Development of Adverse Outcome Pathway for PPARγ Antagonism Leading to Pulmonary Fibrosis and Chemical Selection for Its Validation: ToxCast Database and a Deep Learning Artificial Neural Network Model-Based Approach.

Exposure to certain chemicals such as disinfectants through inhalation is suspected to be involved in the development of pulmonary fibrosis, a lung disease in which lung tissue becomes damaged and scarred. Pulmonary fibrosis is known to be regulated by transforming growth factor ß (TGF-ß) and peroxisome proliferator-activated receptor gamma (PPARγ). Here, we developed an adverse outcome pathway (AOP) to better define the linkage of PPARγ antagonism to the adverse outcome of pulmonary fibrosis. We then conducted a systematic analysis to identify potential chemicals involved in this AOP, using the ToxCast database and deep learning artificial neural network models. We identified chemicals bearing a potential inhalation hazard and exposure hazards from the database that could be related to this AOP. For chemicals that were not present in the ToxCast database, multilayer perceptron models were developed based on the ToxCast assays related to the AOP. The reactivity of ToxCast untested chemicals was then predicted using these deep learning models. Both approaches identified a set of chemicals that could be used to validate the AOP. This study suggests that chemicals categorized using an existing database such as ToxCast can be used to validate an AOP and that deep learning approaches can be used to characterize a range of potential active chemicals for an AOP of interest.

Internal Concentration and Time Are Important Modifiers of Toxicity: The Case of Chlorpyrifos on Caenorhabditis elegans.

The internal concentration of chemicals in exposed organisms changes over time due to absorption, distribution, metabolism, and excretion processes since chemicals are taken up from the environment. Internal concentration and time are very important modifiers of toxicity when biomarkers are used to evaluate the potential hazards and risks of environmental pollutants. In this study, the responses of molecular biomarkers, and the fate of chemicals in the body, were comprehensively investigated to determine cause-and-effect relationships over time. Chlorpyrifos (CP) was selected as a model chemical, and Caenorhabditis elegans was exposed to CP for 4 h using the passive dosing method. Worms were then monitored in fresh medium during a 48-h recovery regime. The mRNA expression of genes related to CYP metabolism (cyp35a2 and cyp35a3) increased during the constant exposure phase. The body residue of CP decreased once it reached a peak level during the early stage of exposure, indicating that the initial uptake of CP rapidly induced biotransformation with the synthesis of new CYP metabolic proteins. The residual chlorpyrifos-oxon concentration, an acetylcholinesterase (AChE) inhibitor, continuously increased even after the recovery regime started. These delayed toxicokinetics seem to be important for the extension of AChE inhibition for up to 9 h after the start of the recovery regime. Comprehensive investigation into the molecular initiation events and changes in the internal concentrations of chemical species provide insight into response causality within the framework of an adverse outcome pathway.

Changes in the expression of cyp35a family genes in the soil nematode Caenorhabditis elegans under controlled exposure to chlorpyrifos using passive dosing.

In order to use sensitive molecular-level biomarkers for the evaluation of environmental risks, it is necessary to establish a quantitative dose-response relationship. Passive dosing is regarded as a promising new technique for maintaining a constant exposure condition of hydrophobic chemicals in the assay medium. The main goals of the present study were (1) to quantitatively compare gene expression results obtained using the passive dosing method and the conventional spiking method and (2) to investigate changes in gene expression with respect to the free concentration and exposure duration using passive dosing. Chlorpyrifos (CP), which is oxidized by the cytochrome P450 monooxygenases, was selected as a model chemical, and the expression of cytochrome P450 subfamily protein 35A gene series (cyp-35a1-5) was analyzed by quantitative real-time PCR on soil nematode Caenorhabditis elegans. Whereas the free concentration of CP rapidly decreased and the expression of cyp genes varied with the volume of exposure medium and the test duration when the spiking method was used, the free concentration in the assay medium was stable throughout the experiment when the passive dosing method was used. In addition, the level of gene expression increased with exposure time up to 8 h and with increasing CP concentration. The observed increased gene expression could be explained by increasing body residue concentration of CP with exposure time. In conclusion, quantitative dose-response relationships for gene expression biomarkers could be obtained for highly hydrophobic chemicals when the constant exposure condition is provided and the free concentration is used as the dose-metric.

