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Invitro Toxicity of Nano Particles

Invitro Toxicity of Nano Particles

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Toxicology in Vitro 19 (2005) 975–983www.elsevier.com/locate/toxinvit0887-2333/$ - see front matter
2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.tiv.2005.06.034
In vitro toxicity of nanoparticles in BRL 3A rat liver cells
S.M. Hussain
a,
¤
, K.L. Hess
b
, J.M. Gearhart
c
, K.T. Geiss
d
, J.J. Schlager
a
a
Applied Biotechnology, Air Force Research Laboratory/HEPB, Wright-Patterson AFB, OH, USA
b
Geo-Centers, Inc., Wright-Patterson AFB, OH, USA
c
Alion Science & Technology, Wright-Patterson AFB, P.O. Box 31009, Dayton, OH 45431-0009, USA
d
Human E 
 V 
ectiveness Directorate, Air Force Research Laboratory, Wright-Patterson AFB, OH, USA
Received 31 March 2005; accepted 17 June 2005Available online 25 August 2005
Abstract
This study was undertaken to address the current de
W
cient knowledge of cellular response to nanosized particle exposure. Thestudy evaluated the acute toxic e
ects of metal/metal oxide nanoparticles proposed for future use in industrial production methodsusing the invitro rat liver derived cell line (BRL 3A). Di
erent sizes of nanoparticles such as silver (Ag; 15, 100nm), molybdenum(MoO
3
; 30, 150nm), aluminum (Al; 30, 103nm), iron oxide (Fe
3
O
4
; 30, 47nm), and titanium dioxide (TiO
2
; 40nm) were evaluatedfor their potential toxicity. We also assessed the toxicity of relatively larger particles of cadmium oxide (CdO; 1
m), manganeseoxide (MnO
2
; 1–2
m), and tungsten (W; 27
m), to compare the cellular toxic responses with respect to the di
erent sizes of nano-particles with di
erent core chemical compositions. For toxicity evaluations, cellular morphology, mitochondrial function (MTTassay), membrane leakage of lactate dehydrogenase (LDH assay), reduced glutathione (GSH) levels, reactive oxygen species (ROS),and mitochondrial membrane potential (MMP) were assessed under control and exposed conditions (24h of exposure). Resultsshowed that mitochondrial function decreased signi
W
cantly in cells exposed to Ag nanoparticles at 5–50
g/ml. However, Fe
3
O
4
, Al,MoO
3
and TiO
2
had no measurable e
ect at lower doses (10–50
g/ml), while there was a signi
W
cant e
ect at higher levels (100– 250
g/ml). LDH leakage signi
W
cantly increased in cells exposed to Ag nanoparticles (10–50
g/ml), while the other nanoparticlestested displayed LDH leakage only at higher doses (100–250
g/ml). In summary the Ag was highly toxic whereas, MoO
3
moderatelytoxic and Fe
3
O
4
, Al, MnO
2
and W displayed less or no toxicity at the doses tested. The microscopic studies demonstrated that nano-particle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregularshape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited signi
W
cant deple-tion of GSH level, reduced mitochondrial membrane potential and increase in ROS levels, which suggested that cytotoxicity of Ag(15, 100nm) in liver cells is likely to be mediated through oxidative stress.
2005 Elsevier Ltd. All rights reserved.
Keywords:
Nanoparticles; In vitro toxicity; Oxidative stress
1. Introduction
Nanotechnology involves the creation and manipula-tion of materials at nanoscale levels to create productsthat exhibit novel properties. Recently, nanomaterialssuch as nanotubes, nanowires, fullerene derivatives(buckyballs) and quantum dots have received enormousattention to create new types of analytical tools forbiotechnology and life sciences (Bruchez etal., 1998;Taton etal., 2000; Cui etal., 2001). Nanomaterials, whichrange in size from 1 to 100nm, have been used to createunique devices at the nanoscale level possessing novelphysical and chemical functional properties (Colvin,2003; Oberdörster, 2004). Although nanomaterials arecurrently being widely used in modern technology, there
*
Corresponding author. Address: Applied Biotechnology, Air ForceResearch Laboratory/HEPB, Area B, R ST, BDL 837, Wright-Patter-son Air Force Base, AFB, Dayton, OH-45433-5707, USA. Tel.: +1 937904 9517; fax: +1 937 904 9610.
E-mail address:
 saber.hussain@wpafb.af.mil(S.M. Hussain).
 
