Physicochemical characterizations of aerosol particles. The PhIP, MeIQx and TDA aerosol particle samples were synthesized via an easy method through a compressor nebulizer, and their morphology and size were investigated using TEM measurements, as shown in Fig. 2a-c. PhIP is rod-like with a uniform particle size of 5 ± 0.6 µm, and MeIQx reveals a strip-like morphology with a size of 1.5 ± 0.2 µm. In contrast, TDA presents an irregular round morphology with a size of 20–40 nm. Furthermore, the zeta potentials of PhIP, MeIQx and TDA were investigated in the dispersion solution (Fig. 2d-f). The obtained PhIP particles are negatively charged with a zeta potential of − 11.7 mV, while MeIQx and TDA are − 5.6 and − 0.2 mV, respectively, indicating that PhIP is more stable in the dispersion solution than MeIQx and TDA. In addition, the particle sizes of PhIP, MeIQx and TDA were determined by dynamic light scattering (DLS) to be 588 nm, 623 nm and 162.1 nm (Fig. 2g-i), respectively. Both the TEM and DLS results exhibit the fabrication of aerosol particles for the evaluation of their adverse effects.
In vitro cytotoxicity with CCK-8 assay. To assess the toxicity of PhIP, MeIQx and TDA aerosol particles in tumor and normal cells, the standard Cell Counting Kit-8 (CCK-8) assay was applied. Tumor cell HepG-2 and normal liver cell L02 were incubated with PhIP, MeIQx and TDA particles at various concentrations (from 0.5 to 4 µg/mL) for different times (24 h, 48 h, 72 h), and the cell viability results are shown in Fig. 3(A). A significant toxicity effect occurred on L02 cells, which was enhanced gradually with increasing dosage from 0.5 to 4 µg/mL after the incubation time was extended from 24 h to 72 h for PhIP (Fig. 3Ad-f). In contrast, PhIP did not show remarkable cytotoxic effects on HepG2 cells, even at concentrations up to 4 µg/mL after 72 h of incubation (Fig. 3Aa-c). As shown in Fig. 3A, MeIQx sample also presented toxicity to L02 cells, while there was weak cell damage through TDA incubation. The half maximal inhibitory concentration (IC50) of PhIP was 1.77 µg/mL, which was less than that of TDA (552.1 µg/mL) and MeIQx (6.01 µg/mL). This can further support that PhIP can harm the liver even at small amounts, while L02 cells show a strong tolerance to TDA. For further assessment the cell damage of PhIP to HepG-2 and L02 cells, they were incubated with PhIP particles at higher concentrations (from 1 to 50 µg/mL) for 24 h. The results displayed that the particulate PhIP did not show remarkable cytotoxic efects on the cancer liver cell lines HepG-2, even at concentration up to 50 µg/mL (Fig. 4a), while presented significant apoptosis on the normal cells L02 under same condition (Fig. 4b). It can be concluded that PhIP aerosol particles can damage normal liver cell with the exposure from low to high dosage .
Furthermore, to evaluate toxicity to the lung, A549 and HelF cells were measured for cell toxicity after incubation with PhIP, MeIQx and TDA aerosol particles at various concentrations for different times (Fig. 3Ba-f). All three samples showed negligible effects on the survival of both A549 and HelF cells, even at concentrations up to 4 µg/mL for 24 and 48 h incubation (Fig. 3Ba and b, d and e). After prolonging to 72 h incubation, there are weaker cell toxicity for HelF cells rather than A549 cells (Fig. 3Bc and f). The results implyed that the three typical chemicals of cooking fumes do not damage lung cells despite those chemicals directly breathing into the lung under low dose and less incubation time. Considering the above results, one can conclued that only low dose PhIP aerosol particles can harm the liver instead of the lung.
In vitro cytotoxicity with flow cytometry and ROS formation. Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell, such as size and granularity, simultaneously as the cell flows in suspension. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time31. The apoptosis ratio was detected with flow cytometry for PhIP, MeIQx and TDA in HepG-2 and L02 cells and is shown in Fig. 5. The apoptosis ratios of PhIP, MeIQx and TDA on HepG2 cells were approximately 95.90%-97.08%, in comparison with that of the control group at 94.35%, while the apoptosis ratios of PhIP on L02 cells at approxmately 52.86% were significantly lower than those in the control group and MeIQx- and TDA-treated groups, with the values at 73.83%, 74.80% and 71.27%, respectively. The results indicated that L02 cell proliferation was inhibited and apoptosis was activated after treatment with PhIP. Conversely, there was little effect on HepG2 cells exposed to PhIP, MeIQx and TDA. The results for the adverse effect of PhIP to L02 cells are almost identical to those of the CCK-8 results.
To investigate whether exposure to PhIP, MeIQx and TDA samples caused direct or indirect damage to the cellular tissues, cells were observed under TEM. PhIP, MeIQx and TDA can intrude into the cytoplasm with their initial particle size in HepG-2 cells, while PhIP and MeIQx can also be observed in the nucleus with reduced particles in L02 cells. This phenomenon indicates that exposure to PhIP and MeIQx resulted in L02 cell damage (Fig. 6a). To understand the mechanism by which PhIP, MeIQx and TDA induce apoptosis, we stained HepG-2 and L02 cells with DCF-DA to investigate the generation of ROS. Dichlorodihydrofluorescein diacetate (DCF-DA) has been widely used in detection of reactive oxygen species (ROS), which was commonly used as a standard to determine the toxicity of pollutants in the medical fields32. As revealed in Fig. 6b, no obvious green fluorescence intensity was found in the control group. In contrast, a strong green fluorescence signal can be found in both PhIP and MeIQx samples, while a negligible green signal can be detected after incubation with TDA. The data maybe deduce that PhIP and MeIQx aerosol particles bring biological adverse effects through ROS.
In vivo toxicity with mice weight and organs staining. To further assess the in vivo toxicity of PhIP, we mimicked the cooking environment and sprayed aerosols with various concentrations of PhIP (10, 20, 50, 100 µg/mL) onto male nude Balb/c mice. Considering that the cooking environment is a continuous accumulation process, it is essential to assess the damage to the mice within a period of time. The body weights of the mice were monitored every two days. As indicated in Fig. 7, no obvious body fluctuation was found in the control group or the A group (10 µg/mL). In contrast, the body weights of mice showed obvious decreases as the concentration increased from 10 to 20, 50 and 100 µg/mL with the cubation time lasting to 30 days, demonstrating the nonnegligible adverse consequence of the increasing dose of PhIP on mice (Fig. 7). Specifically, the body weights of mice decreased to 20.0 g for 100 µg/mL PhIP incubation under 30 days, in comparison with the increased gradually body weights to 27.8 g found in the control group.
Additionally, hematoxylin and eosin (H&E) staining of major organ tissues further confirmed the influence of PhIP on mice33. As Fig. 8A-C shows, there was no obvious tissue damage or inflammation in major organs, including the heart, liver, lung, spleen and kidney, when the administration dosage was low (10 µg/mL), even up to 50 µg/mL. As expected, distinct inflammation in the liver and lung was observed once the concentration of PhIP increased to 100 µg/Ml (Fig. 8D). However, the heart, spleen and kidney, may be less sensitive to PhIP exposure than the liver and lung, because no significant damage was observed in this study up to 100 µg/mL. These results confirmed that PhIP can cause irreversible damage to the liver and lung under a continuous accumulation process. The biological adverse effects from PhIP to mice tissues was consistent with the reduced body weight of mice.