Prior to nanotoxicological assessment, physicochemical characterization of the nanoparticles being examined is required owing to the potential implications of physical and chemical properties in the manifestation of toxicity36,37. Physiochemical analyses were performed, following the standard methods, to demonstrate that the fabricated AgNP suspension was indeed eligible for this study. The EDX qualitative analysis validated that the primary particles of AgNPs are composed of nearly 100% elemental silver (the components of a TEM (transmission electron microscope) grid contributed to the detection of carbon and copper signal peaks) (Fig. 1a). As visualized under a transmission electron microscope (Fig. 1b), the synthetic citrate-capped AgNPs exhibited a spherical structure, with an actual mean size of 18.2 ± 8.7 nm (Table 1). The number-weighted size distribution data (Fig. 1c) indicates that the dimensions of these AgNPs in aqueous suspensions, though appearing rather uniform, are greater than the actual sizes of the particles; the average hydrodynamic diameter of the particles in these suspensions is 32.8 ± 4.4 nm (Table 1). Data pertaining to the other relevant physicochemical parameters of the examined AgNPs and the measurements of these parameters are summarized in Table 1.
Figure 1Chemical composition, morphology and size distribution of synthetic citrate-capped AgNPs. (a) Analysis of the elemental composition of the synthetic AgNPs by energy-dispersion X-ray spectrometry (EDX). (b) Shape and actual size of the synthetic AgNPs visualized under a transmission electron microscope (TEM). (c) Differential number-weighted particle-size distribution of the aqueous AgNP suspension measured using dynamic light scattering (DLS).
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Table 1 Physico-chemical properties of the synthetic AgNPs and their measurements.Full size table
Our study began with an investigation of the adverse effects of AgNPs at the organismal level. D. melanogaster, the model organism of choice for this study, was administered with conventional diets with or without citrate-capped-AgNP supplementation during the foraging (first, second, and early third instars) and wandering (late third instar) stages. We found a dose-dependent increase in mortality among larvae exposed to AgNPs on the 4th day after exposure (Fig. 2a), the time point at which the untreated control larvae almost all molted into the third-instar stage. In the meantime, the level of body-length heterogeneity of the surviving larvae appeared to increase with increases in dosage (Fig. 2b and c). In addition to impaired growth, dietary AgNPs further contributed to a dose-dependent increase in the duration of the larval stage, leading to delayed onset of the pupation process (Fig. 2d). As shown in Fig. 2e, the percentage of larvae that survived the effects of the AgNPs and eventually entered the pupal stage decreased in a dose-dependent manner. Altogether, these results suggest that dietary AgNPs cause developmental toxicity in Drosophila larvae.
Figure 2Effects of dietary AgNPs on developing larvae. (a) Stacked bar chart showing the relative number of viable and dead larvae on the 4th day post-exposure to 0, 10, 20, 30, 40 and 50 μg/ml of AgNPs. Each group contained 100 larvae in total. (b) Stereomicroscopic image of the external morphological appearance of surviving larvae on the 4th day post-exposure. (c) Stacked bar chart demonstrating the distribution of the body-length (BL) heterogeneity among the surviving larvae (i.e., relative percentage of viable larvae (derived from data shown in Fig. 2a) with respect to the range of body lengths) after 4 days of exposure. (d) Cumulative percentage of the surviving larvae (relative to the final pupal number) capable of undergoing pupation between the 4th and 8th day after hatching. (e) Percentage of surviving larvae that eventually turned into pupae (relative to the number of larvae in each group). For the experiments shown in Figs 2d and e, each vial was loaded with 100 larvae, and each group contained 300 larvae in total. **(P < 0.01) and ***(P < 0.001) denote significant differences between the control group and exposure groups.