Prediction of ecotoxicity of heavy crude oil: contribution of measured components.

A prediction model for estimating the ecotoxicity of the water-accommodated fraction (WAF) and water-soluble fraction (WSF) of heavy crude oil is proposed. Iranian heavy crude oil (IHC), one of the major components of the Hebei Spirit oil spill in Korea in 2007, was used as a model crude oil for the preparation of the WAF and the WSF. Luminescence inhibition of Vibrio fischeri was chosen as the model ecotoxicity test for evaluating the baseline toxicity of aromatic hydrocarbons in the IHC. The measured concentration of each chemical species in WAF and WSF agreed well with the predicted soluble concentration calculated using Raoult's law from the measured amount in the IHC. This indicates that the toxic potential of an oil mixture can be evaluated from the dissolved concentration of each species, which in turn, may be predicted from the composition of the crude or weathered oils. In addition, the contribution of each species in the mixture to the apparent luminescence inhibition by the WAF and the WSF was assessed using a concentration-addition model. The relative contributions of benzene, toluene, ethylbenzene, xylenes (BTEX), polycyclic aromatic hydrocarbons (PAHs), and alkylated PAHs in luminescence inhibition were estimated to be 76%, 2%, and 21%, respectively. It was further identified that C3- and C4-naphthalenes were the most important aromatic hydrocarbons responsible for baseline toxicity. This indicates that alkylated PAHs would be the major components of oil-spill residue. Further research is needed to evaluate the fate and ecotoxicity of alkylated PAHs.

Ecotoxicological evaluation of chlorpyrifos exposure on the nematode Caenorhabditis elegans.

To investigate the effects of chlorpyrifos (CP), an organophosphorus insecticide, on the soil nematode Caenorhabditis elegans, the toxicity of the insecticide on the molecular, biochemical, and physiological levels were investigated upon sublethal exposure, and an acute toxicity test was conducted using lethality as an endpoint. To assess the molecular-level effect, stress-related gene expression was investigated, and the neurotoxicity indicator, acetylcholinesterase (AChE) activity was assessed as the biochemical-level response. Growth, reproduction and development were also studied as physiological-level responses. The overall results indicate that CP exposure leads to the alteration of the expression of some stress genes, such as of heat shock protein, metallothionein, vitellogenin and C. elegans p53-like protein genes; the inhibition of AChE activity; and the retardation of development. These data suggest that the toxicity of CP on C. elegans occurred in multiple biological organizations; nevertheless this is not sufficient to conclude that there is a casual relationship between them. Thus, direct experimental demonstrations of the wider relationships between the molecular/biochemical effects of CP exposure and their consequences at higher levels of biological organization are needed to fully understand the effects of this compound on C. elegans.

Comparative study of oxidative stress caused by anthracene and alkyl-anthracenes in Caenorhabditis elegans.

Oxidative stress was evaluated for anthracene (Ant) and alkyl-Ants (9-methylanthracene [9-MA] and 9,10-dimethylanthracene [9,10-DMA]) in Caenorhabditis elegans to compare changes in toxicity due to the degree of alkylation. Worms were exposed at 1) the same external exposure concentration and 2) the maximum water-soluble concentration. Formation of reactive oxygen species, superoxide dismutase activity, total glutathione concentration, and lipid peroxidation were determined under constant exposure conditions using passive dosing. The expression of oxidative stress-related genes (daf-2, sir-2.1, daf-16, sod-1, sod-2, sod-3 and cytochrome 35A/C family genes) was also investigated to identify and compare changes in the genetic responses of C. elegans exposed to Ant and alkyl-Ant. At the same external concentration, 9,10-DMA induced the greatest oxidative stress, as evidenced by all indicators, except for lipid peroxidation, followed by 9-MA and Ant. Interestingly, 9,10-DMA led to greater oxidative stress than 9-MA and Ant when worms were exposed to the maximum water-soluble concentration, although the maximum water-soluble concentration of 9,10-DMA is the lowest. Increased oxidative stress by alkyl-Ants would be attributed to higher lipid-water partition coefficient and the π electron density in aromatic rings by alkyl substitution, although this supposition requires further confirmation.

Successful Stenting for Bronchial Stenosis Resulting from Blunt Airway Trauma.

