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Syddansk Universitet

Silver nanoparticles cause osmoregulatory impairment and oxidative stress in Caspian kutum (Rutilus kutum, Kamensky 1901) Masouleh, Fatemeh F.; Amiri, Bagher M.; Mirvaghefi, Alireza; Ghafoori, Hossein; Madsen, Steffen Published in: Environmental Monitoring and Assessment DOI: 10.1007/s10661-017-6156-3 Publication date: 2017 Document version Peer reviewed version

Citation for pulished version (APA): Masouleh, F. F., Amiri, B. M., Mirvaghefi, A., Ghafoori, H., & Madsen, S. S. (2017). Silver nanoparticles cause osmoregulatory impairment and oxidative stress in Caspian kutum (Rutilus kutum, Kamensky 1901). Environmental Monitoring and Assessment, 189(9), [448]. https://doi.org/10.1007/s10661-017-6156-3

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Download date: 28. nov.. 2018

Silver nanoparticles cause osmoregulatory impairment and oxidative stress in Caspian kutum (Rutilus kutum, Kamensky 1901)

Fatemeh F. Masouleh1, Bagher M. Amiri1, Alireza Mirvaghefi1, Hossein Ghafoori2 & Steffen S. Madsen *3

1

Department of Fisheries, Faculty of Natural Resources, University of Tehran, 31585-

4314, Karadj, Iran 2

Department of Biology, Faculty of Science, University of Guilan, 41335-19141, Rasht,

Iran 3

Department of Biology, University of Southern Denmark, Campusvej 55, DK-5230

Odense M, Denmark

Running title: Effects of silver nanoparticles on Caspian kutums juveniles

* Corresponding Author: Steffen S. Madsen, Department of Biology, Campusvej 55, 5230 Odense M, Denmark, E-mail: [email protected]

Acknowledgements

The authors acknowledge the Department of Biology of University of Southern Denmark and Department of Biochemistry of University of Guilan, Iran for providing necessary lab facilities to carry out the work successfully. This research was supported by the University of Tehran (grant # 6/31/2703010). SSM was supported also by a grant from The Danish Research Council for Independent Research (DFF-4181-00020).

1

Abstract

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Silver nanoparticles (AgNPs) are increasingly used in several industrial and household

4

products because of their antibacterial and antifungal properties. Hence, there is an inevitable

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risk that these chemicals may end up in aquatic biotopes and have adverse effects on the fauna.

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In order to assess potential health effects on aquatic organisms, this study evaluated the effects

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of waterborne AgNP exposure for 7 days on a set of critical stress parameters in juvenile

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Caspian kutum (Rutilus kutum), an economically important fish in the Caspian Sea. The

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applied level 11 µg/l of AgNP is high compared to reported water concentrations and

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corresponds to 40% of the 96 hour LC50-value, initially determined to be 28 µg/l. Gill heat

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shock protein 70 (hsp70) mRNA expression, Na+/K+-ATPase activity and enzymatic activities

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of liver superoxide dismutase (SOD), glutathione peroxidase (Gpx), lactate dehyrogenase

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(LDH) and alkaline phosphatase (ALP), and whole body cortisol and thyroid hormones (T3

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and T4) were measured as endpoints. Gill hsp70 mRNA expression increased and gill Na+/K+-

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ATPase activity decreased in AgNP-exposed fish compared to controls. The specific activities

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of all liver enzymes decreased significantly compared to controls. Whole body cortisol and

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thyroid hormones decreased compared to control. In conclusion, the study demonstrates that

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AgNPs cause oxidative stress and gill osmoregulatory disruption in Caspian kutum juveniles.

19 20

Keywords: nanoparticles, Caspian kutum, Hsp70, gill, liver, stress

21 22 23 24 25 26 27 28 29 30 31 32 33 1

34

Introduction

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Silver nanoparticles (AgNPs) are widely used as biocides in commercial and industrial

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products such as textiles, food storage containers, paints, home appliances and personal care

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products (Maynard et al. 2006). The global production and use of nanomaterials are rapidly

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increasing, and it has been estimated that the annual production of e.g. nanosilver increased

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from 4 to 563 tonnes per year from 2004 to 2008 (Musee et al. 2011). Hence, there is great

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concern about the release and fate of these particles once they leak into sewage treatment

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plants and pouring into rivers. Concentrations of AgNps in surface waters are obviously very

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variable worldwide but have been reported in the range of 0.008 ng/l to 619 ng/l (Gottschalk et

44

al. 2011; Musee et al. 2011). When these particles end up in the aquatic environment they may

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have adverse ecotoxicological impact on aquatic organisms (Schultz et al. 2012). Accordingly,

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AgNP cytotoxicity and genotoxicity effects have been reported in many studies, and AgNP

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toxicity has been reported in a broad range of concentrations from µg/l to mg/l in aquatic

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organisms. In addition to concentration, the toxicity also depends on the type and size of

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particles, species and age of animals and duration of exposure. The mechanisms of toxicity are

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generally unknown and need to be examined in detail (Nebeker et al. 1983; Bianchini et al.

