The The doses of BaP and NNK
The present study was designed to explorethe probability of protective application of nanoparticles againstenvironmental carcinogen induced toxicity. The doses of BaP and NNK thereference carcinogen in the study, were optimized for MTT assay in A549 cells.Exposure to both carcinogen used in this study, caused significant reduction incell viability.
(Xin-jiang W., et al 2003). Different doses of BaP and NNK (1000 µM,100 µM, 10 µM, 1.0 µM & 0.1 µM) were tested in A-549 cells to determine themost suitable dose for MTT assay in the study after 6h, 12h & 24h exposure.25 µM and 10 µM concentration were selected for further experimentation ascytotoxicity & sub-cytotoxic dose respectively.After the dose optimization experiments,we found that 25 µM/ml is most common and suitable cytotoxic dose for allcarcinogens; in this particular dose all carcinogens are showing approximate50% or less than 50% of cell viability. 10µM/ml was selected as sub-cytotoxicdose, because at this dose all carcinogens showed their toxic effects whereasthe viability of the cells was still good (~60-75%).
Cytotoxicity dose – 25µM Sub-cytotoxic dose- 10µMFor MTT assay the cytotoxic dose were usedfor the evaluation of protective potential of Nanoparticles. Whereas, for ROSand MN assays, the sub cytotoxic dose was used, because at this dose thesecarcinogens induced high amount of ROS generation and genetoxicity withoutkilling the cells.TiO2NPs, MWCNT and SWCNT were also evaluatedfor their safer doses, i.
e. their sub-toxic doses. 0.1, 0.
5 and 1.0 µg/ml doseswere found safe and induced least toxic effects, whereas after 10.0 µg/ml dosebecame toxic for all nanoparticles, as shown by us earlier also (SrivastavaR.K.
, et al 2013 & 2011). In order to evaluate the protectiveeffects of nanoparticles, A549 cells were Pre-expsed Post-exposed andco-exposed to some sub-toxic doses of MWCNTs, SWCNTs and TiO2NPs, (0.1, 0.5 and1.0 µg/ml) along with cytotoxic and sub-cytotoxic doses of BaP and NNK.The protective potential of SWCNTs, MWCNTs and TiO2NPswas tested using MTT, ROS and Micronucleus assays in A549 cells.
Cell viability assay results depicted very slightcytotoxicity upon exposure of cells to SWCNTs, MWCNTs and TiO2NPs leadingto slight reduction in cell viability. Upon exposure of cells to BaP and NNK, asignificant reduction in cell viability of A549 cells was observed. However,the pre-exposure and co-exposure of A549 cells to NNK and BaP (25µM cytotoxic dose) along with 0.
1 µg/ml, 0.5 µg/ml and 1 µg/ml of SWCNTs,MWCNTs and TiO2NPs showed a significant increase (P ? 0.05)in cell viability as compared to only BaP and NNK exposed cells, at all timeperiods. Increase in cell viability upon Pre-exposure and co-exposure (nanoparticleand carcinogen) as compared to the exposure to carcinogen alone, indicatesreduction of BaP and NNK induced toxicity (cyto-protection) by nanoparticles(SWCNTs, MWCNTs and TiO2NPs).
At all the doses (0.1 µg/ml, 0.5 µg/ml and 1 µg/ml) of SWCNTs, MWCNTsand TiO2NPs offeredprotection and raised the viability of a549 cells as compared to viability inonly BaP and NNK exposed cells upon pre-exposure and co-exposure.However, no cytoprotection was observed uponpost-exposure of nanoparticles to carcinogen exposed cells at all doses (0.1 µg/ml, 0.5 µg/ml and 1 µg/ml).
Reactive oxygen species (ROS) are chemically reactive speciescontaining oxygen, which are formed as by-products during mitochondrial aerobicrespiration. Under conditions of environmental stress, the levels of ROS areknown to increase dramatically (Devasagayamet al, 2004), resulting in significant damage to various cellularstructures such as membranes, cell organelles, and may also lead to genotoxicity.When the cellular ROS levels exceed the anti-oxidative ability of a cell, it undergoesoxidative stress and may lead to cell death (Niu et al, 2011).
