and Mn NPs are readily taken up by R3-1 cells in a matter of hours. In each case, the NPs are found both as agglomerates and as single particles in lysosomes, the cell cytoplasm, and occasionally a NP is found inside a mitochondrion. The Cu NPs, on the other hand, are not found inside the cell cytoplasm or in the lysosomal compartment. Very likely, this is because the Cu NPs dissolve rapidly over the time frame of the uptake experiments. Finding a large percentage of the Au, TiO2
and Mn NPs inside lysosomes suggests that these NPs get into the cellular endosomal system, since late endosomes are thought to merge with the lysosomal system [14
]. In the case of uptake of Au and TiO2
NPs, there is no indication that the NPs get into the cytoplasm by rupturing the lysosomes, since the lysosomes generally have a non-active appearance. Heterolysosomes, as defined morphologically, are activated by uptake of Mn NPs; nevertheless, the distribution of the Mn NPs is very similar to that seen with Au and TiO2
NPs, suggesting a common mechanism of entry into lysosomes.
Ag NPs appear to enter the cells by a different yet undefined mechanism. They are evenly dispersed widely throughout R3-1 cells in a matter of minutes, appearing in the nucleus in as little as 12 minutes. Interestingly, the particles that get in are small compared to the primary particle size and are smaller than the functional diameter of the nuclear pore complex of ~39 nm. Our sonication method does not change the particle size based on observations of the same Ag particles on TEM grids after sonication with no cells present. This may indicate that only very small Ag particles get into the R3-1 cells and that the smaller Ag NPs rapidly traverse membranes. It might also suggest that Ag dissolution in cell media and in the presence of the cell membranes contributes to the reduction in particle size even at short times (12–30 minutes). The presence of the tiny particles in the nucleus may contribute in some yet undescribed way to the rapid and very potent cell death caused by Ag NPs. Greater than 80% of the R3-1 cells detach from the slides within one hour of Ag exposure.
All of the NP types, except Cu, are occasionally found in mitochondria. They appear not to be restricted to the mitochondrial intermembrane space, but also to be found inside the matrix. While the literature contains suggestions of transfer of components between endosomes and mitochondria [15
], the NPs should have been found in the mitochondrial intermembrane space if this process had occurred. Furthermore, it is likely that many more NPs would be found in mitochondria since large numbers of NPs are transferred to lysosomes. Mitochondria are dynamic structures, moving within the cell while undergoing fusion and fission [5
]. Based on our observations, we suggest it more likely that NPs are occasionally seen inside mitochondria because they were on the edge of a mitochondrion just before it fused to another, thus trapping the NP inside the resultant fused structure.
The mitochondrial electron transport chain is the largest source of ROS in mammalian cells, with most being produced as super oxide at complexes I and III [5
]. The super oxide is converted to H2
by intramitochondrial Mn super oxide dismutase. Mitochondrial uptake of Ca2+
has been reported to increase ROS production; however, several possible mechanisms for this have been proposed and the exact mechanism by which it occurs is not apparent [5
]. We have found that both Mn2+
and Mn NPs increase ROS production by mitochondria. Mn2+
generally binds to Ca2+
binding sites with an affinity greater than that of Ca2+
], so it may be that the increased ROS observed after Mn2+
uptake by mitochondria represents an effect similar to that seen with Ca2+
. However, almost all of the Mn NPs are outside the mitochondria and the mechanism by which this increases ROS production is not clear. One possibility is that Mn ions leach from the particles over time increasing local Mn concentrations and the soluble Mn contributes to the damage and death observed in cells which have sequestered Mn NPs.
While Au, TiO2
, and Mn NPs were all found inside lysosomes after uptake by the cell, only the Mn NPs led to the appearance of heterolysosomes. This is likely to be an effect distinct from that of the NPs on the mitochondria. It is possible that this is caused by H2
production inside the lysosome due to Mn NP dissolution enhanced by the acidity of the lysosome [19
]. While Mn NPs did not dissolve rapidly in normal cell media, the acidic environment of the lysosome drives production of H2
according to equations for Mn similar to those shown previously for Cu in equation 3
This would contribute to an intracellular ROS increase. Similar equations for Ag and Au yield significant positive changes of free energy, making the production of H2
by oxidation of Au and Ag NPs energetically unfavorable, even at low pH [13
production by Mn NPs in our experiments was significantly greater than the increase caused by dissolved Mn2+
acting at the mitochondrial level; however, any dissolved Mn2+
released from lysosomes could be sequestered by mitochondria and contribute in an independent way to the total ROS produced in or near the mitochondria. This is important because ROS production in or near mitochondria has been shown to increase the probability of induction of the mitochondrial permeability transition (MPT) and this has been shown to induce apoptosis [20
]. Clearly shown in is an increase in condensed, distorted nuclei, and R3-1 cell condensation, all morphological signs of apoptotic cell death. After 72 hours PALAS Mn exposure it is likely there is high localized Mn concentration in the cytoplasm that may be in close proximity to mitochondria due to dissolving Mn NPs which would promote apoptosis via the MPT.