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2002; Grosell et al. 2002). The toxicity of AgNPs has been suspected to arise in part from the

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release of Ag+-ions but there is also evidence that AgNP and Ag+ may have independent

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effects (Beer et al. 2012; Völker et al. 2013).

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AgNPs may alter tissue morphology after accumulation, penetrate into cells and cause

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DNA damage, cause changes in gene expression, oxidative stress and impair protein synthesis

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(Reidy et al. 2013; Katuli et al. 2014). Heat shock proteins (HSP), as biomarkers of various

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stressors, are highly conserved chaperones involved in cellular stress response and assist in

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refolding of denatured proteins. The main role of these proteins is cytoprotection (Hartl and

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Hayer-Hartl 2002) and cellular recovery from stress (Iwama et al. 1998). Although the stress

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response pattern of these proteins is related to stress types, animal species and tissue, the

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duration and timing of stressors are also important (Lee and Hahn 1988). One of the well-

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known HSP families, HSP70 (70kDa), a critical mediator of tolerance against threatening

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environmental conditions, is taking part in the repair and detoxification mechanism of the cell

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(Barnes et al. 2002) and has been considered as a stress biomarker in toxicological research

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(Ahamed et al. 2010). Na+/K+-ATPase, an important enzyme as the main engine in ion

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transportation across the gill epithelium, has a key role in ion regulation and osmoregulation. It 2

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has been shown that Ag (AgNP and Ag+) is able to inhibit Na+/K+-ATPase activity and disrupt

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osmoregulation by impairment of Na+ and Cl- uptake in freshwater fish and invertebrates (e.g.

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Wood et al. 1999; Völker et al. 2015). The liver is the major detoxification tissue affected by

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AgNPs, and acts as a protective organ against reactive oxygen species (ROS) mediated

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oxidative stress by antioxidant enzymes like SOD and GPx (Lee et al. 2012). Two other

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important liver biomarker enzymes, lactate dehydrogenase (LDH) and alkaline phosphatase

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(ALP) have been considered as metal cytotoxicity indices. Also, the primary stress hormone

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cortisol as the end product of hypothalamic-pituitary-interrenal (HPI) axis and thyroid

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hormones (T3 and T4) that may act in concert with cortisol and modify the pattern and

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magnitude of the stress response in fishes (Ostrander 2000) are important to consider as

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indicators of general stress in fish (Peter 2011).

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The Caspian kutum (Rutilus kutum, Kamensky 1901) is an important endemic

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commercial fish in the Caspian Sea. For the last 25 years, juvenile fish have been stocked into

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the rivers leading to south coast of the Caspian Sea (Sharyati 1993) but for unknown reasons

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the reported recapture rates are very poor (5.5%, Ghaninejad et al. 2001). Following breeding

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in captivity, juvenile kutums cannot be directly released to the Caspian sea with 13 ppt salinity,

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so the practice is to stock them into the rivers to enable them to gradually acclimate to the

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salinity of the sea. One contributing factor to the poor recapture rates may be that during their

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freshwater residence they are exposed to several pollutants like AgNPs, which may affect their

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osmoregulatory capacity and plasticity. Unfortunately, the exact concentration range of AgNPs

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in these rivers is not known but metal concentrations have been reported to be lower in the

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Caspian Sea than in the affluent rivers. Research is therefore needed to elucidate how kutums

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are affected by AgNPs both when in the freshwater environment and during gradual exposure

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to increasing salinity. The present study is a first step in toxicological studies of silver

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nanoparticles in Caspian kutums and was designed to investigate several general and specific

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stress responses in juvenile Caspian kutums, when exposed to waterborne AgNPs in their

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freshwater rearing environment.

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Materials and Methods

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Fish

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3

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Juvenile Caspian kutums (Rutilus kutum, 0.96 ± 0.1 g, 43 ± 4 mm) were provided by Ansari

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fish propagation center in Rasht (Guilan, Iran) and transferred to the University of Tehran,

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where they were kept in de-chlorinated tap water (salinity: 0.3 ppt, pH: 7.6, Total ammonium:

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0.1 and hardness: 114±9 mg CaCO3/l) at 12 °C under natural photoperiod for 2 weeks before

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starting the experiment. Fish were fed daily with food pellets, 3% of body weight daily (Beyza

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Company, Shiraz, Iran). Feeding was withheld from 48 hours before experimentation was

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initiated.

