Assessing cellular and genomic damage from environmental nickel using a GFP containing strain of Caenorhabditis elegans

Nickel is a naturally found mineral that has become widely used for many electronic devices. As use and subsequent discarding of nickel containing products continues exposure to nickel increases. Nickel can cause external superficial symptoms but if it enters the body the potential exists for genomic damage to occur which could lead to mutation and cancer. Nickel can act by generation of reactive oxygen species, interacting with DNA and altering chromatin wrapping. By utilizing a strain of C. elegans with a ced-1::gfp fusion protein, that detects apoptotic cells in the germ line, the deleterious effects of nickel can be analyzed. Analyses of varying concentrations of a water-soluble and an insoluble form of nickel have been done. Insoluble nickel is held to be more hazardous because while soluble easily enters and exits the cell insoluble nickel can remain in the cell for extended periods allowing for much greater damage. The results of this study were inconclusive about the effects of soluble versus insoluble nickel. Previous testing using the C. elegans germline to assess the effects of nickel have used 12 hour exposures (Kezhou et al. 2010). Tests completed in this study exposed animals to lower concentrations of nickel for their full development. A rise in cell deaths is seen as nickel concentration increases which was quantified with ced-1::gfp and Syto12. Analysis using a strain with resistance to heavy metals had no significant increase in engulfments when exposed to nickel, which shows nickel exposure to have been the cause of increased engulfments seen in the wild-type. A lack of increased engulfments in a strain with a mutation to cep-1, the C. elegans homolog of p53, indicated damage from nickel is recognized by the p53 damage pathway.  M.S

Comparative genomics of Pseudomonas fluorescens subclade III strains from human lungs

Abstract Background While the taxonomy and genomics of environmental strains from the P. fluorescens species-complex has been reported, little is known about P. fluorescens strains from clinical samples. In this report, we provide the first genomic analysis of P. fluorescens strains in which human vs. environmental isolates are compared. Results Seven P. fluorescens strains were isolated from respiratory samples from cystic fibrosis (CF) patients. The clinical strains could grow at a higher temperature (>34 °C) than has been reported for environmental strains. Draft genomes were generated for all of the clinical strains, and multi-locus sequence analysis placed them within subclade III of the P. fluorescens species-complex. All strains encoded type- II, −III, −IV, and -VI secretion systems, as well as the widespread colonization island (WCI). This is the first description of a WCI in P. fluorescens strains. All strains also encoded a complete I2/PfiT locus and showed evidence of horizontal gene transfer. The clinical strains were found to differ from the environmental strains in the number of genes involved in metal resistance, which may be a possible adaptation to chronic antibiotic exposure in the CF lung. Conclusions This is the largest comparative genomics analysis of P. fluorescens subclade III strains to date and includes the first clinical isolates. At a global level, the clinical P. fluorescens subclade III strains were largely indistinguishable from environmental P. fluorescens subclade III strains, supporting the idea that identifying strains as ‘environmental’ vs ‘clinical’ is not a phenotypic trait. Rather, strains within P. fluorescens subclade III will colonize and persist in any niche that provides the requirements necessary for growth.http://deepblue.lib.umich.edu/bitstream/2027.42/116129/1/12864_2015_Article_2261.pd

Assaying Environmental Nickel Toxicity Using Model Nematodes

Although nickel exposure results in allergic reactions, respiratory conditions, and cancer in humans and rodents, the ramifications of excess nickel in the environment for animal and human health remain largely undescribed. Nickel and other cationic metals travel through waterways and bind to soils and sediments. To evaluate the potential toxic effects of nickel at environmental contaminant levels (8.9-7,600 µg Ni/g dry weight of sediment and 50-800 µg NiCl2/L of water), we conducted assays using two cosmopolitan nematodes, Caenorhabditis elegans and Pristionchus pacificus. We assayed the effects of both sediment-bound and aqueous nickel upon animal growth, developmental survival, lifespan, and fecundity. Uncontaminated sediments were collected from sites in the Midwestern United States and spiked with a range of nickel concentrations. We found that nickel-spiked sediment substantially impairs both survival from larval to adult stages and adult longevity in a concentration-dependent manner. Further, while aqueous nickel showed no adverse effects on either survivorship or longevity, we observed a significant decrease in fecundity, indicating that aqueous nickel could have a negative impact on nematode physiology. Intriguingly, C. elegans and P. pacificus exhibit similar, but not identical, responses to nickel exposure. Moreover, P. pacificus could be tested successfully in sediments inhospitable to C. elegans. Our results add to a growing body of literature documenting the impact of nickel on animal physiology, and suggest that environmental toxicological studies could gain an advantage by widening their repertoire of nematode species

