2 H ipótesis y O bjetivos
5.1 Características generales de la población de casos y controles
An important aspect to consider when interpreting the results of the experiments presented in this chapter is the use of BMDMs as model cells for intestinal macrophages. Cell culture studies have been criticised in general, because it is not be possible to accurately model the
in vivo situation in in vitro studies. Ideally, primary intestinal cells would be used if the goal is
to investigate specific effects in cell culture [275]. However, primary intestinal macrophages are difficult to isolate from mice, and only a relatively small number of macrophages can be recovered per animal [276]. This means that many animals would be necessary to obtain a sufficient amount of cells for the experiments described in this chapter. For animal ethics reasons, and also because of limited availability of Nod2m/m mice, this was not feasible.
Alternatively, an immortalised monocyte cell line, e.g. RAW264.7 macrophages, could have been used. Cell lines with macrophage characteristics have been used extensively to study the
effects of TiO2 exposure on macrophages (Table 1.5). Experiments with BMDMs derived
from Nod2m/m mice were also performed in the study in which the Nod2m/m mice were
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pro-inflammatory cytokines after TiO2 and MDP co-stimulation of BMDMs derived from
Nod2−/− mice [253]. Therefore, it was decided to perform these experiments with BMDMs
to be able to compare the data with these two studies.
2.5.3
Cytotoxicity of titanium dioxide
The cytotoxicity of food-grade TiO2 particles on both WT and Nod2m/m macrophages was
investigated based on changes in metabolic activity with the commonly used WST-1 assay.
The results showed that the TiO2 particles did not cause a decrease in metabolic activity,
even at high concentrations (Figure 2.3). These results are in line with other studies that used the WST-1 assay to assess cytotoxicity of TiO2 particles with diameters larger than
100 nm [186-188, 199]. For example, TiO2 particles with diameters from 40 nm to 300 nm
for 24 h were not cytotoxic to RAW264.7 macrophages [199]. Another study investigated the effects of three different types of TiO2 particles, all with average diameters between 250 nm and 450 nm, on RAW264.7 macrophages with the MTS assay, which is another type of tetrazolium salt assay closely related to the WST-1 assay [201]. The metabolic activity of the macrophages was not affected by any of the three types of particles, even at high particle concentrations.
A second study also investigated potential cytotoxic effects of anatase TiO2 particles with
sizes between 350 nm to 500 nm in TCM on RAW264.7 cells with the MTS assay [198]. In contrast to the study by Xiong and colleagues, the metabolic activity was lower for
macrophages that were treated with 100 µg/mL TiO2 particles for 12 h to 24 h, but not for
6 h. The result reported by Sohaebuddin and colleagues for the 24 h time point is also in contrast to the results from other studies in which no cytotoxicity of TiO2 particles was observed after 24 h incubation [186, 188, 199]. However, the result for the 6 h time point from the study by Sohaebuddin and colleagues is in agreement with the observation from the current study that a relatively short incubation time with anatase TiO2 particles did not affect metabolic activity of macrophages.
Other researchers have used the WST-1 assay to investigate cytotoxic effects of TiO2 particles on immortalised intestinal epithelial cell lines. One study showed that the effect on metabolic activity in colon carcinoma cells was dependent on exposure time [186].
Incubation with TiO2 particles with an average diameter of 320 nm for 24 h or 48 h had no
impact on metabolic activity, but incubation for 72 h led to a decrease in metabolic activity. Gerloff and co-workers published two studies that compared the effect of different types of TiO2 particles on Caco-2 cells [187, 188]. Metabolic activity was only negatively affected by
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TiO2 particles with a primary particle size in the nanometre range, but not by anatase TiO2
particles with an average diameter of 215 nm.
Although there was no detectable effect of TiO2 particle concentration on metabolic activity,
it was observed that BMDMs derived from Nod2m/m mice had lower metabolic activity relative
to untreated WT control cells. It had been shown previously that key cellular signalling processes were not affected in Nod2m/m mice [131]. However, it cannot be excluded that this
mutation affected the cells from these knock-in mice in a way that decreased their viability. In line with this is the observation that during the bone marrow collection consistently fewer
cells were obtained from Nod2m/m mice (see Appendix B).
