Titanium Dioxide Nanoparticles As Guardian against Environmental Carcinogen Benzo[alpha]Pyrene

Polycyclic aromatic hydrocarbons (PAH), like Benzo[alpha]Pyrene (BaP) are known to cause a number of toxic manifestations including lung cancer. As Titanium dioxide Nanoparticles (TiO2 NPs) have recently been shown to adsorb a number of PAHs from soil and water, we investigated whether TiO2 NPs could provide protection against the BaP induced toxicity in biological system. A549 cells when co-exposed with BaP (25 µM, 50 µM and 75 µM) along with 0.1 µg/ml,0.5 µg/ml and 1 µg/ml of TiO2 NPs, showed significant reduction in the toxic effects of BaP, as measured by Micronucleus Assay, MTT Assay and ROS Assay. In order to explore the mechanism of protection by TiO2 NP against BaP, we performed in silico studies. BaP and other PAHs are known to enter the cell via aromatic hydrocarbon receptor (AHR). TiO2 NP showed a much higher docking score with AHR (12074) as compared to the docking score of BaP with AHR (4600). This indicates a preferential binding of TiO2 NP with the AHR, in case if both the TiO2 NP and BaP are present. Further, we have done the docking of BaP with the TiO2 NP bound AHR-complex (score 4710), and observed that BaP showed strong adsorption on TiO2 NP itself, and not at its original binding site (at AHR). TiO2 NPs thereby prevent the entry of BaP in to the cell via AHR and hence protect cells against the deleterious effects induced by BaP.


Introduction
Human exposure to xenobiotics is almost inevitable.Most of the human cancers are caused due to exposure to xenobiotics including PAHs and hence they are ultimately preventable.PAHs are produced during the combustion processes of organic materials during industrial and other human activities, like processing of coal and crude oil, vehicle traffic and cigarette smoke.PAHs may cause carcinogenesis by damaging the DNA and/or a number of proteins [1].The benzo[a]pyrene (BaP) is one of the most common PAHs and is a byproduct of grilled foods, tobacco, cigarette smoke and fuel combustion.BaP has long been correlated to a range of human cancers, predominantly lung and skin cancer [2,3].The carcinogenic properties of BaP in particular are mostly explained by their capability to induce DNA damage.BaP is the only PAH listed in group 1 by the International agency for research on cancer [4], and has thus been broadly considered and constitutes the reference compound for assessing toxicity of exposure to mixtures in the toxic equivalent factors approach [5].
After the enzymatic metabolism, BaP is converted to benzo[a]pyrene-7,8-diol-9,10-epoxides (BPDE), a crucial carcinogenic metabolite of BaP, that reacts primarily with the N2 position of guanine residues and to a minor coverage with the N6 position of adenine residues in DNA [8] to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases [3].
Recently, titanium dioxide nanoparticles have been employed in scavenging the high molecular weight polycyclic aromatic hydrocarbons (PAHs) from the contaminated soils [9].The scavenging capacities of the nanoparticles for PAH and other toxicants could be attributed to their higher affinity towards the xenobiotics due to surface chemistry, large surface area and other intrinsic properties of nanoparticles.Some studies also have shown that titanate nanotube has the capacity to scavenge the PAHs from water sample from the environment [10].The nanoform of TiO 2 for example titanate nanoSheets (TNS) and titanate nano tubes (TNT) have also been synthesized and used as additives for removing harmful compounds from cigarette smoke [11] including nicotine, tar, ammonia, hydrogen cyanide, selected carbonyls and phenolic compounds.Interestingly, TNT exhibits highly efficient reduction capability for most of the harmful compounds.This might be related to the intrinsic properties of TNT [11].TiO 2 is a naturally occurring oxide of http://www.absoluteastronomy.com/topics/Titanium titanium, and is biologically inert at lower doses, whereas, at higher doses it may induce slight toxicity and even apoptosis [12].
Considering this, we designed the present study in order to explore whether the discussed property of the TiO 2 NPs could be exploited in the biological system to safeguard against the deleterious effects of PAHs exposure.We also explored the doses of TiO 2 NPs, at which they provide maximum protection.Further, in silico experiments were also performed using bioinformatics tools, to attain insight of mechanism of protection.

