24-h Exposure Research Articles

Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to β-naphthoflavone (β-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to β-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Assessment of heavy metals and environmental stress conditions on the production potential of polyunsaturated fatty acids (PUFAs) in indigenous microalgae isolated from the Gulf of Mannar coastal waters.

The present study evaluated the effects of heavy metals, viz., lead, mercury, and cadmium, on growth, chlorophyll a, b, c, carotenoids, and PUFA content of marine microalgae Chlorella sp. and Cylindrotheca fusiformis. At 96-h exposure, the IC50 values for Hg2+, Pb2+, and Cd2+ were 0.85mg/L, 2.4mg/L, and 5.3mg/L respectively, in Chlorella sp. In C. fusiformis, IC50 values for Hg2+, Pb2+, and Cd2+ were 0.5mg/L, 1.2mg/L, and 3mg/L respectively. The pigment contents of both microalgae were significantly affected upon heavy metal exposure. In Chlorella sp. and C. fusiformis, the exposed concentrations of Hg2+ averagely decreased the PUFA content by 76.34% and 78.68%, respectively. Similarly, Pb2+-exposed concentrations resulted in 54.50% and 82.64% average reductions in PUFA content of Chlorella sp. and C. fusiformis, respectively. Cd2+-exposed concentrations showed 32.58% and 40.54% average reduction in PUFA content of Chlorella sp. and C. fusiformis, respectively. Among the environmental stress conditions, the dark treatment has increased total PUFA content by 6.63% in Chlorella sp. and 3.92% in C. fusiformis. It was observed that the 50% nitrogen starvation (two-stage) significantly improved the PUFA production from 26.47 ± 6.55% to 40.92 ± 10.74% in Chlorella sp. and from 11.23 ± 5.01 to 32.8 ± 14.17% in C. fusiformis. The toxicity for both microalgae was followed in the order Hg2+ > Pb2+ > Cd2+. Among the two species, Chlorella sp. has shown a high tolerance to heavy metals and can be effectively utilized in PUFA production.

Developmental toxicity and metabolomics analyses of zebrafish (Danio rerio) embryos exposed to Fenoxaprop-p-ethyl.

Fenoxaprop-p-ethyl (FEN) is an aryloxy phenoxy propionate herbicide that has been widely used in paddy fields. Previous studies have indicated that FEN is highly toxic to aquatic organisms, but little is known about the developmental effects of FEN. This study investigated acute and developmental toxicity, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities, and metabolomic analyses in zebrafish embryos after 96h of exposure. FEN exhibited high acute toxicity to zebrafish embryos and larvae. Exposure to FEN could reduce heartbeat and hatching rates and increase malformation rates in embryos. Oxidative damage was also caused in embryos. The results of metabolomics analysis showed that 102 differentially abundant metabolites were found in zebrafish embryos in the 0.05mg/L FEN treatment group, and 60 differentially abundant metabolites were found in the 0.20mg/L FEN treatment group. These differentially abundant metabolites mainly belonged to 9 metabolic pathways, of which folate pathways and ABC transport protein pathways had the greatest impact. These results suggested that FEN induced high acute and developmental toxicity in zebrafish embryos.

Field and laboratory studies of fluorescence-based technologies for real-time tracking of cyanobacterial cell lysis and potential microcystins release

