The experimental protocols performed in this study, including the collection of venous blood samples, were submitted to and approved by the Universidade Federal de Santa Maria (RS, Brazil) Research Ethics Committee under the registration number 0089.0.243.000-07.

The study material was prepared as previously described [16]. Briefly, the material that was used for in vitro assays was new and disposable to avoid Cd²+ contamination. Control samples of the reagents were tested to identify metal contamination. Before use, all glassware was immersed in 20% aqueous HNO₃ (v/v) for 24 h and subsequently rinsed with deionized water.

Both the comet and cell viability assays were performed in triplicate, while six replicates were analyzed to quantify the intracellular Cd²+content. The following screening strategy was performed for all assays: (1) negative control-adjusted leukocyte suspension (LS) (10⁴ cells/mm³) in phosphate-buffered saline (PBS; pH 7.4); (2) positive control-LS plus 4 mM H₂O₂; tests-composed of LS with either 1, 2.5, 5, 10, 15, or 25 μg/mL Cd²+ (CdCl₂ analytical grade, Merck®, Darmstadt, Germany). This concentration range was chosen based on specialized literature data that related the metal concentration with cellular or DNA damage [17-19]. All sample groups were incubated for 1 h at room temperature (25°C) under slow stirring of which the direction was inverted every 10 min. Thereafter, the abovementioned tests were performed, as described below, with the exception of the intracellular Cd²+quantification. In this case, the samples were additionally washed five times with PBS (pH 7.4) and centrifuged at 2000 rpm for 10 min at room temperature.

The cytotoxicity of Cd²+ was evaluated by a cell viability test using the trypan blue method to determine the loss of membrane integrity [20]. Initially, the cells were exposed to the previously established Cd²+ concentration. After the incubation, 100 μL of LS was mixed for 8 min with 100 μL of 0.4% trypan blue solution. The cell viability was determined microscopically (400 x magnification) in a Neubauer chamber, and the following two categories of cells were scored: (1) living cells that appeared uncolored or light blue and (2) dead cells with a blue color. For this assay, 300 cells were counted for each sample analyzed.

The alkaline comet assay (single cell gel electrophoresis assay) was performed to evaluate genotoxicity according to a previously described methodology [16] and in accordance with the comet assay guidelines [21]. The microscope slide analysis was performed, counting 100 cells per slide and classifying them based on damage levels from 0 (no damage) to 4 (maximum damage). The average obtained for each treatment allowed calculating the respective damage rate, considering the DNA damage scale from 0 (100 cellsx0) to 4 (100 cellsx4) as proposed earlier [22].

All data obtained from the tests carried out in this study were evaluated by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test, wherever appropriate, considering p < 0.05 for statistical significance.

One of the challenges in studies on metal toxicology is to determine the quantity of the heavy metal that is capable of entering into the cytoplasm from the cell exposed to the metal. This issue is also applicable to Cd²+ and needs to be elucidated. Furthermore, it is crucial to understand if there is a constant cell entry percentage or if this entry is disproportional considering the different concentrations of exposure. Figure 1 shows an increasing intracellular Cd²⁺ concentration in human leukocytes exposed to CdCl2 at concentrations ranging from 1 to 25 μg/mL, which occurred in a concentration-dependent manner (p < 0.001).

Figure 1

The percentage of Cd cellular influx equivalent to a leukocyte related to the exposure to different CdCl₂ concentrations is shown in the figure 2, which allows evaluating the profile of Cd entry into a leukocyte. As shown in the figure, there is a decrease in the percentage of Cd cellular input, which occurs with a stabilization from 5 to 25 μg/mL. This phenomenon indicates a decay entry percentage but at the same time leads us to suggest that the influx kinetics is of zero-order.

Figure 2

Both Cd exposure and intracellular Cd concentrations were related to the percentage of cell unviability (Fig. 3) and DNA damage index (see figure 4) in human leukocytes to elucidate the Cd concentrations capable of inducing injury in these cells. Our results demonstrated that Cd exposure at concentrations ranging from 5 to 25 μg/mL significantly decreased the leukocyte cell viability compared with the negative control (p < 0.001), in particular at two higher concentrations, 10 and 25 μg/mL.

Figure 3

Figure 4 shows the DNA damage index of leukocytes after CdCl₂ exposure. The comet assay demonstrated that the exposure concentrations of 1 and 2.5 μg/mL, where the intracellular Cd levels were 9.85 and 16.22 pg/cell, respectively, did not cause significant DNA damage, since the damage levels were similar to the damage levels found in the negative control (0.001). On the other hand, at the exposure concentration range from 5 to 25 μg/mL, which is equivalent to the intracellular Cd content from 21.21 to 94.38 pg/cell, we were able to observe higher DNA damage levels, although they were lower than those observed for the positive control (p < 0.001).

Figure 4