Furthermore, the cell viability was determined with PI staining and analysis by flow cytometry (Figure 2.4). Using the trypan blue exclusion assay, Palomäki and colleagues could not detect differences in cell viability between RAW264.7 macrophages and control cells that were both treated with rutile TiO2 particles (average primary particle size 35 nm) [197]. A more sensitive method to assess cell viability is staining with PI and determining the number of cells that are negative for PI. This is often used in conjunction with annexin-V (A5) staining which allows distinguishing early apoptotic (PI− A5+), late apoptotic (PI+ A5+), and dead cells (PI+ A5−). For the experiments reported in this chapter only staining with PI was performed because the flow cytometer that was used for analysis was able to detect signals in four channels. The other three channels were used to detect the macrophage marker F4/80 and the activation molecules CD80 and CD86, respectively. Therefore, it is possible that early apoptotic cells, i.e. cells that would be PI− A5+, were not detected in these experiments. However, it has been shown with PI and A5 double staining of J774A.1 macrophages which
were incubated with 100 µg/mL TiO2 (average particle size 220 nm) that most non-viable
cells were PI+ [195]. Only a small fraction of the non-viable cells was PI− A5+. Möller and colleagues reported that the cell viability was approximately 85 % after 24 h of incubation
with TiO2. In another study in which RAW264.7 macrophages were incubated for 20 h with
100 µg/mL anatase TiO2 particles (average particles size 350 nm to 500 nm in TCM) and the
non-viable cells were also assessed with PI and A5 staining the cell viability was
approximately 50 % [198]. Incubation with TiO2 alone lead to increased apoptosis, but early
and late apoptotic cells were not distinguished. In contrast to these two studies that detected a decrease in cell viability after TiO2 incubation, no increased cell death of RAW264.7 macrophages was detected by PI staining in two other studies after exposure to 10 µg/mL
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and the second two studies might be due to different particle types used, i.e. rutile/anatase or a mixture of both.
Palomäki and co-workers noted that TiO2 particles were more cytotoxic to murine APCs
derived from bone marrow than to cultured RAW264.7 macrophages [197]. This observation
could explain why in the current study exposure to TiO2 particles caused a decrease in cell
viability for all treatments and at both time points. Staining with PI showed that the viability of primary cells from humans, namely PBMCs, was also decreased after incubation with just
5 µg/mL anatase TiO2 for 24 h [215, 217]. However, there was no decrease in cell viability
after TiO2 exposure for human macrophages derived from PBMCs [218]. The latter study
was one of the few studies in which cell viability was investigated with PI and A5 staining after exposure to a range of particles in concentrations up to 50 µg/mL TiO2. Both apoptosis and the amount of dead cells increased with increasing particle concentrations, but the levels were not different to the control cells even for the highest TiO2 concentrations. The observation that increasing TiO2 concentrations resulted in decreased cell viability is in agreement with the results from this study (Figure 2.4).
The baseline viability in the current study was higher in cells recovered after 3 h + 21 h treatment, but the decline in viability was similar compared with the 3 h time point. A possible explanation for the higher baseline viability at the later time point is that the cells have recovered from the acute treatment with TiO2 and started to proliferate in the fresh TCM. This is in line with the observation of de Berardis and colleagues that the cell
growth of a colon carcinoma cell line was not affected by stimulation with TiO2 for up to
72 h [186].
When the results of the WST-1 assay (Figure 2.3) and PI staining (Figure 2.4) were visually compared, it was observed that the results from the two assays did not correspond to each other. Although it could be expected that the observed decrease in cell viability, as assessed by PI staining, would result in a decrease in metabolic activity, as assessed by the WST-1 assay, this does not necessarily have to be the case because the two assays measure fundamentally different parameters (personal communication with Dr Mark McCann, AgResearch, Palmerston North, NZ). A possible explanation why no reduction in metabolic
activity was observed after BMDMs were incubated with TiO2 particles (Figure 2.3) whereas
cell viability decreased with increasing particle concentrations (Figure 2.4) could be that although less viable cells were present at higher particle concentrations these live cells had a higher metabolic activity. This could have been the case because more TCM, and therefore nutrients, per live cell was available at higher TiO2 concentrations. The results from this study
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also corresponded to a previous study that did not observe decreased metabolic activity with
the WST-1 assay after exposure of cultured macrophages to TiO2 particles [199]. A decreased
cell viability of TiO2-exposed cultured macrophages according to PI staining has also been
reported previously [195, 218].