Elevated levels of dissolved microcystins (MCs) in source water due to rapid cell lysis of harmful cyanobacterial blooms may pose serious challenges for drinking water treatment. Catastrophic cell lysis can result from outbreaks of naturally-occurring cyanophages – as documented in Lake Erie during the Toledo water crisis of 2014 and in 2019, or through the application of algaecides or water treatment chemicals. Real-time detection of cyanobacterial cell lysis in source water would provide a valuable tool for drinking water plant and reservoir managers. In this study we explored two real-time fluorescence-based devices, PhycoSens and PhycoLA, that can detect unbound phycocyanin (uPC) as a potential indication of cell lysis and MCs release. The PhycoSens was deployed at the Low Service pump station of the City of Toledo Lake Erie drinking water treatment plant from July 15 to October 19, 2022 during the annual cyanobacteria bloom season. It measured major algal groups and uPC in incoming lake water at 15-min intervals during cyanobacteria dominant and senescence periods. Intermittent uPC detections from the PhycoSens over a three-month period coincided with periods of increasing proportions of extracellular MCs relative to total (intracellular and extracellular) MCs, indicating potential for uPC use as an indicator of cyanobacterial cell integrity. Following exposures of laboratory-cultured MCs-producing Microcystis aeruginosa NIES-298 (120 μg chlorophyll/L) to cyanophage Ma-LMM01, copper sulfate (0.5 and 1 mg Cu/L), sodium carbonate peroxyhydrate (PAK® 27, 6.7 and 10 mg H2O2/L), and potassium permanganate (2.5 and 4 mg/L), appearance of uPC coincided with elevated fractions of extracellular MCs. The PhycoLA was used to monitor batch samples collected daily from Lake Erie water exposed to algaecides in the laboratory. Concurrence of uPC signal and surge of dissolved MCs was observed following 24-h exposures to copper sulfate and PAK 27. Overall results indicate the appearance of uPC is a useful indicator of the onset of cyanobacterial cell lysis and the release of MCs when MCs are present.

Comparative In Vitro Study of the Cytotoxic Effects of Doxorubicin’s Main Metabolites on Cardiac AC16 Cells Versus the Parent Drug

Doxorubicin (DOX; also known as adriamycin) serves as a crucial antineoplastic agent in cancer treatment; however, its clinical utility is hampered by its’ intrinsic cardiotoxicity. Although most DOX biotransformation occurs in the liver, a comprehensive understanding of the impact of DOX biotransformation and its’ metabolites on its induced cardiotoxicity remains to be fully elucidated. This study aimed to explore the role of biotransformation and DOX's main metabolites in its induced cardiotoxicity in human differentiated cardiac AC16 cells. A key discovery from our study is that modulating metabolism had minimal effects on DOX-induced cytotoxicity: even so, metyrapone (a non-specific inhibitor of cytochrome P450) increased DOX-induced cytotoxicity at 2 µM, while diallyl sulphide (a CYP2E1 inhibitor) decreased the 1 µM DOX-triggered cytotoxicity. Then, the toxicity of the main DOX metabolites, doxorubicinol [(DOXol, 0.5 to 10 µM), doxorubicinone (DOXone, 1 to 10 µM), and 7-deoxydoxorubicinone (7-DeoxyDOX, 1 to 10 µM)] was compared to DOX (0.5 to 10 µM) following a 48-h exposure. All metabolites evaluated, DOXol, DOXone, and 7-DeoxyDOX caused mitochondrial dysfunction in differentiated AC16 cells, but only at 2 µM. In contrast, DOX elicited comparable cytotoxicity, but at half the concentration. Similarly, all metabolites, except 7-DeoxyDOX impacted on lysosomal ability to uptake neutral red. Therefore, the present study showed that the modulation of DOX metabolism demonstrated minimal impact on its cytotoxicity, with the main metabolites exhibiting lower toxicity to AC16 cardiac cells compared to DOX. In conclusion, our findings suggest that metabolism may not be a pivotal factor in mediating DOX's cardiotoxic effects.Graphical

Synthesis of Lawsonia inermis-encased silver–copper bimetallic nanoparticles with antioxidant, antibacterial, and cytotoxic activity

