Cell viability restoration by DMTU and NAC in human lung cancer cells-A549 receiving the exposure of TiO₂-NPs or MWCNTs.

<p>(<b>a</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of TiO₂-NPs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and TiO₂-NPs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the cell viability reduced due to TiO₂-NPs exposure. All values are mean±S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). (<b>b</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of MWCNTs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and MWCNTs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the cell viability reduced due to MWCNTs exposure. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment).</p

Effect of DMTU/NAC on the reduction of micronuclei formation in A549 cells exposed to TiO₂-NPs/ MWCNTs.

<p>(<b>a</b>) The cells were exposed to TiO₂-NPs (10 & 50 µg/ml) with or without DMTU (10 mM) and NAC (2 mM) for 24 h. Ethyl methanesulfonate (6mM) was used as a positive control. Each data point represents the mean of three slides. A total 1000 bi-nucleated (BN) cells with well-defined cytoplasm were scored. Parallel sets were also run under identical conditions by exposing the cells only with either of DMTU or NAC. This group was used to compare the data between scavenger treated Vs non-scavenger treated cells. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). (<b>b</b>) The cells were exposed to MWCNTs (10 & 50 µg/ml) with or without DMTU (10 mM) and NAC (2 mM) for 24 h. Ethyl methanesulfonate (6mM) was used as a positive control. Each data point represents the mean of three slides. A total 1000 bi-nucleated (BN) cells with well-defined cytoplasm were scored. Parallel sets were also run under identical conditions by exposing the cells only with either of DMTU or NAC. This group was used to compare the data between scavenger treated Vs non-scavenger treated cells. All values are mean ± S.E. of 3 experiments.^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment).</p

TiO₂-NPs and MWCNTs (50 µg/ml) induced translational changes (HSP27, CYP2E1 and P⁵³) in the absences or presence of DMTU (10 mM)/ NAC (2 mM).

<p><b>a (I)</b> Assessment of altered expressions of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to TiO₂-NPs (50 µg/ml) for 24 h. GAPDH was used as internal control to normalize the data. Lane (1) unexposed control (2) 50.0 µg/ml TiO₂-NPs (3) 50.0 µg/ml TiO₂-NPs + DMTU (10 mM) (4) 50.0μg/ml TiO₂-NPs + NAC (2 mM). <b>(II)</b> Relative quantification (fold change) of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to TiO₂-NPs for 24h. GAPDH was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase^TM FC StandAlone V.4.0 software. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). <b>b (I)</b> Assessment of altered expressions of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to MWCNTs (50 µg/ml) for 24 h. GAPDH was used as internal control to normalize the data. Lane (1) unexposed control (2) 50.0 µg/ml MWCNTs (3) 50.0 µg/ml MWCNTs + DMTU (10 mM) (4) 50.0μg/ml MWCNTs + NAC (2 mM). <b>(III)</b> Relative quantification (fold change) of proteins involved in the induction of oxidative stress (HSP27 and CYP2E1) and genotoxicity (P⁵³) in A549 cells exposed to MWCNTs for 24h. GAPDH was used as internal control to normalize the data. Quantification was done in Gel Documentation System (Alpha Innotech, USA) with the help of AlphaEase^TM FC StandAlone V.4.0 software. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment).</p

Experimental design.

<p>Experimental design showing the exposure protocol of nanoparticles (TiO2-NPs and MWCNTs) and scavenges (DMTU and NAC), study groups and endpoints studied in human lung cancer cells-A549.</p

iAs in drinking water induced CA in 0 day old neonate mouse bone marrow cells after 8–18 day <i>in utero</i> exposure (A-C) and toxicant burden in hair, skin, liver and kidney of nulliparous female mice after 30 day oral administration (D) and rescue by PO administration of selenium and/or curcumin.

<p>Display of RF, CB, CG [A], CF [B], significant changes in formation of CB, CF and RF [C]; data are mean of three different experiments; ± SEM, ‘p’ values are ***<0.001, **<0.01; [D] significant reduction in iAs burden after PO selenium and/or curcumin administration, data are expressed as mean of three different experiments; ± SEM, ‘p’ values are ***<0.001, **<0.01.</p

Effect of DMTU/NAC on the restoration of ROS generated in A549 cells exposed to TiO₂-NPs/ MWCNTs.

<p>ROS generation was detected using ‘2, 7-dichlorofluorescin diacetate (DCFH-DA) dye. (<b>a</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of TiO₂-NPs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and TiO₂-NPs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the levels of intracellular ROS, which was induced by the exposure of TiO₂-NPs. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs TiO₂-NPs exposure). ##P<0.01 (TiO₂-NPs exposure Vs DMTU/NAC treatment). The positive control group was consisting of A549 cells pre-treated (1h) with 500 µM of H₂O₂. (<b>b</b>) In short term group, cells were pre-treated with either of DMTU or NAC for 30 minutes followed by exposure of MWCNTs (10 & 50 µg/ml) for 24 h. In long term group, cells were receiving a co-exposure (24 h) of DMTU/NAC and MWCNTs (10 & 50 µg/ml). Cells were then analyzed for DMTU/NAC mediated restoration in the levels of intracellular ROS, which was induced by the exposure of MWCNTs. All values are mean ± S.E. of 3 experiments. ^**P<0.01 (unexposed control Vs MWCNTs exposure). ##P<0.01 (MWCNTs exposure Vs DMTU/NAC treatment). The positive control group was consisting of A549 cells pre-treated (1h) with 500 µM of H₂O₂.</p

<i>In vitro</i> acute exposure to iAs and/or additives induced (A) cytotoxicity and viability, (B) levels of GSH, (C) time course of Nrf2 expression, (D) time course of NFkB expression, (E) time course of IkB expression, (F) time course of phosphorylated-P38 expression in neonate EpASCs.

<p>Data are expressed as mean of three different experiments; ± SEM, ‘p’ values are ^a <0.001, ^b <0.01, ^c <0.05,^d >0.05 vs. control and ^e <0.001, ^f <0.01, ^g <0.05, ^h >0.05 vs. arsenic.</p

Pathway diagram showing Nrf2 activation and enhanced GSH biosynthesis through curcumin and release of arsenic via selenium and GSH complex.

<p>Pathway diagram showing Nrf2 activation and enhanced GSH biosynthesis through curcumin and release of arsenic via selenium and GSH complex.</p

<i>In utero</i> exposure to iAs and/or additives induced changes in levels of (a) LRKs biomarkers, and (b) Nrf2 and NF-kB expression in neonate EpASCs.

<p>Data are mean of three different experiments; ± SEM, ‘p’ values are ^a <0.001, ^b <0.01, ^c <0.05 and ^d >0.05 vs. control and ^e <0.001, ^f <0.01, ^g <0.05 and ^h >0.05 vs. arsenic.</p

Transmission Electron Microscopy (TEM) analysis of internalization of TiO₂-NPs in A549 cells.

<p>TEM microphotographs showing internalization of TiO₂-NPs (a, b) in human lung cancer cells-A549. Arrows indicates the nanoparticles were adhered on the cell surface between microvilli and pseudopodes within few minutes of time (a) and subsequently internalized in small vacuoles deep in the cytoplasm, when observed at 24 h (b).</p