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The pupal stage (i.e., the metamorphosis stage) generally lasts for 4 days (between the 6th and 9th day post-hatch), after which the adult fly emerges from the pupal case. Notably, even without exposure to AgNPs after pupation, AgNPs ingested during the larval stage continue to exert negative effects on flies during metamorphosis, leading to a dose-dependent prolongment of the pupal period (Fig. 3a). Exposure to higher doses of AgNPs (≥30 μg/ml) caused pupal death and unsuccessful eclosion of some emerging flies. Accordingly, the decrease in the successful eclosion of flies from the pupae occurred in response to increasing dosages of AgNPs (Fig. 3b). Even after successful eclosion, significant numbers of flies that received high doses of AgNPs died within three days (Fig. 3c). Taken together, these results indicate the cumulative toxicity potential of AgNPs on exposed organisms. Furthermore, consistent with the findings of a previous study38, we also found that ingestion of AgNPs during early larval development promoted demelanization (i.e., pigment whitening) of the adult cuticle; the higher the dose, the greater the level of whitening (Supplementary Fig. 1).
Figure 3Effects of dietary AgNPs on the development of pupae and newly emerged adults. (a) Cumulative percentage of the flies eclosed daily (relative to the final eclosion number) between the 8th and 12th day after hatching. (b) Percentage of adult flies that successfully emerged from the pupae (relative to the number of larvae in each group). (c) Relative percentage of the successfully eclosed young adult flies that survived for 3 days after eclosion. For the experiments shown here, each vial was loaded with 100 larvae, and each group contained 300 larvae in total. *(P < 0.05), **(P < 0.01) and ***(P < 0.001) denote significant differences between the control group and exposure groups.
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A number of metallic nanoparticles are difficult for physiological clearance pathways to eliminate and may accumulate in certain organs of the body26,39,40,41. As previously shown in several rodent studies, following ingestion, AgNPs can be translocated through the intestinal barrier and then systemically distributed via the bloodstream or lymphatic system to secondary target organs (e.g., brain, liver, spleen, kidney and testes) where these particles cause functional and/or structural impairment2,26,27,42,43,44. The open circulatory system of Drosophila, similar to the mammalian cardiovascular and lymphatic systems, plays a role in facilitating wide tissue distribution of ingested substances following intestinal absorption. To determine whether the observed effects of dietary AgNPs on larval growth and development, on the duration of the larval and pupal stages, and on the success of adult eclosion might be correlated with the bioaccumulation of AgNPs, we performed an atomic absorption spectroscopic (AAS) analysis to quantitate the level of Ag deposition in the pupated flies that had been exposed to AgNPs. On the 3rd day after pupation, the amount of Ag deposited within the AgNP-treated groups, in comparison with the untreated control, significantly increased as the dose of exposure increased (Fig. 4a and Supplementary Fig. 2a). These data suggest that ingested AgNPs are capable of accumulating in Drosophila tissues for a long time, even when the organisms are not under exposure.
Figure 4Cumulative effects of sublethal AgNP exposure on long-term survival and stress-resistance capacity. (a) Level of Ag deposition within the male pupae exposed to AgNPs (0, 10, and 30 μg/ml) during the larval stages. The pupae in each group, with a total weight of approximately 100 mg, were collected and then subjected to AAS analysis. (b) Long-term survival of the male adult flies that ingested sublethal or lethal doses of AgNPs during the larval stages. (c) Stress-resistance capacity of male adult flies that ingested sublethal doses of AgNPs during the larval stages; 20 mM paraquat (PQ) acted as a systemic stressor here. ##(P < 0.01) and ###(P < 0.001) denote significant differences between exposure groups; ***(P < 0.001) denotes significant differences between the control group and exposure groups.