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Characterization of silver nanoparticles

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Silver nanoparticles (commercial name: Nanocid®) were provided by Nano Nasb Pars co.

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(Tehran, Iran). This product is a water-based colloidal suspension with a concentration of 4000

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mg/l (average size 16.6 nm). The zeta potential of suspension (100 mg/l) was -53.33±7.86 mV

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measured by zetasizer (Malvern, UK). The average hydrodynamic diameter was 54.8 nm. The

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free ionic silver in stock suspension measured by a silver ion selective electrode (ISE, HI4115)

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and was nearly 30% of silver in suspension. The detailed specifications of stock suspension

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have been reported previously by Joo et al. (2013).

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Experimental design and sampling procedures

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Following the Organization for Economic Cooperation and Development (OECD) guideline, a

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96-hour acute toxicity (LC50) test was conducted (OECD Test No. 215) in a static water

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renewal experiment. The LC50 (50% lethal concentration) was calculated using the EPA Probit

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Analysis Program version 1.5 (Aydın and Köprücü 2005). The 96 hour LC50 of AgNPs was

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determined to be 28 µg/l (total silver concentration). Based on this, 40% of the LC50

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concentration (11 µg/l) was used for this experiment. Next, 100 fishes were transferred to 200 l

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tanks and exposed to 11 µg/l AgNPs for 7 days. Fish transferred to new tanks every 42 hours.

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To check if the concentration of Ag changed over time in the tanks, water samples were

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examined every day using atomic absorption spectrophotometer spectrophotometry (AAS,

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Perkin-Elmer Analyst 300).

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After 3, 12, 24, 72 and 168 hours of exposure, the fish were anesthetized with 100 mg/l

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clove oil. The gills and livers of fish were removed and frozen quickly in liquid nitrogen and

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then stored at -80°C for mRNA and enzymatic assays. For Na+/K+-ATPase activity 4

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measurements, gills were frozen in SEI buffer (300 mM sucrose, 5 mM Na2EDTA, 50 mM

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imidazole, pH 7.3) and for hormones and total protein analysis, the whole body was used. The

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samples were stored in -80 ºC until analysis. The experimental treatment was done in triplicate.

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We sampled 2 fish in each tank for each factor (N=6). 300 fishes in 3 tanks served as control

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groups in de-chlorinated tap water during the test.

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RNA extraction and cDNA synthesis

139 140

Total RNA from gill tissues was extracted by the TRIzol reagent (Sigma Aldrich, St. Louis,

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MO, USA) according to manufacturer's protocol. Total RNA purity and concentration were

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determined by using a NanoDrop ND-1000 spectrophotometer in duplicate (NanoDrop

143

Technologies, Wilmington, USA). RNA was treated with the RQ1 DNase kit (Promega

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Biotech AB, Stockholm, Sweden). cDNA was synthesized by using DyNAmo cDNA synthesis

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kit (Finnzymes, Espoo, Finland), according to the manufacturer's recommendations.

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Semiquantitative real-time PCR

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The hsp70 primers were developed based on a consensus sequence constructed from 7

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overlapping ESTs from Rutilus rutilus (Genbank Acc. No.: EG540883.1, EG538984.1,

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EG546387.1, EG540883.1, EG546670.1, EG531871.1, EG545695.1) and using the fathead

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minnow hsp70 sequence as blast querry (AY538777). Beta-actin primers were developed

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based on a partial coding sequence of the R. rutilus beta-actin gene (GenBank Acc. No.

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DQ061948.1) (Table 1). Primers were acquired from Sigma-Aldrich. The primer pairs

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generated a unique amplicon based on melting point analysis and the amplicon size was

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verified by gel electrophoresis. qPCR analysis was performed using a Mx3000p instrument

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(Stratagene, La Jolla, CA, USA) with SYBR Green JumpStart Mastermix (Sigma-Aldrich).

