Rats were given Cd in distilled water at 5 ppm (lower dose) and 50 ppm (higher dose) for 30 days. This resulted in an average daily Cd intake (calculated on the basis of water consumption) of 0.35 ± 0.04 mg/kg and 3.51 ± 0.29 mg/kg in the groups receiving 5 ppm and 50 ppm of Cd, respectively. There was no difference in body mass gain during the period of Cd consumption in all experimental groups (228.4 ± 14.6, 233.2 ± 17.6, 219.9 ± 41.8 at 0 ppm, 5 ppm, and 50 ppm, respectively). Consumption of Cd resulted in a dose-dependent increase in the metal levels in peripheral organs, with the highest levels being observed in the kidneys and liver (Table 1). Substantial levels were noted in the lungs and blood also.
Cd Dose (ppm) Lungs Blood Kidney Liver 0 3.5 ± 1.6 9.4 ± 0.9 12.6 ± 0.4 6.2 ± 1.5 5 6.8 ± 1.2* 21.6 ± 15.5* 413.5 ± 26.3* 195.6 ± 101.0* 50 107.7 ± 5.5*, # 25.6 ± 1.3* 5214.3 ± 188.8*, # 260.6 ± 138.8*, # Note.Data are presented as mean values ± SD. Significance at: *P < 0.05 vs. controls (Cd dose 0 ppm) and #P < 0.05 vs. low (5 ppm) Cd dose.
Table 1. Cadmium Concentration in Tissue (µg/kg)
Histological analysis of lung tissue from a control group (Figure 1D) revealed a normal histological structure (despite minimal hyperemic blood vessels, edema and infiltration noted in some animals) (Figure 1A, B). Treatment with lower Cd doses resulted in more prominent tissue changes ranging from minimal to mild in almost all rats (Figure 1A, B), with perivascular edema and infiltration being particularly noticeable (Figure 1E) and peribronchiolar edema and desquamation of respiratory epithelium exhibiting minimal intensity. Lungs from rats exposed to higher Cd doses (Figure 1F) were more seriously affected and exhibited histopathological scores for almost all parameters ranging from minimal to moderate (Figure 1A, B). Perivascular edema and perivascular, peribronchial, and interstitial inflammatory infiltration were the most remarkable changes associated with this treatment group. A detailed examination of all histopathological scores revealed a dose-dependent increase following Cd consumption (Figure 1C).
Figure 1. Effect of oral Cd intake on the lung. (A) Distribution of histological scores for specified parameters in control and Cd-treated animals. (B) Histopathological scores for observed parameters. (C) Summary of analyzed histopathological scores. (D, E, F) Representative micrographs of lung sections from control animals (D) and animals exposed to cadmium at 5 ppm (E) and 50 ppm (F) stained with H & E. Inset on F: remarkable perivascular infiltration. Abbreviations: ar - arteriole, br - bronchiole, pve - perivascular edema, pvi - perivascular infiltration, pbi - peribronchial infiltration. Bar = 100 µm, 40 µm (inset). Data are expressed as mean values of eight animals per treatment group ± SD. Significance at: *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Cd 0 ppm; #P < 0.05 and ##P < 0.01 vs. Cd 5 ppm.
The number of goblet cells in larger intrapulmonary airways was substantially increased after treatments with Cd (Figure 2B, C) compared to that observed in controls (Figure 2A). Consumption of higher Cd doses resulted in the appearance of goblet cells in intrapulmonary airways with a diameter of less than 300 μm (Table 2 and Figure 2B, C).
Figure 2. Goblet cell numbers in airways of animals exposed to Cd. (A) Control animals. (B) Animals exposed to 5 ppm of Cd. (C) Animals exposed to 50 ppm of Cd. Representative micrographs of intrapulmonary respiratory airways of large (up) and small (down) diameter obtained from sections stained using the AB/PAS method. Bar = 100 μm; arrowheads - goblet cells.
Cd dose (ppm) Goblet Cells (N/mm of luminal perimeter) Small airways Large airways 0 0 ± 0 1.7 ± 0.9 (0-1.9) 5 0 ± 0 10.6 ± 2.5 (8.1-13.1) 50 2.3 ± 3.3 (0-10.1) 58.3 ± 46.2 (34.6-137.1) Note.Small airways-airways with diameter less than 300 µm. Large airways-airways with diameter larger than 300 µm. Data are presented as mean values ± SD. Range is given in parenthesis.
