Cadmium induces lung inflammation independent of lung cell proliferation: a molecular approach

Abstract

Background: Cadmium is one of the inflammation-related xenobiotics and has been regarded as a potent carcinogen. The relationship between inflammation and cell proliferation due to chronic infection has been studied, but the mechanism is not fully clear. Though the mode of cadmium toxicity is well characterized in animal cells, still it requires some further investigations. Previously we reported that cadmium induces immune cell death in Swiss albino mice. In the present study we showed that instead of inducing cell death mechanism, cadmium in low concentration triggers proliferation in mice lung cell and our results reveals that prior to the induction of proliferation it causes severe inflammation.

Methods: Swiss albino mice were treated with different concentrations of cadmium to determine the LD50. Mice were subdivided (5 mice each) according to the exposure period (15, 30, 45, 60 days) and were given sub lethal dose (5 mg/Kg body weight) of cadmium chloride and ibuprofen (50 mg/Kg body weight, recommended dose) once in a week. SEM and histology were performed as evidence of changes in cellular morphology. Inflammation was measured by the expression of Cox-2 and MMPs. Expression of proinflammatory cytokines (Cox-2, IL-6), signaling and cell cycle regulatory molecules (STAT3, Akt, CyclinD1) were measured by western blot, ELISA and immunoprecipitation. Mutagenecity was evidenced by comet assay. Cell proliferation was determined by cell count, cell cycle and DNA analysis.

Results: Prolonged exposure of low concentration of cadmium resulted in up regulation of proinflammatory cytokines and cell cycle regulatory molecules. Though NSAIDs like Ibuprofen reduces the expression of inflammatory cytokines, but it did not show any inhibitory effect on cadmium adopted lung cell proliferation.

Conclusion: Our results prove that cadmium causes both inflammation and cell proliferation when applied in a low dose but proliferative changes occur independent of inflammation.

Figure 1

Dose dependence survival of Cadmium treatment. Each value represents the percentage of died populations in each dose chosen with +/- SEM (n = 5) and p < 0.05, compared to normal. The experimental groups represent the Cadmium treated mice [inject CdCl₂, (i.p.)] at a concentration of 2.5 mg/Kg body weight, 5 mg/Kg body weight, 10 mg/Kg body weight, 20 mg/Kg body weight and 40 mg/Kg body weight and 80 mg/Kg body weight respectively. The sub lethal concentration (5 mg/Kg body weight) was chosen as an applied dose.

Figure 2

Histopathology of lung sections of Swiss albino exposed to Cadmium (5 mg/Kg body weight) showing progressive lung inflammation and prevention by Ibuprofen. A, Histology of lung sections of normal mice. B, Mice were treated with low dose of Cadmium (5 mg/Kg body weight), subsequently for 15, 30, 45, 60 days, granulation and air space enlargement was shown after 45 days which is the sign of inflammatory development. C, Application of Ibuprofen reduces the chance of lung oedema formation throughout the experimental periods. Original magnification (100×).

Figure 3

SDS-PAGE and Gelatin zymography represent the expression of MMP-2. Detection, by gelatin zymography, of matrix metalloproteinase-2 (MMP-2) and (MMP-9) expression at different time points after the induction of Cadmium (5 mg/Kg body weight). Lung cell extracts were prepared at days of 15, 30, 45 and 60 from normal (N), Cadmium treated and Cadmium plus Ibuprofen treated (three individual animals per dose) mice. The zymography was developed and stained as described in Materials and Methods. The picture shows increased expression of MMP-2(72 kD), which was not inhibited by Ibuprofen. Gel is representative of three comparable experiments indicate p < 0.05 with respect to the control.

Figure 4

Cadmium induced expression of Cox-2, IL-6, p-STAT3 and p-Akt. Cell lysate from control and treatment lung were subjected to western blot analysis and ELISA. A, Expression of Cox-2, IL-6, p-STAT3, p-Akt were increased throughout all the experimental period. Though Ibuprofen reduced the expression of Cox-2 and IL-6, but it could not be able to revert the expression of p-STAT3 and p-Akt. B, Bar graphs represent the quantitative densitometric value of the expressed protein. C, OD values showed significant increasement of Cox-2 and IL-6 expression by ELISA. Data is representative of three comparable experiments and indicate p < 0.05 with respect to the control.

Figure 5

Immunohistochemical expression of Cox-2. A, Control lung shows no expression of Cox-2. B, Cox-2 expression was profoundly increased in treated lung. C, Application of Ibuprofen reduces the expression level of Cox-2. D, E, F, Negative staining shows no such expression. Original magnification (100×).

Figure 6

Cadmium causes lung cell proliferation. We evaluated the effect of cadmium on the lung cell count in normal and treated mice. The normal and treated mice were given pyrogen free saline water and different dose of Cadmium (2.5 mg, 5 mg, 10 mg, 20 mg, 40 mg and 80 mg/Kg body weight) respectively for two months. Cell count gradually increased in low dose of CdCl₂ (2.5, 5 mg/Kg body weight), but beyond that (10 mg, 20 mg, 40 mg and 80 mg/Kg body weight) cell death occurred. Data is representative of three independent experiments (p < 0.05).

Figure 7

Comet assay. A, DNA tail formation was increased in higher dose of Cadmium, while no significant change was observed between control and low dose. B, C, Showed tail formation increased significantly in 80 mg/Kg body weight dose of Cadmium. Data is representative of three independent experiments (p < 0.05).

Figure 8

Effect of Cadmium on cell cycle distribution of mice lung cell determined by flowcytometry analysis. B, Percentage of cell increased in S-G₂/M phase in treated set. A, Increased cell number was observed in sub-G₀/G₁ phase in control set, which was the indication of cell proliferation. Data is representative of three independent experiments (p < 0.05).C, No DNA ladder was formed in low dose (5 mg/Kg body weight) CdCl₂ treated lung while compared with control ladder (L) and normal one.

Figure 9

Scanning Electron Microscopy of Cadmium treated lung. A, No lobular appearances were observed under lower magnification. B, Alveolar space was getting narrowed in treated lung, compared to the normal one. C, Lobular appearances were prominent at higher magnification. D, Narrow edicular spaces signify the increasement of cell density. Ibuprofen showed no effect to revert the structural deformity.

Figure 10

Effect of Cadmium on CyclinD1 expression. A, Expression of CyclinD1 was increased throughout all the experimental period. B, Bar graphs represent the quantitative densitometric value of the expressed protein. Data is representative of three comparable experiments indicate p < 0.05 with respect to the control. C, Immunoprecipitation showed the formation CyclinD1-Cdk4 complex, which is an indication of cell cycle progression.