Ficociello G et al. (2020) Environ Res "Assessment of the effects of atmospheric pollutants using the animal model Caenorhabditis elegans."

Buccini G, Ficociello G, Massimi L, Inverni A, Uccelletti D, Canepari S

[Environ Res, 2020]

Air pollution is recognized as the world's largest environmental health risk. In this work we evaluated in vivo the effects of three relevant components of atmospheric dusts (brake dust, wood pellet ash and Saharan dust) employing the animal model Caenorhabditis elegans. Main endpoints of C. elegans such as life span, brood size and oxidative stress were addressed by exposing the nematodes to different dust concentrations. Brake dust and pellet ash affected the life span and increased significantly the oxidative stress of exposed nematodes, while Saharan dust showed no effects. Water soluble and insoluble fractions of these dusts were used to investigate the impact of the single fraction on C. elegans. The two fractions of brake dust and pellet ash exerted different effects on C. elegans endpoints in terms of life span and oxidative stress response. These fractions acted in different ways on the worm susceptibility to infection of two human pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) affecting the sek-1 gene expression. In conclusion, our study showed that C. elegans is a valuable tool to investigate in vivo possible effects of atmospheric dusts.

Trifuoggi M et al. (2018) Environ Monit Assess "Topsoil and urban dust pollution and toxicity in Taranto (southern Italy) industrial area and in a residential district."

Buric P, Palumbo A, Tommasi F, Guida M, Trifuoggi M, Thomas PJ, Crisci A, D'Ambra L, Toscanesi M, Oral R, Siciliano A, Gravina M, Pagano G, Mozzillo M, Lyons DM

[Environ Monit Assess, 2018]

Adverse environmental conditions in the Taranto area (southern Italy) were investigated in studies of air, marine sediment, and human health. The present study aimed at providing unprecedented information on soil pollution and toxicity in a set of sites around recognized pollution sources in the Taranto area, since previous studies were focused on marine or air pollution, or on human health effects. The investigated area included a steel foundry and a power plant, as well as some sites located in an adjacent neighborhood. Surface soil samples and urban dust were collected and submitted to inorganic and organic analyses and tested for toxicity in two invertebrate bioassay models; a sea urchin (Sphaerechinus granularis) and an annelid (Caenorhabditis elegans). Inorganic analysis was carried out using ICP-MS for elemental composition for a total of 34 elements, whose levels were evaluated as a function of bioassay data analyzed through principal component analysis (PCA). Other analyses included asbestos search by powder X-ray diffraction (PXRD) and organic analysis for polycyclic aromatic hydrocarbons (PAHs) and aliphatic compounds (C10-C40). Toxicity bioassays were carried out on a sea urchin (Sphaerechinus granularis), and an annelid (Caenorhabditis elegans). Sea urchin bioassays evaluated effects of topsoil or street dust sample exposures (0.1 to 0.5% dry wt/vol) on developing embryos and on sperm, and scored as (a) % developmental defects, (b) inhibition of fertilization success and offspring damage, and (c) frequencies of mitotic aberrations. C. elegans mortality assay displayed significant toxicity associated with soil samples. The overall effects of samples showed very high toxicity at four out of nine sites. These effects were consistent with the highest levels measured for metals and PAHs. Further studies of health effects related to dust exposures in residential areas are warranted. Graphical abstract .

Nagar Y et al. (2019) Chemosphere "Toxicity assessment of parabens in Caenorhabditis elegans."