Abstract The extract of the medicinal plant Lawsonia inermis, known as henna, was employed to synthesize silver–copper bimetallic nanoparticles (Ag–Cu NPs) in a unique, efficient, and cost-effective method. The shape, size, and structural features of synthesized Ag–Cu NPs were determined by ultra–visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffractometer, field emission scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy methods. The rod-shaped Ag–Cu nanoparticles, averaging 41.66 ± 17.18 nm in size, synthesized from L. inermis, exhibited potent antioxidant activity by inhibiting 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) free radicals. They also displayed significant antibacterial effects against Pseudomonas aeruginosa (28 mm), Staphylococcus aureus (27 mm), Bacillus cereus (26 mm), and Escherichia coli (24 mm). Additionally, these nanoparticles induced notable morphological changes in cancer cells and demonstrated promising cytotoxicity against MDA-MB-231 tumor cells (IC50 = 37.40 µg·mL−1). However, they exhibited biotoxicity in Artemia nauplii, resulting in mortality rates ranging from 3.0% to 32.5%. The LC50 and LC90 values recorded for a 48-h exposure were 1.51 mg·L−1 and 2.59 mg·L−1, respectively. These findings highlight the potential application of L. inermis-derived Ag–Cu NPs in pharmacology and bio-nanomedicine.

Nanostructured silver-boron nitride hybrid materials: Toward high-performance antibacterial hydrogels with enhanced mechanical and thermal properties

Silver nanoparticles (AgNPs) are grown uniformly on the surface of white porous boron nitride nanofibers (BNNFs) via a thermal reduction method to overcome the issue of nanoparticle aggregation. The optical characteristics of the AgNPs are retained, with different colors present depending on reduction temperature. This mainly depends on the white appearance of BNNFs. In addition, compared to boron nitride (BN) nanosheets and nanospheres, abundant pore structure and high specific surface area of the BNNFs enable superior AgNPs loading without agglomeration. The prepared AgNPs-loaded BNNFs (Ag-BNNFs) are then incorporated into poly (vinyl alcohol) (PVA) hydrogels fabricated through cyclic freeze-thaw techniques to develop antibacterial Ag-BNNFs/PVA composite hydrogels. The addition of 3 wt% Ag-BNNFs increases the tensile strength by 73.1% and compressive strength by 92.8% relative to the unmodified PVA hydrogels. Thermal stability also improves considerably, with the maximum PVA decomposition rate shifting from 280.9°C to 323.6°C in the composite hydrogels. Importantly, a potent bactericidal effect against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are achieved, with 99.5% and 100% inhibition after 24 h exposure to the Ag-BNNFs/PVA composite hydrogels, respectively. This work demonstrates a new method of dispersing AgNPs with BNNFs, and the resulting Ag-BNNFs effectively enhance the mechanical properties, thermal stability and antibacterial activity of PVA hydrogels.

High-Humidity-Tolerant Chloride Solid-State Electrolyte for All-Solid-State Lithium Batteries.

Halide solid-state electrolytes (SSEs) hold promise for the commercialization of all-solid-state lithium batteries (ASSLBs); however, the currently cost-effective zirconium-based chloride SSEs suffer from hygroscopic irreversibility, low ionic conductivity, and inadequate thermal stability. Herein, a novel indium-doped zirconium-based chloride is fabricated to satisfy the abovementioned requirements, achieving outstanding-performance ASSLBs at room temperature. Compared to the conventional Li2ZrCl6 and Li3InCl6 SSEs, the hc-Li2+xZr1-xInxCl6 (0.3 ≤ x ≤ 1) possesses higher ionic conductivity (up to 1.4 mS cm-1), and thermal stability (350°C). At the same time, the hc-Li2.8Zr0.2In0.8Cl6 also shows obvious hygroscopic reversibility, where its recovery rate of the ionic conductivity is up to 82.5% after 24-h exposure in the 5% relative humidity followed by heat treatment. Theoretical calculation and experimental results reveal that those advantages are derived from the lattice expansion and the formation of Li3InCl6 ·2H2O hydrates, which can effectively reduce the migration energy barrier of Li ions and offer reversible hydration/dehydration pathway. Finally, an ASSLB, assembled with reheated-Li2.8Zr0.2In0.8Cl6 after humidity exposure, single-crystal LiNi0.8Mn0.1Co0.1O2 and Li-In alloy, exhibits capacity retention of 71% after 500 cycles under 1 C at 25°C. This novel high-humidity-tolerant chloride electrolyte is expected to greatly carry forward the ASSLBs industrialization.