Table 2. Number of Goblet Cells in Pulmonary Airways Following Oral Cd Administration
Consumption of Cd caused no changes in relative lung mass (Figure 3A). Examination of basic oxygen free radical scavenging enzymes in lung tissue homogenates, specifically SOD and CAT, revealed a significant decrease in activity of both enzymes at the higher Cd dose and a decrease in CAT activity at the lower dose (Figure 3B).
Figure 3. Effect of oral Cd intake on the relative lung mass, enzyme activity, and proinflammatory cytokine levels in lung homogenates. (A) Relative lung weight. (B) SOD and CAT activity. (C) MPO content. (D) IL-1β. (E) TNF. (F) IL-6. Data are expressed as mean values of eight animals per treatment group ± SD. Significance at: *P < 0.05 and **P < 0.01 vs. Cd 0 ppm; #P < 0.05 and ##P < 0.01 vs. Cd 5 ppm.
An increase in MPO activity was observed only in response to the higher Cd dose (Figure 3C). Measurement of proinflammatory cytokine concentrations in lung homogenates revealed a dose-dependent increase in IL-1β concentrations (Figure 3D), increased levels of TNF at both Cd doses (Figure 3E), and increased IL-6 at the lower Cd dose (Figure 3F), while lower levels of IL-6 were noted at 50 ppm compared to those in controls.
Higher Cd dose intake resulted in an increase in the number of leukocytes isolated from lung tissue by enzyme digestion (Figure 4A) that resulted from an an increased number of macrophages in the lungs (Figure 4B). No significant change was noted in the number of isolated lymphocytes, while the number of granulocytes decreased following higher Cd dose consumption (Figure 4B).
Figure 4. Effect of oral Cd intake on lung leukocyte activity. (A) Number of lung cells. (B) Absolute number of lymphocytes, macrophages, and neutrophils. (C) NBT reduction. (D) NO production. (E) MPO content. Data are expressed as mean values of eight animals per treatment group ± SD. Significance at: *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Cd 0 ppm.
Examination of the activity of isolated lung leukocytes revealed no changes in NBT reduction capacity (Figure 4C) and NO production (Figure 4D), but the activity of intracellular MPO increased following higher Cd dose consumption (Figure 4E). Measurement of proinflammatory cytokine production by lung leukocytes revealed decreased IL-1β (Figure 5A) and TNF (Figure 5B) at both Cd doses and increased IFN-γ production at a higher Cd dose (Figure 5D), with unchanged levels of IL-6 (Figure 5C) and IL-17 (Figure 5E). No changes in the anti-inflammatory cytokine IL-10 were observed (Figure 5F).
To determine if altered activity of lung leukocytes could potentially influence immunological homeostasis within the lungs, we exposed lung leukocytes isolated from controls and from animals receiving lower and higher Cd doses to a commensal microbe, Gram-positive bacteria S. epidermidis (Figure 6, Panel B). S. epidermidis did not affect cytokine production by lung leukocytes isolated from control rats (0 ppm), with the exception of lower IL-1β levels following S. epidermidis stimulation (Figure 6, Panel A). In contrast to controls, lung leukocytes obtained from animals orally exposed to Cd responded to S. epidermidis exposure by exhibiting a dose-dependent increase in IL-1β and increased levels of TNF, IFN-γ, and IL-17 at the higher dose (Figure 6, Panel B). Measurement of anti-inflammatory/regulatory cytokine IL-10 revealed increased levels of IL-10 at both Cd doses compared to those of controls.
Figure 6. Effect of S. epidermidis exposure on IL-1β, TNF, IL-6, IFN-γ, IL-17, and IL-10 production by Cd-exposed lung leukocytes and stimulation index. (Panel A) Non-stimulated and S. Epidermidis-stimulated cytokine production by lung leukocytes from control animals. (Panel B) Responsiveness of lung leukocytes isolated from control and Cd-exposed animals to S. epidermidis stimulation in vitro. Data are expressed as mean values of eight animals per treatment group ± SD. Significance at: & P < 0.05 vs. non-stimulated; *P < 0.05 and **P < 0.01 vs. Cd 0 ppm; #P < 0.05 vs. Cd 5 ppm.