Mechanism of silver nanoparticles action on insect pigmentation reveals intervention of copper homeostasis

Abstract

Silver nanoparticles (AgNPs), like almost all nanoparticles, are potentially toxic beyond a certain concentration because the survival of the organism is compromised due to scores of pathophysiological abnormalities past that concentration. However, the mechanism of AgNP toxicity remains undetermined. Instead of applying a toxic dose, we attempted to monitor the effects of AgNPs at a nonlethal concentration on wild type Drosophila melanogaster by exposing them throughout their development. All adult flies raised in AgNP doped food showed that up to 50 mg/L concentration AgNP has no negative influence on median survival; however, these flies appeared uniformly lighter in body color due to the loss of melanin pigments in their cuticle. Additionally, fertility and vertical movement ability were compromised due to AgNP feeding. Determination of the amount of free ionic silver (Ag(+)) led us to claim that the observed biological effects have resulted from the AgNPs and not from Ag(+). Biochemical analysis suggests that the activity of copper dependent enzymes, namely tyrosinase and Cu-Zn superoxide dismutase, are decreased significantly following the consumption of AgNPs, despite the constant level of copper present in the tissue. Consequently, we propose a mechanism whereby consumption of excess AgNPs in association with membrane bound copper transporter proteins cause sequestration of copper, thus creating a condition that resembles copper starvation. This model also explains the cuticular demelanization effect resulting from AgNP since tyrosinase activity is essential for melanin biosynthesis. Finally, we claim that Drosophila, an established genetic model system, can be well utilized for further understanding of the biological effects of nanoparticles.

Figure 1. Dietary administration of AgNPs cause cuticular demelanization in Drosophila.

(A) Flies are raised in AgNP doped food at various concentrations from the embryonic stage. When raised in 50 mg/L and above concentrations of nanoparticles, all adult flies (100%) appeared extremely lighter in body color with little or no melanin pigments left in their body. Since the eye color remains unchanged this suggests that AgNPs selectively interferes with the melanin pigmentation. (B) In Drosophila black (b) mutants accumulates excessive melanin pigments. Exposure of black flies to 50 mg/L AgNP effectively eliminates all melanin pigments from their body and the flies turned pale. (C) Sufficient accumulation of silver occurs in AgNP fed flies as determined by Atomic Absorption (AA) analysis. Flies fed with AgNO₃ were used for comparison. (C) Measurement of free silver (Ag+) with ion specific electrodes confirmed that 50 mg/L AgNP solution generates ionic Ag⁺ at a negligible quantity when compared to AgNO₃ solution in the same concentration. This led us to conclude that flies raised in AgNP doped food mostly consumed AgNPs and not free Ag⁺ to display the demelanization effect.

Figure 2. Loss of cuticular pigmentation does not alter the survival and metamorphosis, but female fertility and vertical climbing behaviors are affected.

(A) Demelanized adult females live the same median life span as the controls, when adults were maintained in food without the AgNPs. A second control was set up with Na-citrate, which demonstrates, that citrate coating does not influence the survival. We therefore considered 50 mg/L as a nonlethal concentration for Drosophila. A significant drop in median life span was noted with AgNO₃ feeding. (B) Number of progenies obtained from females raised in AgNP doped food was significantly lower than the controls especially during the peak egg-laying period, which is between 10–30 days (boxed). (C) Metamorphosis proceeds normally in demelanized flies. Percent pupae eclosed as adults was determined from #adult/#pupal ratios. Eclosion ratios are not significantly different between Cit-AgNP fed and unfed controls. * represents p<0.05. (D) AgNP fed flies are slow to climb the same vertical distance with respect to the unfed flies, and this effect was evident starting from a very young age which is sustained throughout life. Climbing performance was more seriously impaired in flies fed with AgNO₃.

Figure 3. AgNP feeding negatively influence the tyrosinase enzyme activity.

(A) Determination of purified mushroom tyrosinase activity (2 mg/ml) ensures a well-designed assay condition. The change in absorbance was recorded at a wavelength of 505 nm every 15 seconds for a total of five minutes. (B) Comparison of tyrosinase progress curves in fly extracts obtained from vehicle control and 50 mg/L Cit-AgNP fed flies showed that tyrosinase activity slows down significantly in AgNP fed flies compared to the control (C) Total tyrosinase activity in 50 mg/L Cit-AgNP fed flies was reduced to about 51% of the control value.

Figure 4. An in gel superoxide dismutase (SOD) activity determination in AgNP fed flies.

(A) Electrophoretic separation of active Cu/ZnSOD and MnSOD enzymes and subsequent detection with the help of an electron donor shows two clear bands of enzyme activity. (B) Densitometric scanning of band intensities helped us to determine the activity ratios between Cu/ZnSOD and MnSOD enzymes, which suggests that the activity of the Cu/ZnSOD was significantly reduced in 50 mg/L Cit-AgNP fed flies with respect to the vehicle control. SOD activity analysis was performed in triplicate (n = 20 male flies/treatment).

Figure 5. Copper transporters mutants are insensitive to demelanization.

Comparative analysis of three Ctr1 transcripts A, B and C expressions by RT-PCR showed no changes in mRNA expression following AgNP feeding with respect to the control flies suggesting that the copper transport machinery was not affected in the presence of Ag⁺. (B) Interestingly reduced synthesis of CTR1A and B proteins in Ctr/+ heterozygotes makes them insensitive to demelanization effect, so their body color remains normal (darker abdominal stripes) as opposed to the AgNP fed fly which appears much lighter in body color. Therefore, intracellular copper transporters are required for transporting AgNPs into the cell.

Figure 6. Schematic of silver and copper interaction in context of copper transport.

Intracellular copper (Cu⁺) transport happens through conserved membrane bound copper transporter 1 (Ctr1) and also with the help of other metal transporters called general importers. Drosophila has three such CTR1 proteins, CTR1A, CTR1B, and CTR1C. Once inside the cell, Cu⁺ is utilized via three main branches to copper dependent proteins (e.g., tyrosinase), through copper chaperones to Cu-Zn superoxide dismutase (Cu/ZnSOD), and to cytochrome C oxidase in the mitochondria. Presence of excess Ag in the extracellular environment might inhibit the process of intracellular entry of Cu because Ag and Cu might be competing for the same copper transporters. Copper dependent tyrosinase is a dual action oxidase, which plays an essential role the conversion of tyrosine to dopa and dopa to dopaquinone, which is ultimately converted to melanin pigments. Thus, AgNPs can cause the demelanization effect through inhibiting Cu transport.