Blunt airway trauma is rare but life threatening. Injuries to other vital organs accompany this type of injury in most cases; therefore, conservative treatment may be considered first. In cases of delayed fibrotic airway stenosis after conservative treatment, surgical treatment or bronchoscopic intervention are therapeutic options. We herein report a case of delayed airway stenosis after a blunt traumatic airway injury that was successfully managed by silicone stenting.

Histone methylation-associated transgenerational inheritance of reproductive defects in Caenorhabditis elegans exposed to crude oil under various exposure scenarios.

As part of a study to explore the long-term effects of the Hebei Spirit oil spill accident, transgenerational toxicity and associated epigenetic changes were investigated in the nematode Caenorhabditis elegans. Under experimental conditions, worms were exposed to Iranian heavy crude oil (IHC) under three different scenarios: partial early-life exposure (PE), partial late-life exposure (PL), and whole-life exposure (WE). Growth, reproduction, and histone methylation were monitored in the exposed parental worms (P0) and in three consecutive unexposed offspring generations (F1-3). Reproductive potential in the exposed P0 generation in the WE treatment group was reduced; additionally, it was inhibited in the unexposed offspring generations of the P0 worms. This suggests that there was transgenerational inheritance of defective reproduction. Comparison of developmental periods of exposure showed that IHC-treated worms in the PL group had a greater reduction in reproductive capacity than those in the PE group. Decreased methylation of histone H3 (H3K9) was found in the IHC-exposed parental generation. A heritable reduction in reproductive capacity occurred in wildtype N2 but was not found in a H3K9 histone methyltransferase (HMT) mutant, met-2(n4256), suggesting a potential role for HMT in transgenerational toxicity. Our results suggest that the reproductive toxicity after IHC exposure could be heritable and that histone methylation is associated with the transmission of the inherited phenotype.

Toxic effects of di(2-ethylhexyl)phthalate on mortality, growth, reproduction and stress-related gene expression in the soil nematode Caenorhabditis elegans.

In this study, di(2-ethylhexyl)phthalate (DEHP) toxicities to Caenorhabditis elegans were investigated using multiple toxic endpoints, such as mortality, growth, reproduction and stress-related gene expression, focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP monitoring. The possible use of C. elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conducting the experiment on the exposure of nematode to field soil. Twenty-four-hour median lethal concentration (LC50) data suggest that DEHP has a relatively high potential of acute toxicity to C. elegans. Decreases in body length and egg number per worm observed after 24h of DEHP exposure may induce long-term alteration in the growth and reproduction of the nematode population. Based on the result from the C. elegans genome array and indicated in the literatures, stress proteins, metallothionein, vitellogenin, xenobiotic metabolism enzymes, apoptosis-related proteins, and antioxidant enzyme genes were selected as stress-related genes and their expression in C. elegans by DEHP exposure was analyzed semi-quantitatively. Expression of heat shock protein (hsp)-16.1 and hsp-16.2 genes was decreased by DEHP exposure. Expression of cytochrome P450 (cyp) 35a2 and glutathione-S-transferease (gst)-4, phase I and phase II of xenobiotic metabolism enzymes, was increased by DEHP exposure in a concentration-dependent manner. An increase in stress-related gene expressions occurred concomitantly with the deterioration on the physiological level, which suggests an increase in expression of those genes may not be considered as a homeostatic response but as a toxicity that might have physiological consequences. The experiment with the soil from the landfill site suggests that the potential of the C. elegans biomarker identified in laboratory conditions should be calibrated and validated for its use in situ.

Effects of soil water saturation on sampling equilibrium and kinetics of selected polycyclic aromatic hydrocarbons.

Passive sampling can be applied for measuring the freely dissolved concentration of hydrophobic organic chemicals (HOCs) in soil pore water. When using passive samplers under field conditions, however, there are factors that might affect passive sampling equilibrium and kinetics, such as soil water saturation. To determine the effects of soil water saturation on passive sampling, the equilibrium and kinetics of passive sampling were evaluated by observing changes in the distribution coefficient between sampler and soil (Ksampler/soil) and the uptake rate constant (ku) at various soil water saturations. Polydimethylsiloxane (PDMS) passive samplers were deployed into artificial soils spiked with seven selected polycyclic aromatic hydrocarbons (PAHs). In dry soil (0% water saturation), both Ksampler/soil and ku values were much lower than those in wet soils likely due to the contribution of adsorption of PAHs onto soil mineral surfaces and the conformational changes in soil organic matter. For high molecular weight PAHs (chrysene, benzo[a]pyrene, and dibenzo[a,h]anthracene), both Ksampler/soil and ku values increased with increasing soil water saturation, whereas they decreased with increasing soil water saturation for low molecular weight PAHs (phenanthrene, anthracene, fluoranthene, and pyrene). Changes in the sorption capacity of soil organic matter with soil water content would be the main cause of the changes in passive sampling equilibrium. Henry's law constant could explain the different behaviors in uptake kinetics of the selected PAHs. The results of this study would be helpful when passive samplers are deployed under various soil water saturations.