Reagents and consumables
Most of the specified chemicals, reagents, diagnostic kits etc were purchased from Sigma Chemical Company Pvt. Ltd. (St. Louis, MO, USA).Cell culture media, PBS, antibiotic-antimycotic were purchased from Hi-Media (Hi-Media Pvt. Ltd., Mumbai, India).
1.1 Titanium dioxide Nanoparticles.Anatase form of TiO 2 NPs (d,25 nm, specific surface area 200-220 m 2 /g) without any coating were purchased from Sigma Aldrich (St. Louis, Missouri, USA, Cat no.637254).Particles were sterilized by heating to 120uC for 2 h and suspended in phosphate-buffered saline (stock: in 1 mg/ ml PBS).The mean hydrodynamic diameter and zeta potential (f) of the TiO2 NPs suspension in complete medium as determined by dynamic light scattering (DLS) measurement was 434.1 nm and 27.83 mV, respectively, as described previously by us [13].
1.2 Cell culture and treatment conditions.A549 cells (lung carcinoma cells) were obtained from the cell bank of NCCS Pune, Maharashtra, India, and were grown in a humidified atmosphere with 5% CO2 at 37uC.The cells were cultured in Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal bovine serum and 1% antibiotic and anti-mycotic solution, according to the standard procedure.Prior to use in the experiments, cell viability was estimated using trypan blue dye exclusion assay following the protocol as described earlier [14] and batches showing viability more than 95% were used for further experiments.

Dose optimization
Different doses of BaP (10 mM, 25 mM, 50 mM and 75 mM) were tested in A-549 cells for the selection of most suitable doses for various assays in our study.

Methodology
3.1 Micronucleus Assay.The genetic damage was assessed by MN assay as described earlier [15].In brief, A549 cells were grown on cover slips for 24 h in 6 well plates.The cells were exposed to different treatment conditions as discussed and incubated for 24h.The cells were fixed in cold fixative and stored at 220uC for at least 30 min.DNA staining was performed using bisbenzimide (1 mg/ml; Hoechst 33258; Sigma, St. Louis, MO, USA) for 4 min.the cells were washed in phosphate buffered saline (PBS), and were mounted on slide for microscopy.5000 cells were analyzed for each condition and results were expressed as MN/1000 cells.MN smaller than one-third the diameter of the nucleus were scored under a fluorescent microscope at 6306 magnification.
3.2 MTT Assay.Percentage cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay as described earlier [16].In brief, the cells (1610 4 ) were allowed to adhere for 24 h in 5% CO2 at 37uC and 20% humidity in 96-well culture plates.After the exposure for 6h, 12h and 24h, MTT (5 mg/ml of stock in PBS) was added (10 ml/ well in 100 ml of cell suspension), and plates were incubated for another 4h.At the end of incubation period, the reaction mixture was carefully taken out and 200 ml of dimethyl sulfoxide was added to each well, the contents were mixed well by pippeting up and down several times.The plates were kept on rocker shaker for 10 min at room temperature and then read at 550 nm using multiwell microplate Reader (Multi Skan, Thermo Scientific).Untreated sets were run under identical conditions and served as basal control.
3.3 ROS Assay.ROS generation was assessed in A549 cells using 2',7'-diclorodihydrofluorescein di-acetate (DCFH-DA, Sigma Aldrich, Missouri, USA) dye as fluorescence agent.ROS generation was performed as described earlier [12].The cells (1610 4 per well) were seeded in 96-well black bottom culture plates and allowed to adhere them for 24h in CO 2 incubator at 37uC.The medium was then aspirated and cells were exposed to different conditions as describes for 2h, 6h, 12h and 24h.On the completion of respective exposure periods, cells were incubated with DCFH-DA (10 mM) for 30 min at 37uC.The reaction mixture was then aspirated and replaced by 200 ml of PBS in each well.The plates were kept on rocker shaker for 10 min at room temperature in the dark.Fluorescence intensity was measured using multiwell microplate reader (Multi Skan, Thermo Scientific) on excitation wavelength at 485 nm and emission wavelength at 528 nm.The data were expressed as percentage of the unexposed control.

