Titanium dioxide nanoparticles as guardian against environmental carcinogen benzo[alpha]pyrene

Abstract

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.

Figure 1. AHR structure as predicted with help of I-TASSER online server.

Figure 2. BaP structure as genrated with help of CORINA online server.

Figure 3. TiO₂ NP structure as constructed with help of DISCOVERY STUDIO 2.5.

Figure 4. Number of micronucleus/1000 cells, induced by various concentrations of BaP after 24 h exposure, *p<0.05 considered as significant.

BaP: Benzo[alpha]Pyrene; MN: Micronucleus.

Figure 5. Identification of cell viability after 6, 12 & 24 h of exposure to various concentrations of BaP.

*p<0.05 indicates significance. MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; BaP: Benzo[alpha]Pyrene.

Figure 6. Percentage change in ROS generation following 2, 6, 12 and 24 h of exposure to various concentrations of BaP.

*p<0.05 considered as significant. BaP: Benzo[alpha]Pyrene; ROS: Reactive Oxygen Species.

Figure 7. Number of micronucleus/1000 cells after 24 h exposure to 25 µM BaP, to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs, and co-exposure to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs along with 25 µM BaP.

*p<0.05 considered as significant. a– as compared to control, b- as compared to BaP treated. BaP: Benzo[alpha]Pyrene; MN: Micronucleus; TiO₂ NPs: Titanium Dioxide Nanoparticles.

Figure 8. Percentage viability of the cells exposed to 75 µM BaP, to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs and co-exposure to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs along with 75 µM, for 6, 12 & 24 h, as measured by MTT assay.

*p<0.05 indicates significance. a– as compared to control, b- as compared to BaP treated. MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; BaP: Benzo[alpha]Pyrene, TiO₂ NPs: titanium dioxide nanoparticles.

Figure 9. Percentage changes in ROS generation following 6, 12 and 24 h exposure to 50 µM BaP, to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs and co-exposure to 0.1, 0.5 and 1.0 µg/ml of TiO₂ NPs along with 50 µM BaP, as assessed by DCFH-DA dye.

*p<0.05 considered as significant. a– as compared to control, b- as compared to BaP treated. BaP: Benzo[alpha]Pyrene; ROS: Reactive Oxygen Species; TiO₂ NPs: titanium dioxide nanoparticles.

Figure 10. Modeled structures of AHR showing 83.5% of amino acid residues in favored region of Ramachandran plot.

Figure 11. Binding Sites of AHR-BaP complex.

(Pro180, Ser181, Cys183, Gly187, Leu196, Val200, Asn204, Leu259, Pro260, Leu265, Ala269, Thr270, Leu272, Pro274).

Figure 12. Binding Sites of AHR-TiO₂ NP complex.

(Tyr 145, Ser 151, Phe 148, Leu 369, Asn 673. Asn 366, Agr 384, Pro 385, Leu 413, Try 719, Gln383, Phe 406, Glu 488, Pro 665, Gln 667, Tyr 696, Gln 671, Asp 144, Ser 682, Gln 149).

Figure 13. Binding Sites of AHR - TiO₂ NP complex & BaP.

(Gln 666, Try 719, Phe 700, Pro 669, Gln 698, Thr 408, Phe 406, Phe 675, Thr 696).

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 TiO₂NP with AHR. TiO₂ NP bound to AHR blocks the internalization of BaP, preventing its metabolic conversion to BPDE and finally avoiding DNA damage.