Assessment of stress-related gene expression in the heavy metal-exposed nematode Caenorhabditis elegans: a potential biomarker for metal-induced toxicity monitoring and environmental risk assessment.

The toxicity of cadmium, lead, chromium, and arsenite on Caenorhabditis elegans was investigated to identify sensitive biomarkers for environmental monitoring and risk assessment. Effects of these metals on stress-related gene expression, growth, reproduction, and mortality of C. elegans were investigated under laboratory conditions. The possibility of using C. elegans as a biosensor for environmental toxicity monitoring was also tested using a green fluorescent protein transgenic nematode. The 24-h median lethal concentrations of cadmium, lead, chromium, and arsenite in C. elegans were 846, 34, 115, and 92 mg/L, respectively. Cadmium exposure led to an increase in the expression of most of the genes tested. The degree of increase was more than threefold compared to control in heat shock protein 16.2, heat shock protein 70, metallothionein 2, cytochrome P450 family protein 35A2, glutathione-S-transferase 4, superoxide dismutase 1, catalase 2, C. elegans p53-like protein 1, and apoptosis enhancer 1 genes. The lead-, chromium-, and arsenite-exposed nematode, on the other hand, showed little change in gene expression. Alterations in growth and reproduction were observed in cadmium- and chromium-exposed worms. To consider a transgenic nematode as a biosensor for toxicity monitoring, the responses of stress-related gene promoters need to be tested with a variety of metals. The overall results suggest that cadmium exhibits a high level of tolerance compared to the other metals tested. Use of the responses of stress-related gene expression therefore has considerable potential as a sensitive biomarker for the diagnosis of cadmium contamination, and C. elegans seems to be a good biological model for this approach.

A systems toxicology approach on the mechanism of uptake and toxicity of MWCNT in Caenorhabditis elegans.

The increased volumes of carbon nanotubes (CNTs) being utilized in industrial and biomedical processes carries with it an increased risk of unintentional release into the environment, requiring a thorough hazard and risk assessment. In this study, the toxicity of pristine and hydroxylated (OH-) multiwall CNTs (MWCNTs) was investigated in the nematode Caenorhabditis elegans using an integrated systems toxicology approach. To gain an insight into the toxic mechanism of MWCNTs, microarray and proteomics were conducted for C. elegans followed by pathway analyses. The results of pathway analyses suggested endocytosis, phagocytosis, oxidative stress and endoplasmic reticulum (ER) stress, as potential mechanisms of uptake and toxicity, which were subsequently investigated using loss-of-function mutants of genes of those pathways. The expression of phagocytosis related genes (i.e. ced-10 and rab-7) were significantly increased upon exposure to OH-MWCNT, concomitantly with the rescued toxicity by loss-of-function mutants of those genes, such as ced-10(n3246) and rab-7(ok511). An increased sensitivity of the hsp-4(gk514) mutant by OH-MWCNT, along with a decreased expression of hsp-4 at both gene and protein level suggests that MWCNTs may affect ER stress response in C. elegans. Collectively, the results implied phagocytosis to be a potential mechanism of uptake of MWCNTs, and ER and oxidative stress as potential mechanisms of toxicity. The integrated systems toxicology approach applied in this study provided a comprehensive insight into the toxic mechanism of MWCNTs in C. elegans, which may eventually be used to develop an "Adverse Outcome Pathway (AOP)", a recently introduced concept as a conceptual framework to link molecular level responses to higher level effects.

Ecological risk assessment of chemicals migrated from a recycled plastic product.

