ROS production and oxidative DNA damage in smokeless tobacco extracts – exposed fibroblasts. Intracellular ROS levels were measured by carboxy-H2DCFDA fluorescence and flow cytometry after exposure to 0 (Ctr), 0.1%, 1%, 2%, or 4% of Tob for 2 h, 24 h, or 6 d, or 2 h followed by 24 h recovery in Tob-free medium (A). Oxidative DNA damage was measured using the Fpg-FLARE comet assay and depicted as the average comet tail (B) and the distribution of normalized mean tail lengths (C). Tail lengths were normalized by subtracting the mean tail length of the untreated sample from that of the Fpg-treated sample. At least 50 randomly chosen comet tails were analyzed per duplicate sample.

Analysis of the smokeless tobacco extracts – induced secretory phenotype. Concentrated conditioned medium from control (Ctr) or tobacco-treated 82.6 and WI-38 fibroblasts were normalized to the cell number and used for gelatin and casein substrate zymography for detection of MMP-2 and MMP-3 activity, respectively, and Western blottingfor MMP-1, MMP-2, and MMP-3 (A). Conditioned medium was also analyzed for IGFBP4, TIMP-2, and TIMP-1 by Western blotting(B).

Changes in epithelial cell-cell interactions in cocultures with smokeless tobacco extracts – exposed fibroblasts. HaCAT colonies on control (A, C, and E) or 4% Tob-exposed (B, D, and F) 82.6 dermal fibroblasts were immunostained for E-cadherin (A and B), ZO-1 (C and D), and nuclei (DAPI; E and F). Cocultures of HaCAT and 1%, 2%, or 4% Tob-exposed fibroblasts were also harvested for Western blotting(G).

Effects of smokeless tobacco extracts on the growth of human fibroblasts. Fibroblasts were exposed to 0 (control, Ctr), 1%, 2%, 4%, or 6% tobacco extract (Tob) and photographed 4 d later by phase contrast microscopy (A). Cell proliferation was followed by measuring total cell number (B) and [³H]thymidine incorporation (C–E) after tobacco exposure (B and C) or after tobacco exposure followed by replacement with control medium (2-d exposure, D; and 6-d exposure, E). At least 400 cells from randomly chosen fields were quantified for each data point.

Presence of H2AX foci in fibroblasts and skin rafts during smokeless tobacco extracts – exposure and recovery. Fibroblasts were exposed to 0%, 1%, 2%, or 4% of Tob for 6 d (A and C), or 4% tobacco extracts for 2 h, 24 h, or 6 d with or without a 6-d recovery (B and D), and immunostained for γH2AX foci. Hydrogen peroxide – treated fibroblasts were used as a positive control (H₂O₂). Immunostaining was done as described in Materials and Methods (A, B, and E). Quantification was done manually under the epifluorescent microscope (C, D, and F). At least 200 cells from randomly chosen fields were quantified for each data point. Differentiated skin rafts were exposed to 0% or 4% of Tob for 30 d and then subjected to γH2AX immunostaining(E and F). Representative sections for control (Ctr) and 4% Tob-treated samples were quantified (F).

Effect of smokeless tobacco extracts – exposed fibroblasts on keratinocyte proliferation and interstitial and basement membrane invasion. A. Representative fields of HaCAT cells grown in coculture with control, 2%, or 4% Tob-exposed oral fibroblasts. B. HaCAT keratinocytes were seeded onto 82.6 (left) or NHOF (right) fibroblasts treated with indicated concentration of Tob. Keratinocyte proliferation (epithelial fluorescence/cell number) was quantified as described in Materials and Methods after 8 d and expressed as the fold increase over control fibroblasts (Ctr), which received no Tob. Keratinocyte proliferation on replicatively senescent 82.6 fibroblasts (Sen) is shown for comparison. C. DOK keratinocytes were seeded onto 82.6 (left) or NHOF (right) fibroblasts and monitored for proliferation as described for B. D. HaCAT cells were plated in the upper chamber of transwells, with 82.6 (left) or NHOF (right), treated as indicated, in the lower chambers. Keratinocytes were monitored for proliferation, as described for B. E. HaCAT (left) and DOK (right) were seeded on extracellular matrix prepared from 82.6 fibroblasts, treated as indicated, and monitored for proliferation as described in B. F. DOK (dark gray columns) and HaCAT (white columns) keratinocytes were seeded onto collagen-coated filters above wells containing conditioned medium from 82.6 dermal fibroblasts treated with 0% (Ctr) to 4% Tob or replicatively senescent (Sen). After 16 h, the number of keratinocytes that invaded through the collagen gel to the other side of the membrane was quantified. G. DOK, HaCAT, and HCS-3 (light gray columns; invasive positive control) keratinocytes were seeded onto Matrigel-coated filters above wells containing conditioned medium from 82.6 dermal fibroblasts treated with 0% (Ctr) to 4% Tob. The number of keratinocytes that invaded through the Matrigel to the other side of the membrane was quantified. Columns, mean of three to five samples from independently confirmed representative experiments; bars, SD.