Chromosome Doubling Enhances Biomass and Carotenoid Content in Lycium chinense

Lycium chinense, a type of medicinal and edible plant, is rich in bioactive compounds beneficial to human health. In order to meet the market requirements for the yield and quality of L. chinense, polyploid induction is usually an effective way to increase plant biomass and improve the content of bioactive components. This study established the most effective tetraploid induction protocol by assessing various preculture durations, colchicine concentrations, and exposure times. The peak tetraploid induction efficacy, 18.2%, was achieved with a 12-day preculture and 24-h exposure to 50 mg L–1 colchicine. Compared to diploids, tetraploids exhibited potentially advantageous characteristics such as larger leaves, more robust stems, and faster growth rates. Physiologically, tetraploids demonstrated increased stomatal size and chloroplast count in stomata but reduced stomatal density. Nutrient analysis revealed a substantial increase in polysaccharides, calcium, iron, and zinc in tetraploid leaves. In addition, seventeen carotenoids were identified in the leaves of L. chinense. Compared to the diploid, lutein, β-carotene, neoxanthin, violaxanthin, and (E/Z)-phytoene exhibited higher levels in tetraploid strains T39 and T1, with T39 demonstrating a greater accumulation than T1. The findings suggest that the generated tetraploids harbor potential for further exploitation and lay the foundation for the selection and breeding of novel genetic resources of Lycium.

Antimicrobial and antitumor activities of an alginate-based membrane loaded with bismuth nanoparticles and cetylpyridinium chloride.

To evaluate the antitumor and antimicrobial properties of an alginate-based membrane (ABM) loaded with bismuth lipophilic nanoparticles (BisBAL NPs) and cetylpyridinium chloride (CPC) on clinically isolated bacteria and a pancreatic cancer cell line. The BisBAL NP-CPC ABM was characterized using optical and scanning electron microscopy (SEM). The antimicrobial potential was measured using the disk-diffusion assay, and antibiofilm activity was determined through the live/dead assay and fluorescence microscopy. The antitumor activity was analyzed on the pancreatic cell line (Panc 03.27) using the MTT assay and live/dead assay with fluorescence microscopy. After a 24-h exposure (37°C, aerobic conditions), 5 µM BisBAL NP reduced the growth of K. pneumoniae by 77.9%, while 2.5 µM BisBAL NP inhibited the growth of Salmonella, E. faecalis and E. faecium by 82.9%, 82.6%, and 78%, respectively (p < 0.0001). The BisBAL NPs-CPC ABM (at a ratio of 10:1; 500 and 50 µM, respectively) inhibited the growth of all isolated bacteria, producing inhibition halos of 9.5, 11.2, 7, and 10.3 mm for K. pneumoniae, Salmonella, E. faecalis, and E. faecium, respectively, in contrast to the 6.5, 9.5, 8.5, and 9.8 mm obtained with 100 µM ceftriaxone (p < 0.0001). The BisBAL NPs-CPC ABM also reduced bacterial biofilms, with 81.4%, 74.5%, 97.1%, and 79.5% inhibition for K. pneumoniae, E. faecium, E. faecalis, and Salmonella, respectively. Furthermore, the BisBAL NPs-CPC ABM decreased Panc 03.27 cell growth by 76%, compared to 18% for drug-free ABM. GEM-ABM reduced tumoral growth by 73%. The live/dead assay confirmed that BisBAL NPs-CPC-ABM and GEM-ABM were cytotoxic for the turmoral Panc 03.27 cells. An alginate-based membrane loaded with BisBAL NP and CPC exhibits dual antimicrobial and antitumoral efficacy. Therefore, it could be applied in cancer treatment and to diminish the occurrence of surgical site infections.