Satish A, Patel DK, Ram KR, Parveen T, Nagar Y, Thakur RS

[Chemosphere, 2019]

Parabens, the alkyl esters of p-hydroxybenzoic acid such as methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), butylparaben (BuP) are used as a preservative in food, personal care products (PCPs), and pharmaceuticals, due to their antimicrobial properties. Parabens are continuously released into the environment, during washout of PCPs, disposal of industrial waste from the pharmaceutical and paper industries. Parabens have been detected in the indoor dust, wastewater stream, surface water of rivers, and the marine system. Recent eco-toxicological data and the environmental presence of parabens, has raised concerns regarding the safety and health of environment/humans. Thus, to further understand the toxicity of parabens, the present study was carried out in the soil nematode and well established biological model organism Caenorhabditis elegans. In the present study, LC₅₀ of MeP, EtP, PrP and BuP for 72h exposures from L1 larva to adult stage was found to be 278.1, 217.8, 169.2, and 131.88g/ml, respectively. Further exposure to 1/5th of LC₅₀ of parabens yielded an internal concentration ranging from 1.67 to 2.83g/g dry weight of the organism. The toxicity of parabens on the survival, growth, behavior, and reproduction of the C.elegans was found in the order of BuP>PrP>EtP>MeP. Worms exposed to parabens show significant down-regulation of vitellogenin genes, high levels of reactive oxygen species and anti-oxidant transcripts, the latter being concordant with nuclear localization of DAF-16 and up-regulation of HSF-1 and SKN-1/Nrf. Hence, parabens caused endocrine disruption, oxidative stress and toxicity in C.elegans at environment relevant internal concentration of parabens.

Puchalt JC et al. (2020) Sci Rep "Improving lifespan automation for Caenorhabditis elegans by using image processing and a post-processing adaptive data filter."

Puchalt JC, Genoves Martinez S, Ivorra E, Sanchez-Salmeron AJ, Martinez R, Martorell Guerola P

[Sci Rep, 2020]

Automated lifespan determination for C. elegans cultured in standard Petri dishes is challenging. Problems include occlusions of Petri dish edges, aggregation of worms, and accumulation of dirt (dust spots on lids) during assays, etc. This work presents a protocol for a lifespan assay, with two image-processing pipelines applied to different plate zones, and a new data post-processing method to solve the aforementioned problems. Specifically, certain steps in the culture protocol were taken to alleviate aggregation, occlusions, contamination, and condensation problems. This method is based on an active illumination system and facilitates automated image sequence analysis, does not need human threshold adjustments, and simplifies the techniques required to extract lifespan curves. In addition, two image-processing pipelines, applied to different plate zones, were employed for automated lifespan determination. The first image-processing pipeline was applied to a wall zone and used only pixel level information because worm size or shape features were unavailable in this zone. However, the second image-processing pipeline, applied to the plate centre, fused information at worm and pixel levels. Simple death event detection was used to automatically obtain lifespan curves from the image sequences that were captured once daily throughout the assay. Finally, a new post-processing method was applied to the extracted lifespan curves to filter errors. The experimental results showed that the errors in automated counting of live worms followed the Gaussian distribution with a mean of 2.91% and a standard deviation of +/-12.73% per Petri plate. Post-processing reduced this error to 0.54+/-8.18% per plate. The automated survival curve incurred an error of 4.62+/-2.01%, while the post-process method reduced the lifespan curve error to approximately 2.24+/-0.55%.

Duclairoir Poc C et al. (2011) BMC Res Notes "Caenorhabditis elegans: a model to monitor bacterial air quality."

Duclairoir Poc C, Orange N, Lesouhaitier O, Groboillot A, Feuilloley MJ, Morin JP

[BMC Res Notes, 2011]

