Empirical Characterization of False Discovery Rates of Differentially Expressed Genes and Transcriptomic Benchmark Concentrations in Zebrafish Embryos.

High-throughput transcriptomics (HTTr) is increasingly applied to zebrafish embryos to survey the toxicological effects of environmental chemicals. Before the adoption of this approach in regulatory testing, it is essential to characterize background noise in order to guide experimental designs. We thus empirically quantified the HTTr false discovery rate (FDR) across different embryo pool sizes, sample sizes, and concentration groups for toxicology studies. We exposed zebrafish embryos to 0.1% dimethyl sulfoxide (DMSO) for 5 days. Pools of 1, 5, 10, and 20 embryos were created (n = 24 samples for each pool size). Samples were sequenced on the TempO-Seq platform and then randomly assigned to mock treatment groups before differentially expressed gene (DEG), pathway, and benchmark concentration (BMC) analyses. Given that all samples were treated with DMSO, any significant DEGs, pathways, or BMCs are false positives. As expected, we found decreasing FDRs for DEG and pathway analyses with increasing pool and sample sizes. Similarly, FDRs for BMC analyses decreased with increasing pool size and concentration groups, with more stringent BMC premodel filtering reducing BMC FDRs. Our study provides foundational data for determining appropriate experiment designs for regulatory toxicity testing with HTTr in zebrafish embryos.

The use of insect cell line Sf21 for ecotoxicity testing.

Insect cell lines are finding utility in many areas of biology, but their application as an in vitro tool for ecotoxicity testing has been given less attention. Our study aimed to demonstrate the utility and sensitivity of Sf21 cells to commonly used fungicides: Propiconazole and CuSO4, as well as dimethyl sulphoxide (DMSO) an industrial solvent. Sf21 cells were readily cultured from frozen stocks in 3-4 days and showed utility as an invertebrate in vitro acute toxicity test. The data showed the threshold levels of cell survivability against propiconazole and CuSO4. The EC50 values were 135.1 µM and 3.31 mM respectively. The LOAEL (lowest observed adverse effect level) was ≈ 1 µM for propiconazole and ≈ 10 µM for CuSO4. Culturing of Sf21 cells in media containing the solvent DMSO showed that 0.5% DMSO concentration did not effect cell viability. Sf21 cells are sensitive and useful as a robust ecologically relevant screening tool for acute toxicity testing.

Acute Toxicity Assays with the Artemia salina Model: Assessment of Variables.

The use of the brine shrimp Artemia salina (Leach) in acute toxicity assays has great potential due to its simplicity, low cost and reproducibility. In the current study, some of the variables that can influence the reliability of the assay in terms of test organism survival, were evaluated as part of its implementation in our laboratory. The quality and type of water used, the buffer components and other parameters (salinity, pH and dissolved oxygen level), were all evaluated for optimisation purposes. DMSO (dimethyl sulphoxide) was used as the test substance in the toxicity assay, to evaluate the concentration limits as a solvent in sample preparation. Regarding the buffer salinity, pH and dissolved oxygen level, we found that a 25% to 30% deviation from the standard values did not affect the survival of the nauplii (the first-instar larval stage) under assay conditions. In summary, we corroborate the potential use of this model for the prediction of the toxic potential of substances, to inform future testing strategies.

Morphological and Neurological Impairments Induced by Chronic Administration of Dimethyl Sulfoxide in Mice.

We studied the influence of DMSO administered ad libitum with drinking water in concentrations of 0.01, 0.1, and 1% for 4 and 6 weeks on pain sensitivity, motor coordination, and myelin content in the corpus callosum of C57BL/6 mice. After 6-week administration, DMSO in all studied concentrations decreased myelin content in the corpus callosum. Moreover, 4-week administration of 0.1% DMSO and 6-week administration of 1% DMSO increased the latency to fall in the rotarod test by 3.1 (p<0.05) and 5.1 (p<0.001) times, respectively. After 4-week administration of DMSO in concentrations of 0.01 and 0.1%, the latency of the tail flick response increased by 2.1 (p<0.05) and 1.8 times (p<0.001), respectively. Administration of DMSO in concentrations of 0.01 and 1% for 6 weeks led to a decrease of this parameter by 2.7 (p<0.05) and 3.8 times (p<0.01), respectively. Thus, DMSO in all studied concentrations decreased myelin content in the corpus callosum of C57BL/6 mice and modified motor coordination and pain sensitivity of animals.