976
S.M. Hussain et al. / Toxicology in Vitro 19 (2005) 975–983
is a serious lack of information concerning the humanhealth and environmental implications of manufacturednanomaterials. The major toxicological concern is thefact that some of the manufactured nanomaterials areredox active (Colvin, 2003), and some particles transportacross cell membranes and especially into mitochondria(Foley etal., 2002). One of the few relevant studies waswith single-wall carbon nanotubes in mice (Lam etal.,2004).Lam etal. (2004) demonstrated that carbon nanotube products induced dose-dependent epithelioidgranulomas in mice and, in some cases, interstitial in
X
am-mation in the animals of the 7-day post-exposure groups.The recent study byOberdörster (2004)indicated thatnanomaterials (Fullerences C
60
) induced oxidative stressin a
W
sh model. Although limited studies have beenconducted on the toxicity of nanoparticles, there are noreports on the use of invitro models to evaluate potentialtoxicity screening of nanomaterials. The BRL 3A immor-tal rat liver cell line was selected in the present study as aconvenient invitro model to assess nanocellular toxicity.This cell line has been well characterized for its relevanceto toxicity models (Boess etal., 2003). In vivo exposure tonanoparticles is likely to have potential impact on theliver since exposure to these particles is likely to occurthrough ingestion and clearance by the liver (Jani etal.,1990). The toxicity end points (MTT, LDH, ROS andGSH) that were selected in the current study representvital biological functions of the mammalian system aswell as provide a general sense of toxicity in a relativelyshort time. The results described in this paper provide arange of doses that were toxic to these cultured cells anddata pointing to a general mechanism of nanoparticletoxicity. Since little information is available on nanoma-terial toxicity, simple invitro toxicity models and generaltoxicity end points are likely to assist in mechanisticevents after exposure and subsequent toxicity risk assess-ment of nanomaterials.
2. Materials and methods
 2.1. Chemicals
The test materials silver (Ag; 15, 100nm), molybde-num (MoO
3
; 30, 150nm), aluminum (Al; 30, 103nm),iron oxide (Fe
3
O
4
; 30, 47nm), manganese oxide (MnO
2
;1–2
m), and tungsten (W; 27
m) were received fromAir Force Research Laboratory, Brooks AFB, TX.Cadmium oxide (CdO-1000nm) and titanium oxide(TiO
2
-40nm) were purchased from Fluka Chemicals andAltair, Nanomaterials Inc., respectively. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT),
-nicotinamide-adenine dinucleotide-reduced(NADH), reduced GSH, rhodamine 123, Ham’s nutrientmixture F-12 media and gentamycin, were purchasedfrom Sigma Chemical Company (St. Louis, MO).
 2.2. Dispersion of nanomaterials in solution
The dispersion test was conducted in physiologicalphosphate bu
er saline (PBS) or deionized water. Basedon success of homogeneous dispersion studies usingphysical mixing and sonication, stock solutions wereprepared either in PBS or deionized water. The stocksolutions of Fe
3
O
4
(30, 47nm), TiO
2
(40nm) and CdO(1000nm) particles were homogeneously dispersed inPBS while Ag (15, 100nm) particles were suspended indeionized water. From this stock solution di
erent
W
nalconcentrations were prepared in cell growth medium(Ham’s Nutrient Mixture F-12) without serum. It wasnoted that turbidity increased with increasing concentra-tion of nanomaterials. The turbidity intensi
W
ed signi
W
-cantly at the 250
g/ml in all of the nanoparticlessolutions. Another physical observation was that Ag-100rapidly settled out of dispersed solution suspension andconstant mixing was necessary before adding it to theexposure media. Ag-100nm was not homogenously sus-pended in solution as evidenced by nanoparticles settlingto the bottom of the bottle.
 2.3. Cell culture
BRL 3A (ATCC, CRL-1442) immortalized rat livercells were used between passages 10 and 20. BRL 3Acells were grown in culture media with 5% fetal bovineserum. Cells were plated at a density in 6 or 24-wellplates for con
X
uency exposures by 36–48h when dosingwith nanoparticles was initiated. The cells were main-tained in a 5% CO
2
incubator at 37°C.
 2.4. Treatment protocol 
After the monolayer of cells became con
X
uent in 6 or24-well plate, BRL 3A cells were treated with a range of concentrations of nanoparticles suspended in Ham’sNutrient Mixture F-12 without serum for 24h. After the24h treatment, the various toxicity end points were eval-uated in control and nanoparticle-exposed cells.
 2.5. Qualitative observation of external morphology by phase contrast inverted microscopy
BRL 3A cells were exposed as mentioned above atvarious concentrations of nanoparticles for 24h. Aftercompletion of the exposure period, cells (control andexposed) were washed with PBS and observed by phasecontrast inverted microscopy at 100
£
magni
W
cation.
 2.6. Cytotoxicity endpoints
LDH leakage due to membrane damage was assessedby measuring the activity of LDH in the cells andmediaas described elsewhere with some modi
W
cations
 
