Background Engineered nanoparticles (NPs) are being developed and incorporated in a number of commercial products raising the potential of human exposure during manufacture, use and disposal. Although data about the potential toxicity of some NPs have been reported, validated simple assays are lacking for predicting their in vivo toxicity. Objective To evaluate new response-metrics based on chemical and biological activity of NPs for screening assays that can be used to predict NP toxicity in vivo. Methods Two cell-free and two cell-based assays were evaluated for their power in predicting in vivo toxicity of eight distinct particle types with widely differing physico-chemical characteristics. The cell-free systems comprised fluorescence- and electron spin resonance-based assays of oxidant activity. The cell-based systems also used electron spin resonance as well as luciferase reporter activity to rank the different particle types in comparison to benchmark particles of low and high activity. In vivo experiments evaluated acute pulmonary inflammatory responses in rats. Endpoints in all assays were related to oxidative stress and responses were expressed per unit NP surface area to compare the results of the different assays. Results Results indicate that NPs are capable of producing reactive species, which in biological systems can lead to oxidative stress. Copper NPs had the greatest activity in all assays, while TiO2 and gold NPs generally were the least reactive. Differences in the ranking of NP activity among the assays were found when comparisons were based on measured responses. However, expressing the chemical (cell-free) and biological (cells; in vivo) activity per unit particle surface area showed that all in vitro assays correlated significantly with in vivo results (R>0.81), with the cellular assays correlating best (R>0.87). Conclusions Data from this study indicate that it is possible to predict acute in vivo inflammatory potential of NPs with cell-free and cellular assays by using NP surface area-based dose and response metrics, but that a cellular component is required to achieve a higher degree of predictive power.

Studies showed that certain cytotoxicity assays were not suitable for assessing nanoparticle (NP) toxicity. We evaluated a lactate dehydrogenase (LDH) assay for assessing copper (Cu-40, 40 nm), silver (Ag-35, 35 nm; Ag-40, 40 nm), and titanium dioxide (TiO2-25, 25 nm) NPs by examining their potential to inactivate LDH and interference with β-nicotinamide adenine dinucleotide (NADH), a substrate for the assay. We also performed a dissolution assay for some of the NPs. We found that the copper NPs, because of their high dissolution rate, could interfere with the LDH assay by inactivating LDH. Ag-35 could also inactivate LDH probably because of the carbon matrix used to cage the particles during synthesis. TiO2-25 NPs were found to adsorb LDH molecules. In conclusion, NP interference with the LDH assay depends on the type of NPs and the suitability of the assay for assessing NP toxicity should be examined case by case.

There is an urgent need for in vitro screening assays to evaluate nanoparticle (NP) toxicity. However, the relevance of in vitro assays is still disputable. We administered doses of TiO2 NPs of different sizes to alveolar epithelial cells in vitro and the same NPs by intratracheal instillation in rats in vivo to examine the correlation between in vitro and in vivo responses. The correlations were based on toxicity rankings of NPs after adopting NP surface area as dose metric, and response per unit surface area as response metric. Sizes of the anatase TiO2 NPs ranged from 3 to 100 nm. A cell-free assay for measuring reactive oxygen species (ROS) was used, and lactate dehydrogenase (LDH) release, and protein oxidation induction were the in vitro cellular assays using a rat lung Type I epithelial cell line (R3/1) following 24 hr incubation. The in vivo endpoint was number of PMNs in bronchoalveolar lavage fluid (BALF) after exposure of rats to the NPs via intratracheal instillation. Slope analyses of the dose response curves shows that the in vivo and in vitro responses were well correlated. We conclude that using the approach of steepest slope analysis offers a superior method to correlate in vitro with in vivo results of NP toxicity and for ranking their toxic potency.

There is an urgent need for screening assays for evaluating nanoparticle (NP) toxicity, given the increasing number and variety of NPs and the current dispute regarding the value of in vitro assays in predicting in vivo toxicity of NPs. We evaluated several assays by adopting a response metric that is based on NP surface area with the objective to predict in vivo toxicity from in vitro data. Several anatase TiO2 NPs (3 to 100 nm) were examined for this purpose. In addition to in vitro studies where a cell−free assay and a rat lung Type I epithelial cell line (R3/1) were used, we examined several in vivo endpoints by exposing rats to the NPs via intratracheal instillation. In vitro endpoints included ROS induction, lactate dehydrogenase (LDH) release, protein carbonylation, and heme oxygenase 1 (HO−1); in vivo endpoints included tissue levels (lung and plasma) of carbonylation and HO−1 and number of PMNs in bronchovascular lavage fluid (BALF). Several metrics were evaluated to rank the toxicity of these NPs, including but not limited to, ED50 and the steepest slope in the dose−response curve calculated based on two methods. NP toxicity rankings based on these metrics were performed. Correlations between the in vitro and in vivo ranking results were calculated. LDH assay was proved valid. Carbonylation and HO−1 assays were good markers of oxidative stress related toxicity. The steepest slope in the dose−response curve was the best response metric for ranking NP toxicity when both dose and response were based on NP surface area. It was concluded that toxicity rankings by certain in vitro assays were consistent with the in vivo toxicity rankings when proper dose and response metrics were utilized. This abstract is funded by: US DoD MURI grant FA9550−04−1−430.