ROS generation in A549 cells was evaluated by (DCFH-DA)staining to check the effect of SWCNTs, MWCNTs and TiO2NPs on ROSgeneration. Cells exposed to SWCNTs, MWCNTs and TiO2NPs showed slightgeneration of ROS. The NNK exposed cells showed a significant increase in ROSgeneration. However, upon pre-exposure and co-exposure of cells to BaP and NNK(10µM sub-cytotoxic dose) along with 0.
1µg/ml of nanoparticles (SWCNTs, MWCNTs and TiO2NPs),a significant reduction in ROS generation compared to only BaP and NNK exposedcells was observed after 24 hr. These resultsindicate quenching of ROS by nanoparticles. The anti-oxidative effect of nanoparticlesmay be attributed to a direct exchange between ROS and the high ratio ofelectrons which are found on the large surface area of nanoparticles (Yu et al, 2003 and Hochella et al, 2008) resulting in high abilityof nanoparticles to quench ROS. Further,the genotoxicity analysis in A549 cells done by micronucleus assay showed theinduction of a small number of micronuclei upon the exposure of cells toSWCNTs, MWCNTs, and TiO2NPs as compared to the unexposed control.The exposure of cells to a genotoxic dose (10µM) of BaP and NNK led to a dramatic increase in the number ofmicronuclei. However, the pre-exposure and co-exposure of cells to NNK alongwith 0.1µg/ml SWCNTs, MWCNTs and TiO2NPs resulted in asignificant decrease in the number of micronuclei induced, as compared to only BaPand NNK exposed cells.
A micronucleus is an erratic(third) nucleus which forms during anaphase in mitosis or meiosis.These are cytoplasmicbodies, which have a portion of the chromosome that was not carried to theopposite poles during anaphase. Micronuclei are known to form when a cellundergoes genetic damage. Micronuclei formation is therefore used forgenotoxicity assessment of various chemicals and toxicological screeningof potential genotoxic compounds. The reduction in number ofmicronuclei formed upon pre-exposure and co-exposure of cells to BaP and NNKalong with nanoparticles as compared to the exposure of cells to NNK only,clearly indicates reduction in BaP and NNK induced toxicity by thenanoparticles.
Our data from the MTT, ROS, and Micronucleus Assay indicatedthat the attenuation of BaP and NNK induced toxicity by the SWCNTs, MWCNTs andTiO2NPs. Finally, the MTT, ROS and MN assays data depict that SWCNTs, MWCNTsand TiO2NP, significantly attenuate the Environmental Carcinogens inducedtoxicity and considerably increase the cell viability and efficiently reducethe ROS and MN generation. Once a bulk substance is brought to nano-size, it losesits surface atomic coordinates thereby increasing free surface energy. Thestronger binding of the Nanoparticles as well as adsorption of carcinogens onnanoparticles surface could be an effect of nanosized particles in order tominimize high free surface energy, to accomplish the atomic coordination at thesurface and to establish electronic neutrality (Synowczynski j., et al2007). Another probable reason for the reduced toxic effect ofco-exposure could be the direct adsorption of carcinogens onto nanoparticlesitself, rendering carcinogens unavailable to its target molecules. Nanoparticles have been used for the adsorption ofpolycylic aromatic hydrocarbons (PAH) in an environmental scale (Wang, 2007 andYang et al, 2006). Some nanoparticleshave been used to scavenge Polycyclic Aromatic Hydrocarbons (PAHs), etc.
fromcontaminated soils and water (Karnchanasest and Santisukkasaem, 2007). Thescavenging capacity of nanoparticles may be due to their high surface area tovolume ratio and high affinity towards xenobiotics. Nanoparticles thereforeexhibit protective potential against environmental carcinogen induced toxicityin biological systems. Further research is needed to establish the protectiveproperties of nanoparticles and to investigate their role as toxicantscavengers in biological systems.