Seasonal changes in NRF2 antioxidant pathway regulates winter depression-like behavior.

Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

Comparison of cryoprotectants in hematopoietic cell infusion-related adverse events.

BACKGROUND: The standard cryoprotectant for human cellular products is dimethyl sulfoxide (DMSO), which is associated with hematopoietic cell infusion-related adverse events (HCI-AEs) in hematopoietic stem cell transplantation including peripheral blood stem cell (PBSC) transplantation (PBSCT). DMSO is often used with hydroxyethyl starch (HES), which reduces DMSO concentration while maintaining the postthaw cell recovery. The cryoprotectant medium CP-1 (Kyokuto Pharmaceutical Industrial) is widely used in Japan. After mixture of a product with CP-1, DMSO and HES concentrations are 5% and 6%, respectively. However, the safety profile of CP-1 in association with HCI-AEs has not been investigated. STUDY DESIGN AND METHODS: To compare CP-1 with other cryoprotectants, we conducted a subgroup analysis of PBSCT recipients in a prospective surveillance study for HCI-AEs. Moreover, we validated the toxicity of CP-1 in 90 rats following various dose administration. RESULTS: The PBSC products cryopreserved with CP-1 (CP-1 group) and those with other cryoprotectants, mainly 10% DMSO (non-CP-1 group), were infused into 418 and 58 recipients, respectively. The rate of ≥grade 2 HCI-AEs was higher in the CP-1 group, but that of overall or ≥grade 3 HCI-AEs was not significantly different, compared to the non-CP-1 group. Similarly, after propensity score matching, ≥grade 2 HCI-AEs were more frequent in the CP-1 group, but the ≥grade 3 HCI-AE rate did not differ significantly between the groups. No significant toxicity was detected regardless of the CP-1 dose in the 90 rats. CONCLUSIONS: Infusion of a CP-1-containing PBSC product is feasible with the respect of HCI-AEs.

hMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect.

In this study a combined analysis of osmotic injury and cytotoxic effect useful for the optimization of the cryopreservation process of a cell suspension is carried out. The case of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) in contact with dimethyl sulfoxide (DMSO) acting as Cryo-Protectant Agent (CPA) is investigated from the experimental as well as the theoretical perspective. The experimental runs are conducted by suspending the cells in hypertonic solutions of DMSO at varying osmolality, system temperature, and contact times; then, at room temperature, cells are pelleted by centrifugation and suspended back to isotonic conditions. Eventually, cell count and viability are measured by means of a Coulter counter and flow-cytometer, respectively. Overall, a decrease in cell count and viability results when DMSO concentration, temperature, and contact time increase. A novel mathematical model is developed and proposed to interpret measured data by dividing the cell population between viable and nonviable cells. The decrease of cell count is ascribed exclusively to the osmotic injury caused by expansion lysis: excessive swelling causes the burst of both viable as well as nonviable cells. On the other hand, the reduction of cell viability is ascribed only to cytotoxicity which gradually transforms viable cells into nonviable ones. A chemical reaction engineering approach is adopted to describe the dynamics of both phenomena: by following the kinetics of two chemical reactions during cell osmosis inside a closed system it is shown that the simultaneous reduction of cell count and viability may be successfully interpreted. The use of the Surface Area Regulation (SAR) model recently proposed by the authors allows one to avoid the setting in advance of fixed cell Osmotic Tolerance Limits (OTLs), as traditionally done in cryopreservation literature to circumvent the mathematical simulation of osmotic injury. Comparisons between experimental data and theoretical simulations are provided: first, a nonlinear regression analysis is performed to evaluate unknown model parameters through a best-fitting procedure carried out in a sequential fashion; then, the proposed model is validated by full predictions of system behavior measured at operating conditions different from those used during the best-fit procedure.

