Effects of carbon nanofiber on physiology of Drosophila

Abstract

As nanomaterials are now widely utilized in a wide range of fields for both medical and industrial applications, concerns over their potential toxicity to human health and the environment have increased. To evaluate the toxicity of long-term exposure to carbon nanofibers (CNFs) in an in vivo system, we selected Drosophila melanogaster as a model organism. Oral administration of CNFs at a concentration of 1,000 μg/mL had adverse effects on fly physiology. Long-term administration of a high dose of CNFs (1,000 μg/mL) reduced larval viability based on the pupa:egg ratio, adult fly lifespan, reproductive activity, climbing activity, and survival rate in response to starvation stress. However, CNFs at a low concentration (100 μg/mL) did not show any significant deleterious effect on developmental rate or fecundity. Furthermore, long-term administration of a low dose of CNFs (100 μg/mL) increased lifespan and climbing ability, coincident with mild reactive oxygen species generation and stimulation of the antioxidant system. Taken together, our data suggest that a high dose of CNFs has obvious physiological toxicity, whereas low-dose chronic exposure to CNFs can actually have beneficial effects via stimulation of the antioxidant defense system.

Keywords: Drosophila melanogaster; lifespan; reactive oxygen species; toxicity.

Figure 1

Scanning electron microscopic images of carbon nanofiber (CNF). Notes: The diameter of CNFs is approximately 150–200 nm, and the length of CNFs is approximately 3–15 μm. The scale bars correspond to 0.5 μm.

Figure 2

Effects of CNFs on larval and pupal viability in Drosophila. Notes: (A) Larval viability was determined based on the pupa:egg ratio. Oral administration of CNFs at a dose of 100 μg/mL did not significantly affect larval viability (black bar), whereas 1,000 μg/mL of CNF administration significantly reduced larval viability (gray bar) compared to the control (white bar). *P<0.05. (B) Pupal viability was determined based on the eclosed adult:pupa ratio. CNF administration at both 100 and 1,000 μg/mL did not significantly alter pupal viability. (C) Microscopic images of larvae (a) and adult flies (b) reared on CNF-containing food. Larvae and adult flies reared on CNF-containing food showed dark masses on their body cavities (arrows). Arrowheads indicate melanotic spots in segments of larvae. (D) The number of crystal cells in larvae reared on CNF-containing food and visualized by heat shock-induced melanization increased upon administration of 100 μg/mL of CNFs. *P<0.05. (E) The mRNA levels of defensin were analyzed via real-time quantitative polymerase chain reaction. Expression level of defensin was increased upon 100 μg/mL of CNF administration. *P<0.05. Abbreviation: CNF, carbon nanofiber.

Figure 3

Effects of CNF oral administration on adult viability in Drosophila melanogaster. Notes: (A) Chronic administration of CNFs at a dose of 1,000 μg/mL reduced survival time of Drosophila (lines with black triangles; male, P<0.0001; female, P<0.0001). However, the lifespan of flies administered 100 μg/mL of CNFs significantly increased (lines with black circles; male, P<0.0001; female, P<0.0001). (B) Mortality curves showing age-specific mortality rates of flies administered CNFs. The natural log of the mortality rate was plotted using the Gompertz mortality model. Age-specific mortality rates of both male and female flies administered 1,000 μg/mL of CNFs significantly increased, whereas those of flies administered 100 μg/mL of CNFs slightly decreased. (C) Administration of graphene at the same dose as CNF did not decrease the survival rate of Drosophila (male flies, 100 μg/mL, P<0.1617; 1,000 μg/mL, 9.52% increase of mean lifespan, P<0.0001). (D) Age-specific mortality rate of flies exposed to graphene did not increase at early life stages. Abbreviation: CNF, carbon nanofiber.

Figure 4

Effects of CNF ingestion on reproduction and feeding in Drosophila melanogaster. Notes: (A) Reproductive activity of flies was determined based on the number of eggs obtained from a female fly reared on CNF-containing food. Reproductive activity of flies administered 1,000 μg/mL of CNFs significantly decreased from 4 days after eclosion (line with black triangles). Administration of 100 μg/mL of CNFs did not affect reproductive activity of flies (line with black circles). (B) Food intake by flies was assessed by measuring the amount of blue dye-containing food after 12 hours. Food intake by flies administered CNFs significantly decreased in a dose-dependent manner. *P<0.05; **P<0.001. Abbreviations: CNF, carbon nanofiber; OD, optical density.

Figure 5

Effects of CNF ingestion on physical activity of Drosophila. Notes: The physical activity of flies was determined by vertical climbing assay. The physical activity of female flies (A) was not significantly affected by CNF administration. However, the physical activity of male flies (B) significantly decreased at 1 week after eclosion upon 1,000 μg/mL of CNF ingestion. Male flies administered 100 μg/mL of CNFs showed enhanced physical activity at 3 weeks after eclosion. *P<0.05. Abbreviation: CNF, carbon nanofiber.

Figure 6

Effects of CNF ingestion on resistance to starvation stress and body weight. Notes: (A) Survival rate of flies administered 1,000 μg/mL of CNFs significantly decreased under starvation conditions (lines with black triangles, P<0.0001), whereas 100 μg/mL of CNFs had a minor effect or none (lines with black circles). (B) Body weight of newly hatched flies is shown. Newly eclosed flies reared on CNF-containing food were heavier than the control. (C) Body weight of 7-day-old flies is shown. Body weight of male flies administered CNFs for 7 days still significantly increased. *P<0.05; **P<0.001; ***P<0.0001. Abbreviation: CNF, carbon nanofiber.

Figure 7

CNF-induced oxidative stress in Drosophila. Notes: (A) The intracellular reactive oxygen species level was analyzed by measuring the ratio of GSH to GSSG. The GSH/GSSG ratio significantly decreased upon chronic administration of CNFs to Drosophila. Treatment with 20 mM PQ was used as a positive control. (B) The mRNA levels of antioxidant enzymes were analyzed via real-time quantitative polymerase chain reaction. Expression levels of dSOD1, dSOD2, and catalase increased upon 100 μg/mL of CNF administration but decreased upon 1,000 μg/mL of CNF administration. *P<0.05; **P<0.001; ***P<0.0001. Abbreviations: CNF, carbon nanofiber; GSH, glutathione; GSSG, oxidized glutathione; PQ, paraquat.