Recovery of P0 adult nematodes and F1 progeny from eight un-spiked test sediments.

<p><i>C. elegans</i> – blue. <i>P. pacificus</i> – red. Box and whisker plots: box represents the range between the 25^th and 75^th percentile (interquartile range). The line within the box represents the median. The whiskers indicate minimum and maximum values, except where circles and stars represent outliers (>1.5 times interquartile range from median) or extreme outliers (>3 times interquartile range from median), respectively. Letters above the box and whisker plots represent significant groupings based upon Tukey post-hoc comparison tests (p< 0.05). Lower case blue letters, <i>C. elegans</i> groupings; capital red letters, <i>P. pacificus</i> groupings. (A) Recovery of added P0 animals. (B) Recovery of L1/J2 progeny. (C) Fecundity Index, total progeny recovered / total live P0 adult nematodes recovered.</p

Collection sites for test sediment in the Midwest USA.

<p>(1) SR – Spring River, Jasper County, Missouri(2). STJ – St. Joseph River, Michigan(3). P30 – USGS Pond 30 Missouri(4). DOW – Dow Creek Michigan(5). RR2 – Raisin River Site 2, Michigan(6). STM – South Tributary of Mill Creek, Michigan(7). RR3-0 Raisin River Site 3, Michigan(8). WB – West Bearskin Lake Minnesota. Sites are given in order presented in Tables 1-3 and based upon <i>C. elegans</i> survivorship performance. The letter A designates the position of Chicago, IL, USA.</p

Adult lifespan in nickel-spiked sediment – survivorship curves.

<p>Recovery of <i>Cel-fog-2</i> females from WB spiked nickel sediments, WB-0 (blue circles), WB-2 (green triangle), WB-3 (black asterisks), and WB-5 (red squares). Survivability decreases as nickel increases. Yellow-orange dotted line represents a 50% recovery. WB-0 and WB-2 show a 50% reduction around day 16. WB-3 shows a 50% reduction around day 7. WB-5 showed a 50% reduction around day 3.</p

Recovery of P0 adult nematodes and F1 progeny from nickel-spiked water.

<p><i>C. elegans</i> – blue. <i>P. pacificus</i> – red. (A) P0 recovery. (B) F1 recovery. (C) Fecundity ratio.</p

Morphological markers of life-stage and fertility in <i>C. elegans</i>.