DMSO-free highly differentiated HepaRG spheroids for chronic toxicity, liver functions and genotoxicity studies.

The liver is essential in the elimination of environmental and food contaminants. Given the interspecies differences between rodents and humans, the development of relevant in vitro human models is crucial to investigate liver functions and toxicity in cells that better reflect pathophysiological processes. Classically, the differentiation of the hepatic HepaRG cell line requires high concentration of dimethyl sulfoxide (DMSO), which restricts its usefulness for drug-metabolism studies. Herein, we describe undifferentiated HepaRG cells embedded in a collagen matrix in DMSO-free conditions that rapidly organize into polarized hollow spheroids of differentiated hepatocyte-like cells (Hepoid-HepaRG). Our conditions allow concomitant proliferation with high levels of liver-specific functions and xenobiotic metabolism enzymes expression and activities after a few days of culture and for at least 4 weeks. By studying the toxicity of well-known injury-inducing drugs by treating cells with 1- to 100-fold of their plasmatic concentrations, we showed appropriate responses and demonstrate the sensitivity to drugs known to induce various degrees of liver injury. Our results also demonstrated that the model is well suited to estimate cholestasis and steatosis effects of drugs following chronic treatment. Additionally, DNA alterations caused by four genotoxic compounds (Aflatoxin B1 (AFB1), Benzo[a]Pyrene (B[a]P), Cyclophosphamide (CPA) and Methyl methanesulfonate (MMS)) were quantified in a dose-dependent manner by the comet and micronucleus assays. Their genotoxic effects were significantly increased after either an acute 24 h treatment (AFB1: 1.5-6 µM, CPA: 2.5-10 µM, B[a]P: 12.5-50 µM, MMS: 90-450 µM) or after a 14-day treatment at much lower concentrations (AFB1: 0.05-0.2 µM, CPA: 0.125-0.5 µM, B[a]P: 0.125-0.5 µM) representative to human exposure. Altogether, the DMSO-free 3D culture of Hepoid-HepaRG provides highly differentiated and proliferating cells relevant for various toxicological in vitro assays, especially for drug-preclinical studies and environmental chemicals risk assessment.

Dimethyl sulfoxide for cryopreservation of alginate encapsulated liver cell spheroids in bioartificial liver support; assessments of cryoprotectant toxicity tolerance and dilution strategies.

The Bioartificial Liver (BAL) is an extra-corporeal liver support designed to support the function of the Liver in patients with impaired liver function. The BAL biomass consists of alginate encapsulated liver spheroids (AELS). To facilitate rapid delivery of a BAL to patients the AELS are cryopreserved using a DMSO-containing cryoprotectant solution. This study assesses toxicity of DMSO in AELS at concentrations and temperatures relevant to the cryopreservation and recovery process of a cellular biomass. Additionally, it develops a process to remove DMSO from AELS before delivery of cell product to patients. Exposure of AELS to DMSO, at a concentration of 12% (v/v) for 10 min did not have a negative effect on the viability of the AELS up to 24 h after exposure, irrespective of the exposure temperature between 37 C and 0 C. Evidence of toxicity was only seen with exposure to 40% (v/v) DMSO, which was more notable at warm temperatures. Post-Thaw removal of DMSO was measured by determining the DMSO concentration of the post-thaw washes using refractometry. Washing AELS 3 times in tapering concentrations of Glucose supplemented DMEM at an AELS:wash ratio of 1:2 was sufficient to reduce DMSO to undetectable levels (<1%). The study demonstrated that the thawing method minimised DMSO toxicity to the BAL biomass, and the post-thaw washing protocol successfully removed all the DMSO present in the cryopreserved BAL. Thereby enabling effective cryopreservation of the BAL for future clinical translation.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

High cytotoxicity of betulin towards fish and murine fibroblasts: Is betulin safe for nonneoplastic cells?

BACKGROUND: Betulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells. The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination). RESULTS: The results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells. CONCLUSIONS: The results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

Incorporation of dimethyl sulfoxide into experimental hydrophilic and hydrophobic adhesive resins: evaluation of cytotoxic activities.