UNLABELLED: ABSTRACT: BACKGROUND: Low environmental air quality is a significant cause of mortality and morbidity and this question is now emerging as a main concern of governmental authorities. Airborne pollution results from the combination of chemicals, fine particles, and micro-organisms quantitatively or qualitatively dangerous for health or for the environment. Increasing regulations and limitations for outdoor air quality have been decreed in regards to chemicals and particles contrary to micro-organisms. Indeed, pertinent and reliable tests to evaluate this biohazard are scarce. In this work, our purpose was to evaluate the Caenorhaditis elegans killing test, a model considered as an equivalent to the mouse acute toxicity test in pharmaceutical industry, in order to monitor air bacterial quality. FINDINGS: The present study investigates the bacterial population in dust clouds generated during crop ship loading in harbor installations (Rouen harbor, Normandy, France). With a biocollector, airborne bacteria were impacted onto the surface of agar medium. After incubation, a replicate of the colonies on a fresh agar medium was done using a velvet. All the replicated colonies were pooled creating the "Total Air Sample". Meanwhile, all the colonies on the original plate were isolated. Among which, five representative bacterial strains were chosen. The virulence of these representatives was compared to that of the "Total Air Sample" using the Caenorhaditis elegans killing test. The survival kinetic of nematodes fed with the "Total Air Sample" is consistent with the kinetics obtained using the five different representatives strains. CONCLUSIONS: Bacterial air quality can now be monitored in a one shot test using the Caenorhaditis elegans killing test.

Sulston JE et al. (1977) Worm Breeder's Gazette "mounting techniques for Nomarski microscopy."

McIntosh D, Kimble JE, Sulston JE

[Worm Breeder's Gazette, 1977]

1. New Zeiss immersion oil (brown bottles) is toxic to C. elegans, whereas old oil (containing PCB, white bottles) is innocuous. New oil stops worms pumping immediately upon contact, while the PCB oil does not. L2s mounted on agar surrounded with new oil, fail to produce progeny. In the short term, this may not matter; indeed, the oil repels the worms and helps to stop them escaping. For long-term viability, 18 mm cover slips can be used so that the oil can be kept away from the agar: edges are sealed with Voltalef oil. J.E.K. mounts the worm on a 12 mm slab and fills in the edges with 2% agar. 2. Rapid demounting (a) Brief inspection. Trimming and sealing not necessary. (b) Stable mount. The worm is mounted under a 13 mm circular cover slip; the agar is not trimmed but covered with a square of Saran Wrap containing an 11 mm hole. Seal is good enough to prevent drying overnight. Very little immersion oil is applied to avoid wetting the plastic. Saran Wrap and cover slip can be removed quickly, leaving worm accessible on the agar (watch while lifting cover slip). Useful for:-laser microbeam operations, rapid fixation of transitory states. 3. Invertible mount - Horrible method: Developed for watching lateral cells through flips of later lethargi. 2% celloidin/amyl acetate spread on horizontal microscope slide (~0.1 ml/cm2); air dried under dust cover several days. 60 layer of 1% agarose cast on 22 x 40 mm cover slip (cellulose tape spacers); allowed to dry to ~40 ( edges just receeding); minute drop of buffer added with worm, covered with 10 mm square of the celloidin film, whose centre has been coated with bacteria as usual. Edges allowed to dry; flooded with immersion oil (new will do). Nomarski only slightly degraded through agarose; restraint of worm poor, dehydrates in a few hours. More details from J.E.S.

Douglas, C.D. et al. (2011) International Worm Meeting "The effects of nickel toxicity upon survival, growth, and reproduction in nematodes."

Rudel, D., Ingersoll, C.G., Douglas, C.D., Besser, J.M.

[International Worm Meeting, 2011]

