Smokeless Tobacco And Change In Intracellular Oxidation States By Laser Scanning Confocal Microscopy

The concentration-dependent effects of STE on the intracellular oxidized states of human keratinocytes were determined using confocal microscopy.40 The intensity of fluorescence reflects the intracellular oxidized state. When cells were treated with concentrations of STE at 250 and 300^g/ml, the color and fluorescence intensity of nuclei changed, possibly because of a change

FIGURE 7.1 Changes in intracellular redox states of oral human keratinocytes after treatment with smokeless tobacco extract (STE). Cells (70% confluent) were treated with STE for 24h. After 24h, medium was replaced with fresh medium containing 5|m 2,7-DCF, and fluorescence intensity was measured 5min later at 513nm with a confocal laser scanning microscope. A: control; B: 100|g/ml; C: 150|g/ml; D: 200|g/ml; E: 250|g/ml; F: 300|g/ml; G: 3mM H2O2 (positive control).

FIGURE 7.1 Changes in intracellular redox states of oral human keratinocytes after treatment with smokeless tobacco extract (STE). Cells (70% confluent) were treated with STE for 24h. After 24h, medium was replaced with fresh medium containing 5|m 2,7-DCF, and fluorescence intensity was measured 5min later at 513nm with a confocal laser scanning microscope. A: control; B: 100|g/ml; C: 150|g/ml; D: 200|g/ml; E: 250|g/ml; F: 300|g/ml; G: 3mM H2O2 (positive control).

in the oxidized states of the cells and the accumulation of the dye in the nuclear fractions. A concentration-dependent effect was observed. Approximately 70% confluent primary NHOK grown in six-well plates were treated with different concentrations (0 to 300|g/mg) of STE and incubated at 37°C in an atmosphere of 95% air and 5% carbon dioxide for 24h. After 24h of incubation with STE, the overall intracellular oxidized states of cells were measured by laser scanning confocal microscopy using the dye 2,7-dichlorofluorescin diacetate (2,7-DCF) as the fluorescent probe. The cells were incubated with STE for 24h prior to exposure to 2,7-DCF. Cells were also treated with hydrogen peroxide as a positive control. The dye is incorporated into the cells and is converted to a fluorescent metabolite by oxidation. The fluorescence intensity for each point was measured by confocal laser scanning microscopy according to the method of Ohba et al.41 An excitation wave-length of 513 nm was used. Relative fluorescence intensity was calculated using untreated control cells as the standard.40

The intensity of fluorescence increased with increasing concentrations of STE. Intensities were approximately 3.0-, 3.2-, 3.9-, and 4.6-fold higher than the control cells when the cells were treated with 150, 200, 250, and 300^g/ml of STE, respectively (Figure 7.1). Intracellular oxidized state increased with increasing concentration of STE treatment. The highest level of intracellular fluorescence produced by STE was comparable with that which was observed in cells treated with 3 mM H2O2. The dye 2,7-DCF can be oxidized by any peroxidase and hyperperoxide (including H2O2), which indicates production of highly reactive forms of oxygen, which in turn leads to DNA damage, cell death, and may lead to cancers of the oral cavity.40'41

7.13 SMOKELESS TOBACCO INDUCES PROTEIN KINASE C (PKC) ACTIVATION IN HUMAN ORAL KERATINOCYTES

Changes in the redox state of cells are thought to induce modifications of cellular signaling molecules, including protein kinase.40 PKC is involved in signaling pathways that mediate the regulation of many cell processes, including cell differentiation, cell survival, cytoskeletal function, gene expression, secretion, and cell-cell interactions. Studies by Fox et al.42 have demonstrated that STE causes time- and concentration-dependent loss of cellular adhesiveness of HT 1080 cells to a variety of matrices. Cells that lost their adhesiveness could regain that function by removal of the STE. Our studies have shown that incubation of keratinocytes in culture with STE results in the increased activity of PKC.40 Concentrations in protein kinase C activity in cytosol were measured after keratinocytes were exposed to STE, and time-dependent variations were observed. The cells were treated with 0 to 250^g/ml of STE for 1, 3, and 24h. After exposure of keratinocytes to STE for 1h, increases of 113, 121, and 146% occurred in cytosolic PKC activity with respect to control values for STE treatments of 100,150, and 250^g/ml, respectively. Similarly, after exposure to STE for 3h, increases of 113, 122, and 170% occurred in cytosolic PKC activity with respect to control values at these three same concentrations, respectively. Keratinocytes exposed to STE for 24h exhibited no significant changes in cytosolic PKC activity with respect to controls. PKC activity was also measured in solubilized particulate fractions for the three time points, but no significant differences were observed with respect to the control values. A growing body of evidence indicates that free radicals and ROS may be involved in mediating signal transduction through interaction with PKC. Thus, the current results support a role for both ROS and PKC with respect to the tissue-damaging effects of smokeless tobacco.40-43

Aloe and Your Health

Aloe and Your Health

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