This study evaluated the cytotoxicity of methacrylate-based resins containing dimethyl sulfoxide (DMSO). DMSO was incorporated into hydrophobic (R2) and hydrophilic (R5) resins at weight concentrations of 0, 0.01, 0.1, 1, 5, or 10 w/w %. Resin discs (n = 10/group) were prepared. Human gingival fibroblasts (HGF-1) were exposed to resin eluates for 24 h. Furthermore, dentin barrier test was performed using 3-D cultures of odontoblast-like cells (SV40 transfected pulp derived cells) with dentin slices of 400 µm thickness (n = 8). After acid etching of dentin, DMSO-modified resins were applied into the cavity part of the device and light-cured for 20 s. Cell viability (%) was assessed by MTT and analyzed spectrometrically. Data were analyzed by ANOVA and Tukey test (α = 0.05). Resin eluates showed statistically significantly lower % cell viability for all neat and DMSO-modified resins than seen for the negative control. Moreover, DMSO-R5 eluates resulted in significantly lower % cell viability than DMSO-R2 emulates. The dentin barrier test showed that DMSO-R2 did not result in significantly lower % cell viability, whereas incorporation of 1-10 w/w % DMSO into R5 resulted in significantly lower % of cell viability. Incorporating DMSO into hydrophilic self-etching resins may increase cytotoxicity. The biocompatibility is not influenced by the addition of DMSO into hydrophobic resin.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

(-)-Epicatechin protects from amebic liver abscess development in hamster.

In this work the effect of (-)-epicatechin on the development of amebic liver abscess in hamsters was evaluated. (-)-epicatechin is a flavonoid present in plants that possesses various biological properties, including its activity against some protozoal parasites; however its antiamebic activity in a living model had not been evaluated. Syrian golden hamsters were intrahepatically inoculated with 1x106E. histolytica trophozoites, three days after inoculation they received nine intraperitoneal doses of (-)-epicatechin (10 mg/100 g) every 48 h. Animals without treatments and treated with metronidazole were included as controls. Macroscopic characteristics of the hepatic abscess, histopathological analysis of the tissue and the levels of inflammatory cytokines were determined. (-)-epicatechin produced a decrease in liver abscess progression being observed only 9.49% of damage compared to 84% shown by untreated animals. During treatment with (-)-epicatechin hepatic tissue showed signs of liver repair and absence of amoebae. Additionally, (-)-epicatechin produced a modulating effect on inflammatory cytokines TNF-α, IL-1ß and IL-10. All these events observed in animals treated with (-)-epicatechin could contribute to the elimination of trophozoites and liver healing.

Antenna regeneration as an ecotoxicological endpoint in a marine amphipod: a proof of concept using dimethyl sulfoxide and diflubenzuron.

Regeneration is a widely spread process across the animal kingdom, including many species of marine crustaceans. It is strongly linked to hormonal cycles and, therefore, a great endpoint candidate for toxicology studies. We selected the amphipod Parhyale hawaiensis as test organism, already used in ecotoxicological studies and able to regenerate its body appendages. We are proposing a protocol to use the antenna regeneration as a toxicity endpoint. First, we evaluated differences in time of completion of regeneration in males and females after the amputation of one antenna of 6 months old animals. Then we compared the influence of different testing volumes in the regeneration process (100 and 5 mL). We used as testing substances, dimethyl sulfoxide (DMSO) and diflubenzuron, a chitin synthesis inhibitor. The most suitable protocol consisted of volumes of 5 mL in 12-well microplates, with 1 organism per well, 12 organisms per concentration (1:1 females/males) and test time duration of around 5 weeks. DMSO accelerated regeneration time with a NOEC of 0.06%. Diflubenzuron inhibited the time necessary to its completion with a NOEC of 0.32 µg L-1. We conclude that the Parhyale hawaiensis antenna regeneration protocol proposed here is a potential tool in ecotoxicology, but more studies are required for its validation not only to verify its utility for testing chemicals but also environmental samples.