The process occurring with the Cu NPs was consistent with previous observations by our lab that the presence of cells (R3-1 cells, HL60 cells, or red blood cell ghosts) increases the rate of H2
production (data not shown). It is believed that the concentration of O2
is around 3 fold higher [21
] in the membranes of cells than in the media bathing them (i.e. O2
partitions into the membrane phase); however, it is lower in mitochondrial membranes due to oxidative phosphorylation. Dissolution of Cu particles causes alkalinization of the medium coupling H2
production with oxidation (data not shown). Furthermore, experimentally the addition of 80 units of Mn super oxide dismutase (MnSOD) which would convert super oxide to H2
does not increase the observed signal in our Amplex Red tests (data not shown), suggesting the observed ROS were already in the H2
form and were not formed by conversion of super oxide. These observations support the hypothesis that oxidation of Cu and any Cu+
, which may be present in the NP, and dissolution of the NPs work together to drive the consumption of protons and direct formation of H2
Our use of H2
specific Amplex Red indicates that H2
production was not increased in R3-1 cells exposed to Ag NPs. Other investigators have shown that Ag increases ROS production using DCF-DA [22
]. The type of Ag used (pure silver versus silver with carbon coating), the concentration of Ag, the cell type specificities, and the assay being used to measure ROS (DCF-DA versus Amplex Red) could all contribute to these observed inconsistencies in ROS production.
There are many possible ways in which NPs and other toxicants damage cells. We have focused these studies on cell morphology effects of NPs paired with redox reactions as summarized in .
We have found that where the free energy change for giving up electrons is negative, NPs containing oxidizable components, such as neutral Cu, Cu+, or neutral Mn, can be induced to give up electrons and form ROS. In the case of Cu, which dissolves readily, it is likely that the Cu NPs, which can have different shapes and therefore many ways of associating with the cell surface, are more easily oxidized when interacting with the cell because the cell membrane contains higher O2 concentration than the medium. Rapid dissolution of the particles while passing electrons from Cu on to O2 produces a high rate of H2O2 production through which the cell exterior is damaged. This seems to be what causes initial cell death upon Cu NP exposure (60% by 22 hours shown in ). The lack of morphological damage to the cell interior is likely due to the fact that the Cu NPs dissolve so rapidly that they are not effectively transported as particles into the cell interior. Mn, on the other hand, dissolves much more slowly and like Au, TiO2, and Ag, is found in lysosomes. With time, and exposure to the acidic lumen of the lysosome, heterolysosomes are observed at 48 hours, followed by the morphological evidence of cell death observed at 72 hours post Mn. In addition, dissolved Mn2+, sequestered by mitochondria, like Ca2+, then increase ROS production by the mitochondrial ETC. This could contribute to long-term damage to mitochondria and the observed changes in cell morphology associated with apoptosis. Au, TiO2, and Ag NPs exposed cells do not show morphology as evidence of redox-induced cell death.
Cu and Mn NP entry, localization and effect on cellular ROS production correlate with cellular time-of-death in exposed R3-1 cells. Mn increases ROS, dissolution occurs in lysosomes and Mn NPs cause R3-1 cell death over a period of days whereas Cu increases ROS, dissolution is rapid at the cell surface, cell death is observed within hours and then the surviving R3-1 cells recover over time. Future studies are required related to Cu exposure and upregulation of antioxidants or other pro-survival mechanisms allowing some cells to adapt. Ag particles rapidly enter these cells and cause immediate R3-1 cell detachment without significantly increasing H2
production in these experimental conditions. Still other particles like Au and TiO2
do not dissolve, accumulate inside the cells and do not cause the R3-1 cells to die. We have used cell death as an end point in these studies in order to make sense of localization and entry into this particular cell model. As observed by others previously [23
], it is important to relate uptake and ROS production findings with each particular NP type and to consider that in vivo
NP concentration may not be sufficient for cell death but in the absence of death, the passage of NPs, or ions derived from NPs, through this cell type may be key to entry into the circulation and beyond after inhalation exposure.