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As mentioned above, AgNPs appear to have the potential for long-term bioaccumulation within exposed flies, which introduces the possibility that accumulated AgNPs may exert chronic adverse effects on the lifespan and healthspan of Drosophila. To address this issue, we performed a longevity assay to assess whether dietary AgNPs decrease the lifespan of adult flies exposed to AgNPs during the larval stage. As shown in Fig. 4b and Supplementary Fig. 2b, AgNP exposure led to decreased lifespan, regardless of sex, in a dose-dependent manner. These results indicate that constant exposure of developing larvae to AgNPs, even at relatively low doses, can shorten the lifespan of the adult. As the healthspan of an organism is usually positively correlated with its lifespan, we investigated whether AgNP exposure also affects the health of adult flies. For this investigation, we carried out the paraquat challenge assay (which is a stress-resistance measurement that is usually an integral part of healthspan studies) to evaluate the stress tolerance capacity of adult flies that had been exposed to low doses of AgNPs (i.e., 10 and 20 μg/ml) during the larval stage. As shown in Fig. 4c and Supplementary Fig. 2c, the survival trends of these AgNP-treated adults were almost comparable to those of the untreated control group. Treatment with paraquat alone led to a significant decrease in survival compared to the untreated control. Intriguingly, pretreatment with low doses of AgNPs, regardless of sex, was shown to cause a sharp decline in the survival trends of flies challenged with equivalent amounts of paraquat in a dose-dependent manner. Thus, this result suggests that flies pre-exposed to AgNPs, even at low doses, are more prone to stress. Based on the above results, we concluded that dietary exposure to AgNPs during the larval stage not only interferes with larval growth, development and survival but also exerts long-lasting adverse effects on the lifespan and healthspan of adult flies following eclosion. In view of these conclusive findings, we aimed to further explore the toxicity mechanisms of AgNPs at the cellular and molecular levels.
Multiple lines of in vitro evidence have indicated that the generation of ROS is the mechanism underlying the cytotoxicity of AgNPs8,45,46. ROS act as an upstream regulator of various cellular responses, such as apoptosis, DNA damage and autophagy activation. In contrast, organismal effects of ROS generation in response to AgNP exposure remain elusive and have yet to be characterized on a system-wide scale. First, we determined the importance of ROS in dietary AgNP-induced in vivo adverse effects. As shown in Fig. 5a, dietary exposure to 30 μg/ml of AgNPs during the larval stage strongly elevated DHE fluorescence signals (indicative of superoxide generation) in a variety of Drosophila tissues (including gut, brain, salivary gland, wing disc, and eye/antenna discs) dissected from 3rd-instar wandering larvae, suggesting that systemic and massive ROS production resulting from dietary AgNP exposure account for the adverse effects detectable at the organismal level.
Figure 5AgNP-induced generation of ROS and activation of the Nrf2-dependent antioxidant pathway. (a) The confocal microscopy image indicating systemic generation of superoxide (assessed by DHE staining) in multiple tissues (brain, gut, salivary gland, wing disc, and eye/antenna discs) of the wandering 3rd-instar larvae exposed to 30 μg/ml of AgNPs. (b) Dose-dependent enhancement of the GFP signal in adult Nrf2/ARE-reporter flies by dietary exposure to sublethal or lethal doses of AgNPs during larval stages [mRE-RFP flies were used as the unresponsive control].
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For the maintenance of cellular redox homeostasis, multiple antioxidant pathways might be activated in response to oxidative stress. Of these pathways, the Nrf2-ARE pathway is evolutionarily conserved in organisms from yeast to mammals. Massive ROS production can lead to nuclear translocation of Nrf2, which assists in the transcriptional regulation of a variety of antioxidant genes (e.g., sod1, sod2, cat, gclc, gstD and gstE) located downstream of the antioxidant response element (ARE)47. Therefore, we suggested that the Nrf2-dependent antioxidant pathway is activated in response to AgNP-induced ROS. To confirm this hypothesis, we subjected the Nrf2-ARE transgenic reporter fly line to AgNP exposure during the larval stage. As demonstrated in Fig. 5b, AgNPs elevated the levels of Nrf2 signaling activity (i.e., green fluorescence signal) in adult head and trunk segments in a dose-dependent manner. In contrast, such exposure had no effect on the transgenic flies harboring the mutant version of the antioxidant response element (mRE) (i.e., non-responsive control). In summary, these results imply that the Nrf2/ARE-dependent antioxidant system can be activated to attenuate AgNP-induced oxidative damage.