Glucocorticoids and serum- and glucocorticoid-inducible kinase 1 are potent regulators of CFTR in the native intestine: implications for stress-induced diarrhea.

Nongenomic glucocorticoid (GC) and serum- and glucocorticoid-inducible kinase 1 (SGK1) signaling regulate ion transport, but CFTR has not been investigated in the intestine. We examined GC, SGK1, and phosphatidylinositol 3-kinase (PI3K) kinase signaling of CFTR ion transport in native intestine and the role of GCs on mRNA, protein, surface expression, and cyclic guanosine monophosphate (cGMP)-elicited diarrhea. Rats were treated with dexamethasone (DEXA; 2 mg/kg ip) or DMSO for 1, 4, and 24 h. Cyclic adenosine monophosphate (cAMP)-activated ion transport was examined in the presence or absence of SGK1 and PI3K inhibitors. Phosphorylation of SGK1, phosphoinositide-dependent kinase 1, and Akt kinases was confirmed by immunoblots using phosphor-specific antibodies. Tissue lysates were analyzed by mass spectrometry. CFTR and SGK1 mRNA were measured by quantitative PCR. Changes in total and surface CFTR protein were determined. The role of GC in cGMP-activated CFTR ion transport was examined. GC synergistically increased CFTR ion transport by SGK1 and PI3K signaling and increased CFTR protein without altering SGK1 or CFTR mRNA. GC induced highest levels of CFTR protein at 4 h that were associated with marked increase in surface CFTR, phosphorylation of the ubiquitin ligase neural precursor cell expressed developmentally downregulated 4-like (Nedd4-2), and 14-3-3ε, supporting their roles in surface retention and stability. Coimmunoprecipitation of CFTR, Nedd4-2, and 14-3-3ε indicated that assembly of this complex is a likely effector of the SGK and Akt pathways. Mass spectrometry identified phosphorylated peptides in relevant proteins. GC-SGK1 potently regulates CFTR in the intestine and is implicated in diarrheal disease.NEW & NOTEWORTHY This is the first study to examine the mechanisms of glucocorticoid, serum- and glucocorticoid-inducible kinase 1, and nongenomic kinase signaling of CFTR in the native intestine. We identified unique and druggable intestine-specific factors of the pathway that are targets for treating stress-induced diarrhea.

Cellular and molecular effects of silymarin on the transdifferentiation processes of LX-2 cells and its connection with lipid metabolism.

Fibrosis process in the liver is a clinical condition established in response to chronic lesions and may be reversible in many situations. In this process, hepatic stellate cells (HSCs) activate and produce extracellular matrix compounds. During fibrosis, the lipid metabolism is also altered and contributes to the transdifferentiation of the HSCs. Thus, controlling lipid metabolism in HSCs is suggested as a method to control or reverse the fibrotic condition. In the search for therapies that modulate lipid metabolism and treat liver diseases, silymarin has been identified as a relevant natural compound to treat liver pathologies. The present study aimed to evaluate the cellular and molecular effects of silymarin in the transdifferentiation process of HSCs (LX-2) from activated phenotype to a more quiesced-like cells , also focusing on understanding the modulatory effects of silymarin on lipid metabolism of HSCs. In our analyses, 100 µM of silymarin reduced the synthesis of actin filaments in activated cells, the synthesis of the protein level of α-SMA, and other pro-fibrotic factors such as CTGF and PFGF. The concentration of 150 µM silymarin did not reverse the activation aspects of LX-2 cells. However, both evaluated concentrations of the natural compound protected the cells from the negative effects of dimethyl sulfoxide (DMSO). Furthermore, we evaluated lipid-related molecules correlated to the transdifferentiation process of LX-2, and 100 µM of silymarin demonstrated to control molecules associated with lipid metabolism such as FASN, MLYCD, ACSL4, CPTs, among others. In contrast, cellular incubation with 150 µM of silymarin increased the synthesis of long-chain fatty acids and triglycerides, regarding the higher presence of DMSO (v/v) in the solvent. In conclusion, silymarin acts as a hepatoprotective agent and modulates the pro-fibrogenic stimuli of LX-2 cells, whose effects depend on stress levels in the cellular environment.

