In Caenorhabditis elegans nanoparticle-bio-interactions become transparent: silica-nanoparticles induce reproductive senescence - PubMed (original) (raw)
In Caenorhabditis elegans nanoparticle-bio-interactions become transparent: silica-nanoparticles induce reproductive senescence
Adam Pluskota et al. PLoS One. 2009.
Abstract
While expectations and applications of nanotechnologies grow exponentially, little is known about interactions of engineered nanoparticles with multicellular organisms. Here we propose the transparent roundworm Caenorhabditis elegans as a simple but anatomically and biologically well defined animal model that allows for whole organism analyses of nanoparticle-bio-interactions. Microscopic techniques showed that fluorescently labelled nanoparticles are efficiently taken up by the worms during feeding, and translocate to primary organs such as epithelial cells of the intestine, as well as secondary organs belonging to the reproductive tract. The life span of nanoparticle-fed Caenorhabditis elegans remained unchanged, whereas a reduction of progeny production was observed in silica-nanoparticle exposed worms versus untreated controls. This reduction was accompanied by a significant increase of the 'bag of worms' phenotype that is characterized by failed egg-laying and usually occurs in aged wild type worms. Experimental exclusion of developmental defects suggests that silica-nanoparticles induce an age-related degeneration of reproductive organs, and thus set a research platform for both, detailed elucidation of molecular mechanisms and high throughput screening of different nanomaterials by analyses of progeny production.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Translocation of fluorescently labelled nanoparticles (NPs) into organs and tissue of the nematode worm Caenorhabditis elegans.
Young adult hermaphrodites were placed onto agar plates and fed on a bacterial lawn that contained labelled silica-NPs or polystyrene- NPs, respectively. After 16 h of incubation animals were collected and prepared for analysis of nanoparticle distribution by epifluorescence microscopy. Single fluorescence staining was inverted using Adobe Photoshop in order to visualize nanoparticle localization (A, C, D, lower panels). Corresponding nematode anatomy is obtained by differential interference contrast (A, C, D, upper panels). (A) Red-labelled silica-NPs were detectable in the lumen of pharynx and intestine. (B) Schematic representation of Caenorhabditis elegans hermaphrodite anatomy. (C) Red (YO, carboxy)-labelled polystyrene-NPs were detectable in the intestine (lumen and tissue), proximal gonad (white arrow), pharynx, spermatheca and cytoplasm of early embryos. (D) Green(YG)-labelled polystyrene-NPs showed a similar translocation to intestine and gonad, but were not detectable in other tissues. Bars, 10 µm.
Figure 2. Untreated controls and worms fed on nanoparticles exhibit an identical life span.
Worms were cultivated on agar plates overgrown with bacterial lawns that were prepared as indicated. Life span of hermaphrodites was monitored at 25°C as described in Results. (A) Survival curves show no difference in life-expectancy between control (dark blue) and nanoparticle-exposed worms. (B) A corresponding box-plot shows that mean life span of particle-fed worms ranges between 8.6 (±0.45; n = 73) to 10.3 (±0.5; n = 61) days. Untreated nematodes (dark blue) have a mean life span of 9.54 (±0.44; n = 71) days. Analysis by Log-Rank-Test corroborated lack of significant differences in life span. Circles depict two individual worms with abnormally long life spans. Values represent means +/− SD from three independent experiments.
Figure 3. Untreated control worms and Caenorhabditis elegans fed on nanoparticles exhibit an identical accumulation of lipofuscin with age.
(A) Representative micrographs show increased autofluorescence of lipofuscin in an untreated nematode worm over time. Single fluorescence staining was inverted using Adobe Photoshop. (B, C) Quantitative analysis of fluorescence signals in the intestine from untreated (white) and nanoparticle(NP)-treated worms. Nematodes fed on 0.5 mg/ml polystyrene-NPs (light grey) or 2.5 mg/ml silica-NPs (black), respectively. Specimens were prepared for microscopy and micrographs were acquired at the indicated times. Average fluorescence intensity was measured within regions-of-intrest (ROI) drawn around intestines of each adult hermaphrodite worm. Values represent means +/− SD from three experiments (n = 47, B; n = 30, C). Bars, 50 µm.
Figure 4. Reproductive life span of Caenorhabditis elegans fed on silica-NPs is reduced in comparison to untreated control worms.
L4 larvae were placed on OP50 E. coli plates. The bacterial lawn was supplemented as indicated. Adult worms were transferred onto fresh (identically prepared) agar plates daily and residual embryos and larvae were counted as progeny. (A) Progeny production in control (white), and silica-NPs-fed hermaphrodites (light gray, gray, black). (B) Curves show average daily progeny production in control worms and worms fed on silica-NPs. (C) Ratio (%) of the bag of worms (BOW) phenotype that was observed during the progeny production experiments. BOW is significantly elevated in nematodes that fed on silica-NPs. Values represent means (B) +/− SD (A, C) from three experiments (n = 96). *, p<0,05; **, p<0,01; ***, p<0,001.
Figure 5. Developmental defects are not responsible for reduced progeny production in Caenorhabditis elegans fed on silica-NPs.
L4 larvae were placed on OP50 E. coli plates. The bacterial lawn was supplemented as indicated when development into adult hermaphrodite worms was completed (arrow). Adult worms were transferred onto fresh (identically prepared) agar plates daily and residual embryos and larvae were counted as progeny. (A) Progeny production in control (white), and silica-NPs-fed hermaphrodites (light gray, gray, black). (B) Curves show average daily progeny production in control worms and worms fed on silica-NPs. (C) Ratio (%) of the bag-of-worms (BOW) phenotype that was observed during the progeny production experiments. BOW is significantly elevated in nematodes that fed on silica-NPs. Values represent means (B) +/− SD (A, C) from three experiments (n = 60). *, p<0,05; **, p<0,01; ***, p<0,001.
Figure 6. Ethosuximide rescues silica-nanoparticle-induced decrease of progeny production and BOW phenotype.
Hermaphrodites were placed onto bacterial lawn on agar at larval stage L4. The bacterial lawn was supplemented as indicated. Adult worms were transferred onto fresh (identically prepared) agar plates daily and residual embryos and larvae were counted as progeny. (A) Progeny production (from left to right) in control (white), silica-NP-fed (anthracite), ethosuximide treated (dark gray) and silica-NP/ethosuximide co-treated worms (black, and light gray). (B) Curves show average daily progeny production in control worms and worms fed with silica-nanoparticles and/or ethosuximide. (C) Ratio (%) of the bag-of-worms (BOW) phenotype that was observed during the progeny production experiments. Values represent means (B) +/− SD (A, C) from three experiments (n = 36). Significance was tested by one-way ANOVA and Tukeys-Post-hoc-test.
Figure 7. Bulk silica-particles do not induce the BOW phenotype.
Hermaphrodites were placed onto bacterial lawn on agar at larval stage L4. The bacterial lawn was supplemented as indicated. Adult worms were transferred onto fresh (identically prepared) agar plates daily for a time period of 7 days. Columns show the ratio (%) of the bag-of-worms (BOW) phenotype that was observed during the indicated treatments. BOW is exclusively elevated in nematodes that fed on silica-nanoparticles. Values represent means +/− SD from eight experiments (n = 96). *, p<0.05.
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