OBJECTIVES: Potential environmental risks caused by chemicals that could be released from a recycled plastic product were assessed using a screening risk assessment procedure for chemicals in recycled products. METHODS: Plastic slope protection blocks manufactured from recycled plastics were chosen as model recycled products. Ecological risks caused by four model chemicals -di-(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), cadmium (Cd), and lead (Pb)- were assessed. Two exposure models were built for soil below the block and a hypothetic stream receiving runoff water. Based on the predicted no-effect concentrations for the selected chemicals and exposure scenarios, the allowable leaching rates from and the allowable contents in the recycled plastic blocks were also derived. RESULTS: Environmental risks posed by slope protection blocks were much higher in the soil compartment than in the hypothetic stream. The allowable concentrations in leachate were 1.0×10(-4), 1.2×10(-5), 9.5×10(-3), and 5.3×10(-3) mg/L for DEHP, DINP, Cd, and Pb, respectively. The allowable contents in the recycled products were 5.2×10(-3), 6.0×10(-4), 5.0×10(-1), and 2.7×10(-1) mg/kg for DEHP, DINP, Cd, and Pb, respectively. CONCLUSIONS: A systematic ecological risk assessment approach for slope protection blocks would be useful for regulatory decisions for setting the allowable emission rates of chemical contaminants, although the method needs refinement.

Involvement of Caenohabditis elegans MAPK Signaling Pathways in Oxidative Stress Response Induced by Silver Nanoparticles Exposure.

In the present study, toxicity of silver nanoparticles (AgNPs) was investigated in the nematode, Caenohabditis elegans focusing on the upstream signaling pathway responsible for regulating oxidative stress, such as mitogen-activated protein kinase (MAPK) cascades. Formation of reactive oxygen species (ROS) was observed in AgNPs exposed C.elegans, suggesting oxidative stress as an important mechanism in the toxicity of AgNPs towards C. elegans. Expression of genes in MAPK signaling pathways increased by AgNPs exposure in less than 2-fold compared to the control in wildtype C.elegans, however, those were increased dramatically in sod-3 (gk235) mutant after 48 h exposure of AgNPs (i.e. 4-fold for jnk-1 and mpk-2; 6-fold for nsy-1, sek-1, and pmk-1, and 10-fold for jkk-1). These results on the expression of oxidative stress response genes suggest that sod-3 gene expression appears to be dependent on p38 MAPK activation. The high expressions of the pmk-1 gene 48 h exposure to AgNPs in the sod-3 (gk235) mutant can also be interpreted as compensatory mechanisms in the absence of important stress response genes. Overall results suggest that MAPK-based integrated stress signaling network seems to be involved in defense to AgNPs exposure in C.elegans.

Oxidative stress-related PMK-1 P38 MAPK activation as a mechanism for toxicity of silver nanoparticles to reproduction in the nematode Caenorhabditis elegans.

In the present study, a toxic mechanism of silver nanoparticles (AgNPs) was investigated in the nematode, Caenorhabditis elegans, focusing on the involvement of oxidative stress in reproduction toxicity. Initially, AgNPs were tested as potential oxidative stress inducers, and increased formation of reactive oxygen species (ROS) was observed in AgNP-exposed C. elegans. Subsequently, the potential upstream signaling pathway activated in response to AgNP exposure was investigated, paying special attention to the C. elegans PMK-1 p38 mitogen-activated protein kinase (MAPK). Increased PMK-1 p38 MAPK gene and protein expressions were observed in C. elegans exposed to AgNPs. Expression of the p38-dependent transcription factor genes and glutathione S-transferase (GST) enzyme activity was also investigated in wildtype (N2) and pmk-1 mutant (km25) C. elegans exposed to AgNPs. The results indicated that AgNP exposure led to increased ROS formation, increased expression of PMK-1 p38 MAPK and hypoxia-inducible factor (HIF-1), GST enzyme activity, and decreased reproductive potential in wildtype (N2) C. elegans; whereas in the AgNP-exposed pmk-1 (km25) mutant, ROS formation and HIF-1 and GST activation were not observed, and decreased reproductive potential was rescued. These results suggest that oxidative stress is an important mechanism of AgNP-induced reproduction toxicity in C. elegans, and that PMK-1 p38 MAPK plays an important role in it. The results also suggest that GST and HIF-1 activation by AgNP exposure are PMK-1 p38 MAPK-dependent, and that both play an important role in the PMK-1 p38 MAPK-mediated defense pathway to AgNP exposure in C. elegans.

Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans.

In this study, the effect of organophosphorous (OP) pesticide, fenitrothion (FT), on the non-target organism was investigated using the soil nematode, Caenorhabditis elegans. Toxicity was investigated on multiple biological levels, from organism to molecular levels, such as, immoblity, growth, fertility, development, acetyl cholinesterase (AChE) activity and stress-response gene expressions. FT may provoke serious consequences on the C. elegans population, as it induced significant developmental disturbance. As expected, FT exposure inhibits AChE activity of C. elegans. The increased expression of the cytochrome p450 family protein 35A2 (cyp35a2) gene was also observed in FT exposed worms. To experimentally demonstrate the relationships between organism-level effects and the cyp35a2 gene expression in FT-exposed C. elegans, the integration of the gene expression with biochemical-, and organism level endpoints were attempted using a C. elegans cyp35a2 RNA interference (RNAi) and cyp35a2 mutant (gk317). The 24 h-EC50s of C. elegans on FT exposure were in the order of cyp35a2 RNAi in cyp35a2 mutant (gk317)>cyp35a2 mutant (gk317)>cyp35a2 RNAi in wildtype (N2)>wildtype (N2). The higher EC50 values of cyp35a2 RNAi and cyp35a2 mutant (gk317) compared to that of wildtype C. elegans strongly supported that cyp35a2 gene plays an important role in the toxicity of FT towards C. elegans. The experiments with cyp35a2 RNAi also indicated that the development disturbance and decreased AChE activity, which were observed in FT exposed wildtype C. elegans were significantly rescued in the cyp35a2 RNAi C. elegans. Overall results suggest that the cyp35a2 may be an important gene for exerting FT toxicity in C. elegans.

Maintaining the Constant Exposure Condition for an Acute Caenorhabditis elegans Mortality Test Using Passive Dosing.

OBJECTIVES: Maintaining the constant exposure to hydrophobic organic compouds in acute toxicity tests is one of the most difficult issues in the evaluation of their toxicity and corresponding risks. Passive dosing is an emerging tool to keep constant aqueous concentration because of the overwhelming mass loaded in the dosing phase. The primary objectives of this study were to develop the constant exposure condition for an acute mortality test and to compare the performance of the passive dosing method with the conventional spiking with co-solvent. METHODS: A custom cut polydimethylsiloxane (PDMS) tubing loaded with benzyl butyl phthalate (BBP) was placed in each well of a 24-well plate containing assay medium. The rate of the release of BBP from PDMS was evaluated by measuring the change in the concentration of BBP in the assay medium. The efficiency of maintaining constant exposure condition was also evaluated using a simple two-compartment mass transport model employing a film-diffusion theory. An acute mortality test using 10 C. elegans in each well was conducted for the evaluation of the validity of passive dosing and the comparative evaluation of the passive dosing method and the conventional spiking method. RESULTS: Free concentration in the assay medium reached 95% steady state value within 2.2 hours without test organisms, indicating that this passive dosing method is useful for an acute toxicity test in 24 hours. The measured concentration after the mortality test agreed well with the estimated values from partitioning between PDMS and the assay medium. However, the difference between the nominal and the free concentration became larger as the spiked concentration approached water solubility, indicating the instability of the conventional spiking with a co-solvent. CONCLUSIONS: The results in this study support that passive dosing provides a stable exposure condition for an acute toxicity test. Thus, it is likely that more reliable toxicity assessment can be made for hydrophobic chemicals using passive dosing.

Ecotoxicological investigation of CeO(2) and TiO(2) nanoparticles on the soil nematode Caenorhabditis elegans using gene expression, growth, fertility, and survival as endpoints.