Nuclear factor of activated T cells as a marker of in vivo low-dose dibenzo[a,h]anthracene exposure.

We previously demonstrated that particulate matter ≤2.5 µm (PM2.5) suppresses the immune response in the spleen in vivo. Although PM2.5 includes the polycyclic aromatic hydrocarbon (PAH) such as dibenzo[a,h]anthracene (DBA), it is unclear whether PAH has a direct effect on the responses of splenocytes. In our study, the concentration of DBA used was approximately 0.8 µm, which is much lower than concentrations used in other toxicological studies of DBA. Although exposure to high concentrations of DBA is implicated in carcinogenesis, the effects of low doses of DBA on immune cells in vivo remain unclear. Here, we investigated the effects of low DBA doses on mouse splenocytes in vivo. Mice were administered dimethyl sulfoxide or DBA (0.4 or 0.8 µm) intratracheally. Twenty-four hours after treatment, the mice were killed and their splenocytes were collected. DBA treatment enhanced mitogen-induced cell proliferation and cytokine production in the mouse splenocytes. Furthermore, DBA enhanced splenic CD4+ and CD8+ cell proliferation and cytokine production. The nuclear factor of activated T cells (NFAT) was activated in CD4+ cells. DBA also activated nuclear factor-kappa B and CCAAT enhancer-binding protein pathways in CD11b+ cells. DBA-enhanced splenocyte activation was Toll-like receptor 2-, 4-, 9- and MyD88-independent. These results suggest that NFAT represents a promising marker for evaluation of the effects of DBA on T cells and T-cell-dependent antibody responses.

Assessment of sublethal ecotoxicity of solvents on larvae of a model native amphibian (Lithobates pipiens).

Carrier solvents are used frequently in toxicity testing to assist hydrophobic chemicals into solution, but such solvents may have toxic effects on test subjects. Amphibians are model organisms in toxicity studies; however, little is known about the direct effects of solvents on native amphibians. Following modifications to standardized guidelines for native species, we used acute 96-hour exposures to assess the direct effects of three common solvents on survival, differences in morphology and occurrence of abnormalities of northern leopard frog larvae (Lithobates pipiens). The solvents, dimethyl sulfoxide (DMSO), ethanol (ETOH) and acetone (ACE) were used at nominal concentrations ranging from 1 to 100 µL/L. We also conducted a 30-day exposure to assess the direct chronic effects of DMSO at 1 and 5 µL/L, on larval growth, development and sex differentiation, but found no effects. Acute exposure to solvents also had no effect on the survival of larvae, but we found significant abnormalities in tadpoles acutely exposed to 100 µL/L ACE. Acute exposure to DMSO and ETOH had further concentration-dependent effects on larval morphological traits. Our study suggests that DMSO and ETOH at ≤20 µL/L may be used as solvents in amphibian ecotoxicological studies, but ACE should be limited to ≤50 µL/L in ecotoxicity studies and perhaps much less (≤10 µL/L) in studies with other amphibians, based on a review of existing literature. We emphasize pilot studies when using solvents on acute and chronic ecotoxicity tests, using native amphibians.

UBE2W down-regulation promotes cell apoptosis and correlates with hypospermatogenesis.

Ubiquitin conjugating enzyme (E2) is crucial for mediating N-terminal ubiquitination. Recent study reports that UBE2W is involved in male infertility. However, the correlation between UBE2W expression and hypospermatogenesis is unclear. The present study is to explore the biological role of UBE2W and its association with hypospermatogenesis. Results showed that the sexpression levels of UBE2W in mouse testes were gradually elevated from 2 to 10 weeks, while were significantly deceased in the testes with hypospermatogenesis. When UBE2W expression was successfully down-regulated in spermatogenic cells, the rate of apoptosis was significantly increased and the P53/Bcl-2/caspase 6/caspase 9 signal pathways were activated. Thus, these data indicate that UBE2W down-regulation promotes cell apoptosis and correlates with hypospermatogenesis, which may be helpful for the diagnosis of male infertility.