By using biomarkers, we are able to predict not only the present disease and clinical condition, but also the risk of acquiring disease in the future. Therefore, it is necessary to progress studies of biomarkers for nanomaterials because very little information is available on the biological effects of nanomaterials. Here, we used 2D-DIGE analysis to perform a comprehensive screen of plasma proteins to identify protein biomarkers of nanomaterials. We identified hemopexin (Table 1) as a useful biomarker for analyzing the biological responses associated with exposure to silica nanoparticles. Because 2D-DIGE is a proteomic method, this approach has potential to uncover the as yet unknown biological effects of nanomaterials.
On the other hand, an inherent disadvantage of 2D electrophoresis is the poor resolution of hydrophobic proteins [25, 26]. Hence, it is likely that integral membrane proteins are strongly underrepresented. Isobaric tags for relative and absolute quantification or iTRAQ is a comprehensive gel-free quantitative proteomic method based on mass spectrometry [27, 28]. This approach can be used to generate proteomic profiles that reflect the pathological state of organs and aid in the early detection of diseases [29, 30]. We envisage that a combination of comprehensive proteomic methods would help to identify potential toxicities of nanomaterials during their development and contribute to the establishment of strategies to ensure their safety.
Silica materials with amorphous particle morphology are known to cause the hemolysis of mammalian red blood cells [31, 32]. For this reason, we investigated the possibility that silica nanoparticles could induce hemolytic activity. While the exact mechanism is still under investigation, most reports agree that the hemolytic activity of silica particles is related to surface characteristics such as area and curvature [33–35]. However, we found that nSP70 at 0.8 mg/mouse does not induce hemolytic activity through elevation of haptoglobin and hemopexin (Figure 3). We are continuing investigations to understand the biological mechanisms associated with the elevation of hemopexin after exposure to silica nanoparticles.
We then examined the effects of surface modification of silica nanoparticles on the production of hemopexin. Compared to controls, hemopexin was not induced by nSP70-N but was induced to a significantly higher level by nSP70-C, which was a similar level to that induced by unmodified nSP70 (Figure 4A). On the other hand, the plasma levels of haptoglobin (Figure 4B) and SAA (Figure 4C) in mice treated with nSP70-C were significantly lower than those in mice treated with nSP70, as reported previously . These results suggest that the production of acute-phase proteins depends on the characteristics of the nanomaterials and that nSP70-C induces some biological effects associated with the elevation of hemopexin. Increased hemopexin levels have been found in patients with diabetes mellitus and are associated with some malignancies, such as malignant melanoma and breast cancer [20, 36, 37]. Elevated hemopexin levels have also been found in inflammatory psychiatric disorders, such as major depression, schizophrenia, and mania . Taken together, these findings suggest that hemopexin might be associated with these diseases so it is possible that the induced elevation of hemopexin by both nSP70 and nSP70-C is related to the induction of inflammatory responses. Therefore, we are currently analyzing not only the mechanisms underlying the differences in production of hemopexin, haptoglobin, and SAA induced by nSP70-C and nSP70, but also the relationship. It could be speculated that there are differences between the fates of the injected nSP70 and nSP70-C. Therefore, there is a need to evaluate the distribution or accumulation of the injected nanoparticles in the liver where acute-phase proteins are known to be produced. An understanding of these mechanisms will advance the use of biomarkers for different purposes and improve the predictive value of these biomarkers.
Hemopexin is one of the acute-phase proteins released from the liver, and its production is known to be regulated by cytokines. For instance, interleukin (IL)-6 and IL-22 induce the hepatic production of circulating SAA [39, 40]. Furthermore, it is conceivable that instead of inflammatory cytokines, small silica particles act directly on the liver to induce the release of acute-phase proteins. However, nSP70, at this dose, did not induce any significant elevation of liver injury or dysfunction markers, such as ALT or AST. Therefore, it is unclear why nanomaterials induce the production of acute-phase proteins. We are currently analyzing the detailed mechanism by which silica particles induce acute-phase proteins.