In silico study
4.1 Preparation and Validation of AHR.PDB structure of aryl hydrocarbon receptor (AHR Uniprot entry -P35869) is not available in the PDB databank.I-TASSER [17] online server was utilized for the ab-initio modeling to build the 3D structure of AHR as shown in Figure 1, and validated by the approach of Ramacharndran Plot by using RAMPAGE [18].
AHR 3D structure has been submitted in Protein Model Data Base, (PMDB ID-PM0078981) [19], a Protein Data Base which collects three dimensional protein models obtained by structure prediction methods.The Accelrys Discovery studio 2.5 program was found to be the most suitable software for the designing of TiO 2 anatase crystal structure.
After the construction of Unit cell of TiO 2 anatase by using the anatase lattice parameters, a surface was created and this unit cell was extended in the desired directions (axis) creating a new surface of TiO 2 comprising [1,0,1] of 5 unit cells in 6direction and 2 unit cells in the Z direction.This gave a surface of dimensions 1.89160.378261.9004nm 3 as shown in Figure 3.
4.4 Docking Study.All the in silico docking analyses were performed using PatchDock [21].The AHR was docked with the BaP, as well as TiO 2 NP.The resultant pdb file obtained after AHR and TiO 2 NP docking was used as AHR-TiO 2 NP complex, and was docked with BaP by uploading the receptor and molecules in PatchDock Server, an automatic server for molecular docking.Clustering RMSD was chosen as 4.0 A ˚.

Statistical analysis
All the experiments were performed in triplicates and were repeated twice.The final results were expressed as mean of the values obtained from all experiments.The standard error of mean (SEM) was also calculated.Statistical analysis was performed by one-way analysis of variance (ANOVA) using Newman-Keuls test to compare all the groups by graph pad prism3.In all the cases, p,0.05 was considered as significant.

Protein Structure Validation
3D structure model of AHR was generated using I-TASSER online server (ab initio modeling), as shown in Figure 1.The model was validated using RAMPAGE by Ramachandran plot approach.The torsion angles of the 3D structure of AHR showed 83.5% amino acid residues in the favored regions as shown in Figure 10.

In Silico Docking Studies of TiO 2 NP and BAP
In the present study, the orientation and binding affinity (in terms of the total docking score and binding residues) of TiO 2 NP and BaP was explored with AHR.TiO 2 NP showed high binding affinity with AHR with a docking score of 12074, as compared to the docking score of BaP with AHR (4600).Docking score of BaP with AHR-TiO 2 NP complex was 4710.
The chemical nature of binding site residues of AHR within a radius of 4Au with TiO 2 NP showed hydrogen bond interaction with Gln667-NE2: O98 bond length 3. Whereas, the BaP when docked with AHR-TiO 2 NP complex was adsorbed at the surface of TiO2 NP, as shown in Figure 13.The chemical nature of binding site residues of AHR-TiO 2 NP complex within a radius of 4Au, showed the hydrophobic interaction with Gln 666, Try 719, Phe 700, Pro 669, Gln 698, Thr 408, Phe 406, Phe 675, Thr 696.