AgNP-induced ROS have been shown to contribute to the occurrence of apoptosis in vitro48. In this study, we performed immunohistofluorescence analysis, by detecting the fluorescence levels of the apoptotic marker “cleaved/active caspase 3” (the executioner of apoptosis), to evaluate whether AgNP exposure causes apoptosis in vivo. As shown in Fig. 6, dietary exposure to 50 µg/ml of AgNPs distinctly induced the activation of caspase 3 (i.e., green fluorescence signal) in the brain, salivary gland, wing disc and gut of the third-instar larvae.
Figure 6AgNP-induced occurrence of systemic apoptosis. Immunofluorescent signals of the apoptotic biomarker “active caspase 3” in multiple tissues (brain, salivary gland, wing disc and gut) dissected from AgNP-exposed 3rd-instar wandering larvae.
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Furthermore, massive ROS generation or impaired antioxidant response can lead to peroxidation of DNA, causing DNA strand breaks49. To investigate whether AgNPs possess genotoxic potential in vivo, we investigated the expression level of gamma-H2AX, a biomarker of double-stranded DNA breaks, in the nuclear foci of larval tissues. Based on the immunofluorescence images (Fig. 7), we determined that AgNPs cause DNA damage in larval brain, salivary gland, and gut, which suggests that AgNPs systemically cause DNA damage in vivo following ingestion.
Figure 7AgNP-induced occurrence of systemic DNA damage. Immunofluorescent signals of the double-stranded-DNA-break biomarker “γ-H2AX” in multiple tissues (brain, salivary gland, and gut) dissected from AgNP-exposed 3rd-instar wandering larvae.
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A growing body of in vitro and in vivo evidence has noted the importance of autophagy in nanoparticle-induced toxicity. When activated at an appropriate stage and to a suitable extent, autophagy helps maintain cellular homeostasis2. However, overactivation of autophagy and/or subsequent disruption of autophagic flux has been observed in response to exposure to various metal nanoparticles (including AgNPs) in vitro2. In contrast, to date, there is insufficient in vivo evidence to address the role of autophagy in the systemic toxicity of AgNPs. To investigate this issue, we crossed a ubiquitous Gal4-driver fly line (daughterless Gal4) with a transgenic fly line carrying UAS-GFP- mCherry-tagged Atg8a to generate a reporter fly line that can be used to monitor the progression of autophagic flux in vivo. As illustrated in Fig. 8a, autophagosomes incorporated with fluorescently labeled Atg8a (the LC3 homolog in Drosophila) will be observed as punctae under a fluorescence microscope. The simultaneous presence of GFP (green fluorescence) and mCherry (red fluorescence) signals leads to the observation of yellow punctae, which represent autophagosomes. Upon fusion of autophagosomes with lysosomes, the acidic lysosomal environment causes quenching of GFP signals, resulting in the punctae merely emitting red fluorescence. Accordingly, red punctae represent autolysosomes. As the experimental scheme shows (Fig. 8b), we obtained the tissues from mid-third-instar AgNP-exposed larvae to evaluate whether ingested AgNPs trigger autophagy activation in vivo; this time-point was selected for the dissection because developmental autophagy occurs during the late-third-instar larval stage. To examine the time-dependent effects of AgNP exposure on the progression of autophagy, first-instar larvae were selected and dietarily exposed to 50 µg/ml of AgNPs for 24, 48 and 72 hrs. Interestingly, we found that AgNPs could enhance autophagy activation in a time-dependent manner in the larval brain (Fig. 8c) and in fat bodies (Fig. 8d). Our results suggest that sharp activation of autophagy in response to AgNP exposure is associated with adverse outcomes identified at the organismal level.
Figure 8AgNP-induced autophagy activation. (a) The illustrative graph of the Atg8-GFP-mCherry reporter for qualitatively and quantitatively examining autophagic flux. (b) The experimental scheme for measuring AgNP-induced autophagy activation and subsequent progression. A time-dependent increase in the number of red fluorescent punctae (i.e., mCherry signals) were found in the (c) brain and (d) fat bodies of AgNP-exposed 3rd-instar wandering larvae.
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