Nickel is one of the most prevalent heavy metal contaminants in the dust floating in the air we breath, in soils, and in many waterways. Contamination continues to increase largely due to human activities like mining, manufacturing, and waste management. Ni2+ is a carcinogen to plants and animals, and a major concern for the health and safety both of humans and food crops. In addition to mutagenesis and cancer, nickel exposure leads to a variety of other deleterious health conditions. In collaboration with the United States Geological Survey, an assay to evaluate the effects of substrate bound nickel and soluble nickel on survival, growth, and fecundity using C. elegans and Pristionchus pacificus has been developed. Synchronized L1 (C. elegans) or J2 (P. pacificus) larvae were added to sediment and water samples in twelve-well tissue culture plates with a defined amount of freshly killed bacteria as a food source. Animals were grown for 96 hours at 20degC and harvested. Harvested animals were assayed for survival, growth, and fertility. Growth in eight sediment-types collected at discrete uncontaminated sites along the Missouri/Mississippi river basin suggests that both nematodes grow well in organic based sediments and less well in more sandy/inorganic based sediments. Growth in sediments spiked with increasing amounts of environmentally relevant levels of sediment-bound nickel showed that C. elegans was highly sensitive to nickel in the environment of the sediment. Treatment was lethal prior to adulthood. Growth in nickel spiked liquid media with freshly killed food and cholesterol, i.e. hard H20 pH 7.5, distilled (DI) H20, SB Media (hard H20), and SB Media (DI H20), showed that C. elegans has a strong tolerance for environmentally relevant levels of soluble nickel. Adult survivors of nickel treatments showed no significant effects on adult length/width measurements or the presence of fertilized eggs in the uterus. As nickel has other known cell stress effects, we will also assay total germline apoptosis as an measure of DNA damage.

Dixon DK et al. (1986) Worm Breeder's Gazette "nuclease hypersensitive sites of the 16 kd heat shock genes of C elegans."

Jones D, Dixon DK, Candido EPM

[Worm Breeder's Gazette, 1986]

Recently our laboratory has cloned and sequenced six hsp16 genes at two chromosomal loci. Regulatory sequences responsible for hsp16 heat induction have also been investigated by cloning the hsp16 genes, and in vitro constructed mutants thereof, into bovine papilloma virus vectors and transfecting the various constructions into mouse C127 cells. By assaying hsp16 heat inducibility in selected C127 clones, it has been shown that C. elegans hsp16 regulatory sequences are correctly recognized in mouse cells and features of these 5' regulatory sequences important for proper heat induction have been delineated. We are now determining if the hsp16 genes in C. elegans share any of the chromatin structural features identified in the heat shock genes of other organisms by examining nuclease hypersensitive sites within the genes and flanking sequences. To facilitate this investigation we have developed a simple and rapid method for preparing large amounts of nuclei from frozen eggs, larvae and adults. These nuclei are essentially free of endogenous nuclease and protease activity and appear to be an excellent substrate for investigating chromatin structure in C. elegans.The starting material consists of viable eggs or worms pelleted and resuspended with one-half the pellet volume of buffer A (250 mM sucrose; 10 mM Tris, pH 8.0; 10 mM MgC12; 1 mM EGTA) and frozen into balls by dripping the slurry slowly into liquid nitrogen. At all stages of the procedure, buffers contain 0.2 mM PMSF and 7 mM mercaptoethanol, added fresh. The frozen balls are stored at -70 C until needed. Worms and larvae should be freed of bacteria by flotation in 30% sucrose, followed by a couple of washes in buffer A prior to freezing. For a typical experiment nuclei are prepared by grinding one to two grams of frozen balls into a fine dust (Espresso grind) using a Waring blender equipped with a small, covered stainless steel cup (30 ml) that has been cooled on dry ice. The grinding usually takes a couple of 15-30 second bursts at the highest speed. Small dry ice chips may be added as a grinding agent and to maintain temperature, but be careful of pressure build up. A chilled mortar and pestle also works, but is less convenient. The worm dust is allowed to warm until it just melts, then 10 ml of ice-cold buffer A is added and the mixture blended at the highest speed for another 15-30 seconds. The homogenate is collected and the blender cup rinsed with two more 10 ml aliquots of buffer A that are pooled with the homogenate. All subsequent steps are carried out at 0-4 C. Nuclei and debris are then pelleted at 4,000 x g for 5 minutes using a swing-out rotor (HB-4). The pellet is resuspended in 10 ml of buffer A containing 0.25% NP-40, plus 0.1% Triton X-100 and homogenized using ten strokes of a Dounce homogenizer with a tight fitting pestle. The debris is removed by a low speed spin (40 x g for 5 minutes) and the supernatant containing the nuclei is carefully aspirated and kept on ice. The debris pellet is reextracted by vortexing at maximum speed for 15-30 seconds in 10 ml of buffer A without detergent. The debris is removed again by a low speed spin and the nuclei pooled. The debris pellet is reextracted one to two more times or until the turbidity of the supernatant drops significantly. The pooled nuclei are pelleted at 4, 000 x g for 5 minutes, resuspended in 10 ml of buffer A and pelleted once again. We routinely obtain the highest yield of nuclei from eggs, with yields in the order of one to two mg of DNA per gram of worms (wet weight). Phase-contrast and fluorescence microscopy (using DAPI and ethidium bromide staining) indicate that the nuclei are clean, with little or no cytoplasmic tags. There is some contamination by shards of cuticle, but this is estimated to be less than 1%. Pelleting the nuclei through 2.5 M sucrose doesn't appear to significantly improve the purity. We have used 18% SDS-PAGE to verify the absence of proteolytic activity during incubations up to 60 minutes at 37 C. Furthermore, all the histones are represented with little or no apparent contamination from ribosomal proteins. Similarly, high percentage agarose gels and Southern blots have confirmed the absence of endogenous nuclease activity and micrococcal nuclease digests reveal a typical nucleosomal banding pattern with no evidence of nucleosome sliding. We are currently using nuclei prepared by this procedure to map nuclease hypersensitive sites within the hsp16 loci from both control and heat shocked eggs and worms. We have been able to obtain data for a variety of nucleases, including DNase I, micrococcal nuclease, S1 and the N. crassa nuclease. These data are beginning to reveal interesting features about chromatin structure and gene regulation in C. elegans and, hopefully, the relative ease with which nuclei may be prepared will encourage other laboratories to undertake similar investigations.