S.M. Hussain et al. / Toxicology in Vitro 19 (2005) 975–983
977
(Hussain and Frazier, 2002). Mitochondrial functionwas evaluated spectrophotometrically by measuringthe degree of mitochondrial reduction of the tetrazo-lium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) to (aqueous insoluble product)formazan by succinic dehydrogenase (Carmichael etal.,1987) with minor modi
W
cation as described elsewherebyHussain and Frazier, 2002. ROS generation wasdetermined using dichlorodihydro
X
uorescein diacetate(H
2
DCFDA) (Wang and Joseph, 1999) with minormodi
W
cations as previously described byHussain andFrazier, 2002. Mitochondrial membrane potential wasdetermined by the uptake of rhodamine 123 (Molecu-lar Probes, Inc., Eugene, OR) according to the methodof Wu etal. (1990). Reduced glutathione (GSH) wasmeasured in 96-well plates using a SpectraMAX Plus190 microplate reader (Molecular Devices, Sunnyvale,CA) according to the procedures described in the Glu-tathione Assay Kit (Cayman Chemical Company, AnnArbor, MI).
 2.7. Statistical evaluation
The data were expressed as mean
§
standard devia-tion (SD) of three independent experiments. Whereverappropriate, the data were subjected to statistical analy-sis by one-way analysis of variance (ANOVA) followedby Dunnett’s method for multiple comparisons. A valueof 
 p
<0.05 was considered signi
W
cant. SigmaStat forWindows version 2.03 software was used for the statisti-cal analysis.
3. Results
The results demonstrated that exposure to Ag nano-particles for 24h resulted in concentration-dependentincrease in LDH leakage and exhibited asigni
W
cant(
 p
<0.05) cytotoxicity at 10–50
g/ml (Fig.1B). It wasnoted that there is a statistically signi
W
cant di
erencebetween di
erent silver particle sizes of 100 and 15nm,
Fig.1. E
ect of nanoparticles on LDH leakage in rat liver cells BRL 3A cells. Cells were treated with di
erent concentrations of nanoparticles for24h. At the end of the incubation period, the LDH assay was performed to assess the LDH leakage as described inSection 2. The percent of LDHactivity was then calculated by dividing the amount of activity in the medium by the total activity (medium and cell lysate). Control cells were cul-tured in nanoparticle-free media were run in parallel to treatment groups. The data are expressed as mean
§
SD of three independent experiments.(
¤
) indicates a statistically signi
W
cant di
erence compared to controls (
 p
<0.05).
µ
g/ml
024681012
   L   D   H   l  e  a   k  a  g  e   i  n   t  o  m  e   d   i  a   (   %   o   f  c  o  n   t  r  o   l   )
020406080100
CdO (1000 nm)
****
µ
g/ml
0102030405060
   L   D   H   l  e  a   k  a  g  e   i  n   t  o  m  e   d   i  a   (   %   o   f  c  o  n   t  r  o   l   )
020406080100
 Ag (15 nm) Ag (100 nm)
******
µ
g/ml
050100150200250300
   L   D   H   l  e  a   k  a  g  e   i  n   t  o  m  e   d   i  a   (   %   o   f  c  o  n   t  r  o   l   )
020406080100
MoO3 (30 nm)MoO3 (150 nm) Al (30 nm) Al (103 nm)Fe3O4 (30 nm)Fe3O4 (47 nm)TiO2 (40 nm)
****
µ
g/ml
050100150200250300
   L   D   H   l  e  a   k  a  g  e   i  n   t  o   M  e   d   i  a   (   %   o   f  c  o  n   t  r  o   l   )
020406080100
Mn (2000 nm)W (27000 nm)
****
ABCD

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