Transcriptional modulation of heat shock proteins and adipogenic regulators in bovine adipocytes following heat exposure

This research endeavored to elucidate the transcriptional modulation of heat shock proteins and adipogenic regulators in bovine subcutaneous adipocytes following thermal exposure. Post-differentiation, mature adipocytes were subjected to three treatments of control (CON), moderate (MHS), and extreme (EHS) heat stress. These treatments consist of thermal conditions at temperatures of 38 °C (CON), 39.5 °C (MHS), or 41 °C (EHS) along with of 3 or 12 h. There was no statistically significant variations observed in the gene expressions of HSP27 and HSP70 when comparing CON with MHS across both exposures. Contrastingly, when comparing CON with EHS, an upregulation (P < 0.01) in HSP27 gene expression was evident for both 3 and 12 h of incubation, while HSP70 gene expression exhibited elevation (P < 0.01) at the 3-h mark, with no change observed at 12 h. Protein quantification, however, revealed an elevation (P < 0.01) in HSP27 and HSP70 for both CON vs. MHS and CON vs. EHS at the 12-h exposure. This trend in protein level mirrored (P < 0.05) that of proliferator-activated receptor-gamma (PPARγ). Elevated (P < 0.05) protein levels of fatty acid synthase (FAS) were exclusively discernible in the CON vs. MHS. Increased (P < 0.01) transcriptional activity of PPARγ, CCAAT/enhancer-binding protein alpha (C/EBPα), stearoyl-CoA desaturase (SCD), and FAS was evident in the CON vs. EHS comparison. Complementary to these molecular findings, an augmented lipid droplet accumulation was observed (P < 0.01) in EHS-exposed adipocytes progressively from day 6 through day 9. Our current study highlights how different levels and lengths of heat stress can impact the activity of important heat-related proteins and factors that play a role in fat development in beef cattle. These findings can help guide strategies to manage how beef cattle are exposed to heat, which can affect fat storage and ultimately the quality of the meat's marbling.

EFFECT OF KAOLIN ON HEMATOLOGICAL AND BIOCHEMICAL PARAMETERS OF CYPRINUS CARPIO L. AGAINST COPPER SULFATE TOXICITY

The present study was aimed to evaluate the effect of kaolin on copper sulfate toxicity and on some hematological and biochemical performance in Cyprinus carpio L. 240 common carp ranged between 85-95g in weight. A total of 120 fish were used to determine the median lethal concentration LC50 of copper sulfate and 120 fish and randomly distributed into six treatments (two replications for each treatment). The LC50 of copper sulfate was 1.82 mg/l for 48h exposure, then added 0.91mg/l of copper sulfate to all treatment groups except control negative .The experimental treatments included adding kaolin: Control negative (without copper sulfate or kaolin); Control positive 0.91mg/l copper sulfate without kaolin.T1, T2, T3 and T4 kaolin were added in the water at levels of 2, 4 , 6 and 8 g/l respectively. Results showed that the values of RBCs count were ranged 1.16 x106 - 1.77x106 cell /mm³ in cont.+ve and T3 respectively. WBCs count were ranged 8.40×10³ in cont.+ve to 13.14×10³ cell/mm³ in T3.The lowest Hb content found in cont.+ve reached 8.35 g/dl, while the highest content found in control-ve attained 12.87g/dl. PCV ranged from 21.75 to 35.60% in cont.+ve and T3 respectively. Results of total protein ranged between 6.6 - 18.19 g/dl in T4 and cont.-ve respectively, while albumin ranged from 1.31 to 1.53 g/dl in cont.+ve and cont.-ve respectively. Globulin level recorded the lowest value in T4 which was 5.15g/dl whereas the highest level in cont.-ve which was 16.67 g/dl. In conclusion, this investigation indicated that kaolin has reduced the toxicity effects of copper sulfate in common carp and the best level can be use was 6g/l (T3).