Clark-Maquire SD et al. (1990) Worm Breeder's Gazette "Transformation Rescue of mei-1, a Gene that Affects Meiosis and"

Clark-Maquire SD, Mains PE

[Worm Breeder's Gazette, 1990]

The one-cell C. elegans embryo must support two distinct patterns of cell division. Shortly after fertilization, the embryo undergoes the two meiotic divisions; and about twenty minutes later, the first mitotic cleavage occurs. Essentially the same cytoplasm must support both the meiotic and mitotic spindles, which differ in many aspects, including location, morphology and chromosomal alignment. Clearly, the activities of the gene products that govern the features that distinguish meiosis from mitosis are carefully regulated during this time. We have identified maternal effect mutations in four loci that disrupt either the meiotic or the mitotic divisions. The dominant ts gain-of-function (gf) mutations mei-1(ct46) I and mel-26(ct61) I and recessive loss-of-function (lf) mutations of zyg-9 II show similar defects in the first mitotic division, and these mutations are strong enhancers of one another's defects. If mutations of mei-1 and mei-2 result in recessive meiotic abnormalities but act as dominant suppressors of the mitotic defects of mei-1(ct46gf and mel-26(ct61gf). We are focusing our attention on mei-1, which can be mutated to affect either meiosis or mitosis. The molecular cloning of this gene has been straightforward, since it maps between lin-10 and lin-28, both of which have been located on the physical map. A total of 14 overlapping cosmids (provided by Alan Coulson and John Sulston) span the region, and we have used these to rescue the recessive maternal- effect lethality of mei-1(lf) by germline transformation. The individual cosmids were co-injected with a plasmid containing the dominant rol-6 mutation (described by Craig Mello) into mei-1(lf) /+ hermaphrodites (with appropriate markers in cis), and transgenic lines were established. Segregants homozygous for mei-1 that rolled were then tested for fertility. Since in previous mapping experiments recombinants between mei-1 and either lin-10 or lin-28 were easily obtained, we started testing cosmids in the center of the region and worked our way out. Naturally the rescuing activity was very near one end. Two overlapping cosmids, T01G9 (which also includes lin-10) and F54A4 provide mei-1+ activity. Injecting F54A4 directly into homozygous mei-1 hermaphrodites does not result in even transient rescue. We tested F54A4 cut with various restriction enzymes for rescuing activity in order to identify enzymes that did not cut within the gene, as described by Kim and Horvitz [Genes Devel. 4: 357 (90)]. The SalI digest was positive. Since only one of the four fragments produced by this enzyme was also cut by every enzyme that abolished rescuing activity, we could predict which SalI fragment should rescue. Being dubious about taking the (negative) results of failure to rescue too seriously we injected the other three gel-purified fragments anyway (since we can inject faster than we can analyze). However, only the predicted 10 kb SalI fragment contained mei-1+ activity. We are repeating this strategy with additional enzymes to whittle the clone down to a more convenient size.