Discussion
The present study was designed to explore the probability of protective application of nanoparticles against environmental carcinogen induced toxicity.The doses of BaP, the reference carcinogen in the study, were optimized for MN, MTT and ROS generation assays in A549 cells.The BaP exposure caused significant reduction in cell viability, which was dependent on period of exposure and was highest with 75 mM at 24h (10.416.494%).This effect could be attributed to enhanced production of ROS as a result of BaP exposure which was found to be highest with 50 mM at 12h (246.961.178%).Further, ROS generation might have caused the DNA damage which was highest with 25 mM at 24h (53.3364.41MN/1000cells).Which is in accordance with the results of past toxicological studies of BaP [22].The doses of 25 mM for MN assay, 75 mM for MTT assay and 50 mM for ROS generation assay were selected, as maximum effects were observed at these doses in respective assays.
In order to evaluate the protective effect of nanoparticles, A549 cells were co-exposed to some non-toxic doses of TiO 2 NPs (0.1, 0.5 and1.0 mg/ml) along with BaP.
At all doses, the TiO 2 NPs offered protection and raised the viability of A549 cells as compared to viability in only BaP exposed cells as measured by MTT assay after co-exposure.The protective effect was slightly higher with 0.1 mg/ml concentration of TiO 2 NPs than 0.5 mg/ml and 1.0 mg/ml TiO 2 NPs.
Similarly, co-exposure of all three doses of TiO 2 NPs caused significant lowering in ROS production in BaP exposed A549 cells at all time periods.Again, the effect of 0.1 mg/ml concentration of TiO 2 NPs was marginally higher as compared to 0.5 mg/ml and 1 mg/ml TiO 2 NPs.Similar effects were observed in the MN assay also, where coexposure of all three doses of TiO 2 NPs caused significant reduction in MN induction by BaP in A549 cells.Again, the effect of 0.1 mg/ml concentration of TiO 2 NPs was marginally higher as compared to 0.5 mg/ml and 1 mg/ml of TiO 2 NPs.
All three end points used for study depicted clear cut reduction in the toxicity of BaP, which indicated the protective potential of TiO 2 NPs in low dose.
In silico approach was applied in order to explore the probable mechanism by which TiO 2 NP provided protection against BaP induced toxicity.Previous studies have established that AHR is responsible for the entry and regulation of the enzymatic metabolism of BaP to Benzo[a]pyrene -7,8-diol-9,10-epoxides (BPDE), a crucial carcinogenic metabolite of BaP, which reacts primarily with the N2 position of guanine residues and to a minor coverage with the N6 position of adenine residues in DNA [8] as shown in Figure 14A.BPDE forms bulky adducts with DNA which blocks DNA synthesis during replication by high fidelity DNA polymerases [3].
The docking study, performed to determine the binding abilities of BaP and TiO 2 NPs with AHR, revealed that TiO 2 NP bind with much higher molecular docking score with AHR (12074) as compared with docking score of BaP with AHR (4600).This establishes a strong possibility of preferential binding of TiO 2 NP over BaP with AHR, incase both TiO 2 NP and BaP are present together in cell vicinity.Further, to investigate how AHR might respond to BaP when TiO 2 NP is already bound to AHR, the BaP was docked with AHR-TiO 2 complex.The BaP was adsorbed strongly at the TiO 2 NP (score 4710) and not at its original binding site at AHR, as shown in Figure 14B.Previous Studies have also shown a strong adsorption potential of TiO 2 NP towards PAHs and some other chemical carcinogens present in cigarette [9,11].In present case also strong adsorption potential of TiO 2 NP might have caused the shifting in binding position of BaP from AHR to TiO 2 bound to AHR.
Once a bulk substance is brought to nano-size, it loses its surface atomic coordinates thereby increasing free surface energy.The stronger binding of the TiO 2 NP on AHR as well as adsorption of BaP on TiO 2 surface could be an effect of nanosized TiO 2 in order to minimize high free surface energy, to accomplish the atomic coordination at the surface and to establish electronic neutrality [23].Another probable reason for the reduced toxic effect of coexposure could be the direct adsorption of BaP onto TiO 2 nanoparticles itself, rendering BaP unavailable to its target molecules, which requires further in depth analysis.

Conclusion
The present study clearly describes the attenuation of BaP induced toxicity by TiO 2 nanoparticles in A549 cells, along with the probable mechanism of TiO 2 NPs protection against BaP.To the best of our knowledge, this is the very first study suggesting future prophylactic application of nanoparticles as guardian against the chemical carcinogens at the molecular/cellular level.We are in process of further investigating whether TiO 2 NPs are capable of protecting cells against other chemical carcinogens also?And if this process of protection can be further enhanced by modifying the surface chemistry of TiO 2 NPs.Also, we are exploring the capacities of other nanoparticles like CNT, Fullerene etc. for their potential to provide protection against chemical carcinogens.

4 . 2
Preparation of Benzo[a]Pyrene.The SMILES (Simplified Molecular Input Line Entry Specification) notations of the BaP were obtained from the ZINC database (ZINC ID -01530818).The 3D-structure of BaP was generated de novo, via the internet (http://molecular-networks.com/products/corina), by program CORINA on server running in Computer-Chemie-Centrum, Universiy of Erlangen-Nurnburg, Germany.It is a ruleand data-base system, that automatically generates 3D atomic coordinates from the constitution of a molecule as expressed by connection table or linear code as shown in Figure 2. 4.3 Construction of Anatase TiO 2 Nanoparticle.After studying the anatase crystal structure, we found that anatase is the thermodynamically favored phase.According to the anatase lattice parameters, the tetragonal crystal have lengths (A, B and C) as; A = B = 3.782A ˚, C = 9.502.A ˚and angles (alpha, beta and gamma) as; Alpha = beta = gamma = 90 o [20].

Figure 14 .
Figure 14.Role of AHR in BaP internalization.(A) Internalization of BaP in to cell through AHR, metabolic conversion to BPDE and interaction of BPDE with DNA.(B) Preferential binding of TiO 2 NP with AHR.TiO 2 NP bound to AHR blocks the internalization of BaP, preventing its metabolic conversion to BPDE and finally avoiding DNA damage.doi:10.1371/journal.pone.0107068.g014