Effects of nonnutritive sugar inclusion in laboratory diets and attracticidal spheres on survivorship and mobility of 2 Dipteran species, Rhagoletis pomonella (Diptera: Tephritidae) and Drosophila suzukii (Diptera: Drosophilidae).

Native apple maggot fly, Rhagoletis pomonella, and invasive spotted-wing drosophila, Drosophila suzukii, are key pests of apple and small fruit, respectively, in the United States. Both species are typically managed with standard insecticide applications. However, interest in alternative strategies that result in insecticide reductions has led to evaluations of nonnutritive sugars as toxicants for Drosophila species and development of attracticidal spheres for both species. Here, we evaluated the survivorship of R. pomonella and D. suzukii when provided with standard diets that substituted saccharin, sucralose, aspartame, erythritol, dextrose, or mannitol for the sucrose component and compared them with standard diets and water-only controls for up to 15 days. Presence of erythritol and mannitol significantly decreased survivorship of R. pomonella and erythritol significantly decreased the survivorship of D. suzukii. However, mobility trials following a 2 h exposure to aqueous solutions of each sugar treatment resulted in no strong impact on either species. Survivorship after 30 min exposure to erythritol or mannitol alone, or in combination with varying concentrations of sucrose (serving as a phagostimulant) at 30 min and 24 h were evaluated for both species. Only D. suzukii survivorship was affected with decreased survivorship on erythritol:sucrose solutions of 20:0% and 15:5% for 24 h. Based on all results, erythritol appeared most promising, and was integrated into attracticidal spheres as a toxicant but even at the highest concentration, survivorship remained unaffected for either species, thus making this nonnutritive sugar impractical and ineffective as a toxicant substitute in attracticidal spheres.

Toxicity evaluation of silver nanoparticles synthesized from naringin flavonoid on human promyelocytic leukemic cells and human blood cells.

Increasing applications of silver nanoparticles (AgNPs) in multiple products like cosmetics, medicines, drugs, paints, and other new materials have raised concern for their toxic effects on living beings and the surrounding environment. In the present study, cytotoxicity and genotoxicity of AgNPs synthesized using plant flavonoid (Naringin) as a reducing agent were investigated on human promyelocytic leukemic (HL-60) cells and human blood as an in vitro model. The LC50 of AgNPs was found to be 4.85µM. Dose-dependent increase in cell death and caspase activity was observed in the presence of AgNPs. The comet assay showed a 60%-70% (p < .05) increase in tail DNA at 0.48 and 0.96µM AgNPs. CBMN in PBMCs also confirmed the genotoxic potential of AgNPs-induced DNA damage. AgNPs resulted in 1.5-1.54 fold (p < .05) increase in the level of ROS in HL-60 cells after 12h of exposure. AgNP showed toxicity in human cells through ROS generation and cellular damage through membrane dysfunction, caspase activation, apoptosis, and DNA damage.

Are the BPA analogues an alternative to classical BPA? Comparison between 2D and alternative 3D in vitro neuron model to assess cytotoxic and genotoxic effects

BPA is used in a wide range of consumer products with very concern toxicological properties. The European Union has restricted its use to protect human health. Industry has substituted BPA by BPA analogues. However, there is a lack of knowledge about their impacts. In this work, BPA and 5 BPA analogues (BPS, BPAP, BPAF, BPFL and BPC) have been studied in classical SH-SY5Y and the alternative 3D in vitro models after 24 and 96 h of exposure. Cell viability, percentage of ROS, cell cycle phases as well as the morphology of the spheroids were measured. The 2D model was more sensitive than the 3D models with differences in cell viability higher than 60% after 24 h of exposure, and different mechanisms of ROS production. After chronic exposure, both models were more affected in comparison to the 24 h exposure. After a recovery time (96 h), the spheroids exposed to 2.5–40 µM were able to recover cell viability and the morphology. Among the BPs tested, BPFL>BPAF>BPAP and >BPC revealed higher toxicological effects, while BPS was the only one with lower effects than BPA. To conclude, the SH-SY5Y 3D model is a suitable candidate to perform more reliable in vitro neurotoxicity tests.

