBتفاده کرده و نتايBاس گزارش خواهد mean±SDسه آزمايBش جداگانه

شد.

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RESULT CHARTS

CYTOTOXICITY EFFECT

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OXIDATIVE STRESS (ROS)

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MITOCHONDRIAL MEMBRANE POTENTIAL

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LYSOSOMAL MEMBRANE DESTABILIZATION

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RESULTS Our results showed that CuO nanoparticle, in particular, decreased cell viability in a concentration-dependent manner and 382µM CuO nanoparticles caused 50% decrease in human lymphocytes viability (Fig. 1). CuO nanoparticles induced cytotoxicity on human blood lymphocytes was associated with significant increase at intracellular ROS level (Fig. 2) and loss of mitochondrial membrane potential and lysosomal membrane leakage.