In this study, the potential harmful effect of cerium dioxide (CeO(2)), and titanium dioxide (TiO(2)) nanoparticles on the environment was investigated using Caenorhabditis elegans ecotoxicity tests. Multiple toxic endpoints, such as stress-response gene expression, growth, fertility, and survival, were analyzed in C. elegans, in response to the CeO(2) and TiO(2) exposure. To investigate relationship between sizes of nanoparticles and toxicity, C. elegans were exposed to nanoparticles to the different sizes of nanoparticles (15, 45nm for CeO(2) and 7, 20nm for TiO(2)). An increase in the expression of the cyp35a2 gene, decrease in fertility and survival parameters were observed in the 15 and 45nm of CeO(2) and in the 7nm of TiO(2) nanoparticles exposed to C. elegans. Gene knock-down experiment using RNA interference (RNAi) suggested that physiological level disturbances may be related with the cyp35a2 gene expression. Smaller sized nanoparticles (7nm of TiO(2) and 15nm of CeO(2)) seemed to be more toxic than larger sized ones (20nm of TiO(2) and 45nm of CeO(2)) on the observed toxicity. The size-dependent effect in CeO(2) and TiO(2) nanoparticles-induced toxicity needs to be investigated under more detailed experimental settings with the various sizes of nanoparticles. Further studies on the mechanism by which CeO(2) and TiO(2) nanoparticles affect cyp35a2 gene expression, fertility, and survival are warranted to better understand the CeO(2) and TiO(2) nanoparticles-induced ecotoxicity in C. elegans, as are studies with the causal relationships between these parameters. Overall results suggest that CeO(2) and TiO(2) nanoparticles have a potential for provoking ecotoxicity on C. elegans and the data obtained from this study can comprise a contribution to knowledge of the ecotoxicology of nanoparticles in C. elegans, about which little data are available.

A cadmium toxicity assay using stress responsive Caenorhabditis elegans mutant strains.

To test the applicability of Caenorhabditis elegans mutant for toxicity screening, the sensitivity of cadmium (Cd) in C. elegans was investigated on 14 mutant strains using median lethal concentration (LC50) tests, with further analysis on growth and reproduction conducted on five selected strains. The 24h LC50 of Cd observed on the wildtype and mutant strains of C. elegans was in the order of age-1(hx546)>mtl-2(gk125)>sod-3(gk235)>daf-21(p673)>cyp35a2(gk317)>skn-1(or13)>daf-12(rh62rh157)>hsp-16.2(gk249)>daf-18(e1375)>ctl-2(ok1137)>wildtype(N2)>sod-1(or13)>daf-16(mu86)>cep-1(gk138)>cdr-2(ok1996). Compared to the wildtype response, a decreased reproduction potential was observed in mtl-2(gk125), sod-3(gk235), cdr-2(ok1996) and cep-1(gk138) strains. To gain a mechanistic understanding of different sensitivities of the mutant strains, a time-course gene expression analysis was also performed on the five genes. A dramatic increase in the expression of the mtl-2 gene due to Cd exposure confirmed the importance of this gene in C. elegans Cd toxicity. An increased expression of the sod-3 gene at the longer exposure time period (48h) suggests that oxidative stress may not be a direct toxic mechanism, but may rather be a consequence of Cd toxicity. Even though, LC50 values for the age-1(hx546) mutant strain were the highest among the tested strains, the response on the reproduction potential in age-1(hx546) mutant was unchanged compared to the wildtype, and the age-1 gene expression remained unaltered on exposure to Cd, which may be interpreted as the maintenance of age-1 expression level is needed for the exertion of Cd toxicity; however, the role of the age-1 gene in Cd toxicity may not be via a reproduction-related pathway. The overall results suggest that the C. elegans mutant assay seems to be a promising tool for the study of toxic mechanisms, as well as for toxicity screening in ecotoxicological research.

Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics.

In the present study, the ecotoxicity of silver nanoparticles (AgNPs) was investigated in Caenorhabditis elegans using survival, growth, and reproduction, as the ecotoxicological endpoints, as well as stress response gene expression. Whole genome microarray was used to screen global changes in C. elegans transcription profiles after AgNPs exposure, followed by quantitative analysis of selected genes. The integration of gene expression with organism and population level endpoints was investigated using C. elegans functional genomics tools, to test the ecotoxicological relevance of AgNPs-induced gene expression. AgNPs exerted considerable toxicity in C. elegans, most clearly as dramatically decreased reproduction potential. Increased expression of the superoxide dismutases-3 (sod-3) and abnormal dauer formation protein (daf-12) genes with 0.1 and 0.5 mg/L of AgNPs exposures occurred concurrently with significant decreases in reproduction ability. Overall results of functional genomic studies using mutant analyses suggested that the sod-3 and daf-12 gene expressions may have been related to the AgNPs-induced reproductive failure in C. elegans and that oxidative stress may have been an important mechanism in AgNPs toxicity.