Benzo[a]pyrene disrupts LH/hCG-dependent mouse Leydig cell steroidogenesis through receptor/Gαs protein targeting

Steroidogenesis of gonadal cells is tightly regulated by gonadotropins. However, certain polycyclic aromatic hydrocarbons, including Benzo[a]pyrene (BaP), induce reproductive toxicity. Several existing studies have considered higher than environmentally relevant concentrations of BaP on male and female steroidogenesis following long-term exposure. Also, the impact of short-term exposure to BaP on gonadotropin-stimulated cells is understudied. Therefore, we evaluated the effect of 1 nM and 1 µM BaP on luteinizing hormone/choriogonadotropin (LH/hCG)-mediated signalling in two steroidogenic cell models, i.e. the mouse tumor Leydig cell line mLTC1, and the human primary granulosa lutein cells (hGLC) post 8- and 24-h exposure. Cell signalling studies were performed by homogeneous time-resolved fluorescence (HTRF) assay, bioluminescence energy transfer (BRET) and Western blotting, while immunostainings and immunoassays were used for intracellular protein expression and steroidogenesis analyses, respectively. BaP decreased cAMP production in gonadotropin-stimulated mLTC1 interfering with Gαs activation. Therefore, decrease in gonadotropin-mediated CREB phosphorylation in mLTC1 treated with 1 μM BaP was observed, while StAR protein levels in gonadotropin-stimulated mLTC1 cells were unaffected by BaP. Further, BaP decreased LH- and hCG-mediated progesterone production in mLTC1. Contrastingly, BaP failed to mediate any change in cAMP, genes and proteins of steroidogenic machinery and steroidogenesis of gonadotropin-treated hGLC. Our results indicate that short-term exposure to BaP significantly impairs steroidogenic signalling in mLTC1 interfering with Gαs. These findings could have a significant impact on our understanding of the mechanism of reproductive toxicity by endocrine disruptors.

Salinity tolerance in Clarias gariepinus (Burchell, 1822): insight on blood parameter variations and gill histological changes.

This study was designed to evaluate the tolerance of Clarias gariepinus juveniles to a gradual and abrupt increase in salinity over time. To this effect, C. gariepinus juveniles were exposed to three salinity incremental protocols namely 1g L-1day-1, 5g L-1day-1, and 10g L-1day-1. Changes in the hematological parameters and the gill histology of fish were analyzed to determine the impact of osmotic stress on the health status of the fish and its osmoregulatory ability. The result obtained showed that juveniles of C. gariepinus can tolerate salinity stress up to 14g L-1. At 15g L-1 and beyond, all samples died regardless of gradual (i.e., 1g L-1day-1 administered for 15days) or abrupt salinity exposure (i.e., 5g L-1day-1 administered for three days and 10g L-1day-1 administered for two days). Interestingly, more than 90% of the fish survived a direct 10g L-1 exposure for 24h without prior acclimation. The hematological parameters accessed in the fish exposed to 10g L-1 (either gradually or abruptly) showed a significant increase in the white blood cells and a decrease in the red blood cells, packed cell volume, hemoglobin concentration, and all derived blood parameters. The results of the serum biochemistry show a lower total protein and albumin in the salinity-treated fish compared to the control group. However, the serum glucose and the plasma electrolytes (i.e., K+, Na+, and Cl-) were higher in the former group than in the latter. Aside from the stress response expressed in the blood parameters, severe gill degenerations were seen in the histological micrograph obtained for the salinity-treated fish, while the control had a near-normal gill architecture. It was concluded that C. gariepinus could tolerate salinity exposure of 10g L-1day-1 (administered gradually or abruptly) and below without killing the fish within 24h.