<p>Detailed descriptions of <i>P. pacificus</i> vulva and gonad development are available [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B38" target="_blank">38</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B39" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077079#B42" target="_blank">42</a>]. Panels include a DIC micrograph on top and a cartoon of the micrograph below. For body orientation, anterior is to the left and dorsal to the top. Black arrows mark the position of the vulva, the egg- laying organ. Asterisks in the cartoons denote developing embryos <i>in </i><i>utero</i> and are not shown in the micrograph for clarity. White triangles denote the position of oocytes. Tissues are denoted by color: gut, blue; uterus and spermatheca, purple; germ line (including the germ cells, oocytes, and developing embryos), red. Ovals and circles depict easily seen nuclei within tissues. (A) Young adult hermaphrodite. The entire posterior gonad arm is shown. The red arrow outlines the path of gonad arm extension starting proximal to the vulva and terminating with the arrowhead at the distal tip. As an adult, this animal has made sperm, oocytes, and contains embryos, has two fully reflexed and inflated gonadal arms, and a fully everted vulva with a slit like morphology. The black asterisk denotes an egg in the spermatheca that has just been fertilized but has not developed an eggshell yet. In contrast, L4 larvae never contain embryos as they only begin to produce gametes late in the L4-stage. The L4 gonad arms are smaller and not as inflated, and early in L4 have only reached the dorsal side of the animal, but do not reach the center above the vulva until late in L4. (B) Wild type early L4-larva hermaphrodite detail of uterus and vulva. The vulva has a characteristic “Christmas tree” like shape, it is not everted. The uterus is empty and un-inflated. Gametes have not been produced, the gonad arms are skinny and contain relatively few germ cells making the gonad arms difficult to capture in the same focal plane as the vulva. (C) Wild type adult hermaphrodite detail of uterus and vulva. The uterus is full of multicellular embryos. The gold asterisk denotes an embryo with a clearly visible eggshell. The eggshell is present as an oval around the ball of cells. The embryo and shell are separated by a slim cleared liquid-filled space. (D) Adult fog-<i>2</i> female detail of uterus and vulva. <i>fog-2</i> females do not produce sperm and contain no embryos. Hence the uterus and spermatheca remain unexpanded. Unfertilized oocytes stack up in the gonad arms and become compressed, giving a “piano key” phenotype. Eventually pressure may push an oocyte into the uterus and the oocyte will be laid, but without an eggshell. Laid oocytes have a “mushy” appearance and remain single celled until decomposition. The edge of a laid embryo has a refractory appearance due to the eggshell.</p

Recovery of P0 adult nematodes and F1 progeny from nickel-spiked sediments (i.e. WB-0 through WB-5 and SR-0 through SR-5).

<p>Box-and-whisker plots are formatted and labeled as in Figure 3. Scatterplots: P0 recovery for individual wells plotted against sediment nickel concentration. Blue circles, <i>C. elegans</i>; red diamonds, <i>P. pacificus</i>. (A-D, I) WB Ni(II)-spiked sediment series. (A) P0 recovery and sediment treatment. (B) P0 recovery and sediment nickel concentration. (C) F1 recovery and sediment treatment. (D) F1 recovery and sediment nickel concentration. (E-H, J) SR Ni(II)-spiked sediment series. (E) P0 recovery and sediment treatment. (F) P0 recovery and sediment nickel concentration. (G) F1 recovery and sediment treatment. (H) F1 recovery and sediment nickel concentration. (I and J) Fecundity ratio and sediment treatment.</p

Dual Electrochemical and Physiological Apoptosis Assay Detection of in Vivo Generated Nickel Chloride Induced DNA Damage in Caenorhabditis elegans

Environmental nickel exposure is known to cause allergic reactions, respiratory illness, and may be responsible for some forms of cancer in humans. Nematodes are an excellent model organism to test for environmental toxins, as they are prevalent in many different environments. Nickel exposure has previously been shown to impact nematode life processes. In this study, Caenorhabditis elegans nematodes exposed to NiCl₂ featured high levels of programmed cell death (PCD) in a concentration-dependent manner as measured by counting apoptotic corpses in the nematode germ line. A green fluorescent protein (GFP) reporter transgene was used that highlights cell corpse engulfment by fluorescence microscopy. Analysis of the reporter in a <i>p53</i> mutant strain putatively indicates that the PCDs are a result of genomic DNA damage. In order to assay the potential genotoxic actions of NiCl₂, DNA was extracted from nematodes exposed to increasing concentrations of NiCl₂ and electrochemically assayed. In vivo damaged DNA was immobilized on pyrolytic graphite electrodes using the layer-by-layer (LbL) technique. Square-wave voltammograms were obtained in the presence of redox mediator, ruthenium trisbipyridine (Ru­(bpy)₃²⁺), that catalytically oxidizes guanines in DNA. Oxidative peak currents were shown to increase as a function of NiCl₂ exposure, which further suggests that the extracted DNA from nematodes exposed to the nickel was damaged. This report demonstrates that our electrochemical biosensor can detect damage at lower Ni concentrations than our physiological PCD assay and that the results are predictive of physiological responses at higher concentrations. Thus, a biological model for toxicity and animal disease can be assayed using an electrochemical approach