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Reactions were carried out with final volume of 20 µl containing cDNA, 150 nM forward and

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reverse primer, 1× Mastermix. Cycling conditions were 95 °C for 3 min followed by 40 cycles

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of 95 °C for 30 s and 60 °C for 60 s for hsp70 and 64 °C for 30 s for β-actin. To establish the

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amplification efficiency for each primer pair, one pooled cDNA sample was serially diluted

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and analyzed by qPCR. All samples were normalized to the abundance of the housekeeping β-

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actin gene, which did not change significantly between samples. The relative expression of

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hsp70 was calculated using the ΔΔCt method (Pfaffl 2001). 5

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Gill Na+/K+-ATPase activity

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Gill Na+/K+-ATPase activity was determined spectrophotometrically at 25°C using a

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microplate reader (SPECTRAmax PLUS, Molecular Devices, Sunnyvale, CA, USA) according

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to the semi-micro method described by McCormick (1993). Briefly, pieces of gill filaments

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were homogenized in ice cold SEIDM buffer (300 mM sucrose, 20 mM Na2EDTA, 50 mM

171

imidazole, 0.1% Na-deoxycholate, and 10 mM mercaptoethanol) using a teflon

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pestle/Eppendorf tube homogenizer, centrifuged at 5,000×g for 30 sec, then the supernatant

173

was separated and kept on ice until assayed. The ATPase activity was measured in the presence

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or absence of 0.5 mM ouabain. Protein content in the homogenates was measured by a

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modification of the Lowry method (Lowry et al. 1951) and the enzymatic activity was

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normalised to protein content and expressed as µmol ADP/mg protein/h.

177 178

Liver enzyme activities

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The activities of several liver enzymes were measured in liver homogenates. Approximately

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two mg of liver tissue was homogenized in 1 ml phosphate buffer 50 mM (pH 7.4). The

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homogenates were sonicated for 30 s at 10 kHz on ice to release enzymes, centrifuged at 9,000

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g for 20 min and then the supernatant was separated and stored at -80°C until enzymatic assays

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were run. All enzymatic assays were run in triplicate.

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Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by its ability to inhibit

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the photochemical reduction of Nitroblue Tetrazolium (NBT). Superoxide ions reacts with

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NBT to generate a blue colored formazan which can be measured at 560 nm (Beauchamp and

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Fridovich 1971). The reaction mixture contained 0.1 mM Na2EDTA, 13 mM L-methionine, 75

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μM NBT, 0.21 mM riboflavin. Absorbance of reaction mixture with phosphate buffer was read

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at 560 nm after 3 min incubation period in the dark as blank. Control sample contained reaction

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mixture with buffer and the samples were a mix of 970 µL reaction mixture and 30 µl enzyme

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homogenate illuminated with luminescent lamps for 3 min. superoxide onions activity were

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calculated using the following equation (Siddhuraju and Becker 2003):

194 195

% of inhibition

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6

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One unit of SOD activity is defined as the amount of enzyme required to inhibit the reduction

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of NBT by 50%. The values are expressed in units/mg protein.

199 200

Glutathione peroxidase (Gpx, EC 1.11.1.9) activity was measured by a GPx Diagnostic kit (Biorex, Muckamore, UK) according to the manufacturers' protocol.

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Lactate dehydrogenase (LDH, EC 1.1.1.27) and alkaline phosphatase (ALP, EC 3.1.3.1)

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activities were assayed by a Diagnostic Reagent kit (Pars Azmoon, Tehran, Iran) according to

203

the manufacturer's protocol.

204 205

Hormone analyses

206 207

For whole-body cortisol, samples homogenized in 1 ml of phosphate buffer saline (PBS: 137

208

mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH=7.4) then transferred to another tube and 5 ml

209

of laboratory grade ethyl ether was added to each sample and vortexed for 1 min. The samples

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were centrifuged for 10 min at 3,000 rpm and frozen immediately at -20°C for 2 h. The

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unfrozen portion was transferred to a fresh tube and the ethyl ether evaporated under a stream

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of nitrogen. Finally, the lipid extracts were used for the cortisol assays (Sink et al. 2007) using

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a commercial cortisol ELISA kit purchased from Monobind (Lake Forest, CA, USA). The

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measurements were performed according to the manufacturer's protocol.

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For T3 and T4 analyses, samples were first homogenized in PBS. Next, tissue structures

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in the samples were disrupted by intermittent sonication for 3 min on ice and the samples

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vortexed vigorously, then centrifuged at 5,000×g for 10 min at 4°C. The supernatants were

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collected and stored at -80°C for thyroid hormones assay. Commercial T3 and T4 ELISA kits

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were used for the analyses (Pishtaz Teb Co., Tehran, Iran) by following the manufacturer's

220

protocol.

221 222

Statistical analyses

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The Statistical Package for the Social Sciences (SPSS, ver 17.0) was used for statistical

225

analyses. All results are presented as means with standard deviation (SD). All data were tested

226

for normality using the Kolmogorov–Smirnov test. The differences between groups were

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analyzed by two way ANOVA and Tukey HSD. For the hormone data, the differences between

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groups were analyzed by one way ANOVA. P-values