Boyles AL et al. (2017) Environ Int "Systematic review of community health impacts of mountaintop removal mining."

Avanasi R, Thayer KA, McComb S, Blain RB, Boyles AL, Goldhaber SB, Holmgren SD, Masten SA, Rochester JR

[Environ Int, 2017]

BACKGROUND: The objective of this evaluation is to understand the human health impacts of mountaintop removal (MTR) mining, the major method of coal mining in and around Central Appalachia. MTR mining impacts the air, water, and soil and raises concerns about potential adverse health effects in neighboring communities; exposures associated with MTR mining include particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs), metals, hydrogen sulfide, and other recognized harmful substances. METHODS: A systematic review was conducted of published studies of MTR mining and community health, occupational studies of MTR mining, and any available animal and in vitro experimental studies investigating the effects of exposures to MTR-mining-related chemical mixtures. Six databases (Embase, PsycINFO, PubMed, Scopus, Toxline, and Web of Science) were searched with customized terms, and no restrictions on publication year or language, through October 27, 2016. The eligibility criteria included all human population studies and animal models of human health, direct and indirect measures of MTR-mining exposure, any health-related effect or change in physiological response, and any study design type. Risk of bias was assessed for observational and experimental studies using an approach developed by the National Toxicology Program (NTP) Office of Health Assessment and Translation (OHAT). To provide context for these health effects, a summary of the exposure literature is included that focuses on describing findings for outdoor air, indoor air, and drinking water. RESULTS: From a literature search capturing 3088 studies, 33 human studies (29 community, four occupational), four experimental studies (two in rat, one in vitro and in mice, one in C. elegans), and 58 MTR mining exposure studies were identified. A number of health findings were reported in observational human studies, including cardiopulmonary effects, mortality, and birth defects. However, concerns for risk of bias were identified, especially with respect to exposure characterization, accounting for confounding variables (such as socioeconomic status), and methods used to assess health outcomes. Typically, exposure was assessed by proximity of residence or hospital to coal mining or production level at the county level. In addition, assessing the consistency of findings was challenging because separate publications likely included overlapping case and comparison groups. For example, 11 studies of mortality were conducted with most reporting higher rates associated with coal mining, but many of these relied on the same national datasets and were unable to consider individual-level contributors to mortality such as poor socioeconomic status or smoking. Two studies of adult rats reported impaired microvascular and cardiac mitochondrial function after intratracheal exposure to PM from MTR-mining sites. Exposures associated with MTR mining included reports of PM levels that sometimes exceeded Environmental Protection Agency (EPA) standards; higher levels of dust, trace metals, hydrogen sulfide gas; and a report of increased public drinking water violations. DISCUSSION: This systematic review could not reach conclusions on community health effects of MTR mining because of the strong potential for bias in the current body of human literature. Improved characterization of exposures by future community health studies and further study of the effects of MTR mining chemical mixtures in experimental models will be critical to determining health risks of MTR mining to communities. Without such work, uncertainty will remain regarding the impact of these practices on the health of the people who breathe the air and drink the water affected by MTR mining.