Signaling pathways involved in microbial indoor air pollutant 3-methyl-1-butanol in the induction of stomatal closure in Arabidopsis.

Indoor air pollution is a global problem and one of the main stress factors that has negative effects on plant and human health. 3-methyl-1-butanol (3MB), an indoor air pollutant, is a microbial volatile organic compound (mVOC) commonly found in damp indoor dwellings. In this study, we reported that 1mg/L of 3MB can elicit a significant reduction in the stomatal aperture ratio in Arabidopsis and tobacco. Our results also showed that 3MB enhances the reactive oxygen species (ROS) production in guard cells of wild-type Arabidopsis after 24h exposure. Further investigation of 24 h 3MB fumigation of rbohD, the1-1, mkk1, mkk3, and nced3 mutants revealed that ROS production, cell wall integrity, MAPK kinases cascade, and phytohormone abscisic acid are all involved in the process of 3MB-induced stomatal. Our findings proposed a mechanism by which 3MB regulates stomatal closure in Arabidopsis. Understanding the mechanisms by which microbial indoor air pollutant induces stomatal closure is critical for modulating the intake of harmful gases from indoor environments into leaves. Investigations into how stomata respond to the indoor mVOC 3MB will shed light on the plant's "self-defense" system responding to indoor air pollution.

Biochemical Studies to Understand Teratogenicity and Lethality Outcomes in Modified-FETAX.

The frog embryo teratogenesis assay-Xenopus (FETAX) is a standardized test used to assess the toxic and teratogenic effects of xenobiotics. With this test, toxic and/or teratogenic concentrations of xenobiotic substances can be determined using morphological parameters such as lethality, length, and malformations in stage 8-11 Xenopus laevis embryos after 96h exposure. These parameters enable the determination of the median lethal and effective concentrations (LC50 and EC50), minimum concentration to inhibit growth (MCIG), and teratogenic index of the tested chemical to reveal the short-term effects of relatively high concentrations. On the other hand, although FETAX provides quantitative and qualitative data on teratogenicity and toxicity, the biochemical and molecular mechanisms of these effects cannot be explained. Recent studies have tried to elucidate the mechanisms causing malformations and to explain the underlying causes of toxicity and teratogenicity by biochemical marker analysis. This chapter describes methods to analyze modified-FETAX and some detoxification and oxidative stress-related biomarkers during the early embryonic development of X. laevis.

Exploring the modulation of MLH1 and MSH2 gene expression in hesperetin-treated breast cancer cells (BT-474).

The major mortality factor for women globally is breast cancer, and current treatments have several adverse effects. Hesperetin (HSP) is a flavone that occurs naturally with anti-tumor capabilities and has been investigated as a potential treatment for cancer. This study aimed to investigate the cytotoxic and anti-malignant potential of HSP on breast cancer cells (BT-474) and normal cells (MCF-10a). The results indicated that HSP has dose-dependent cytotoxicity in BT-474 and MCF-10a cells. The elevated concentration of HSP lowered cell viability and proliferation. The half-maximal inhibitory concentration (IC50) of HSP in BT-474 cancer cells after a 48-h exposure was 279.2 μM/ml, while the IC50 in normal cells was 855.4 μM/ml. The cytotoxicity of HSP was more significant in cancer cell lines than in normal cell lines and this aspect presents a favorable factor in utilizing the drug for the treatment of breast cancer. The apoptotic effect of HSP in BT-474 cells was investigated, and it was found that the higher the concentration of HSP more the cells underwent apoptosis. Furthermore, the highest concentration of HSP led to overexpression of the MLH1 and MSH2 genes in both breast cancer and normal cell lines. Overall, our study suggests that HSP has an anticancer effect on breast cancer cell lines, and the effect is concentration dependent.