24-h Exposure Research Articles

Dnmt3a Loss-of-Function Enhances DNA Repair in Proliferative HSCs Under Inflammatory Stress

Somatic mutations in DNMT3A can be enriched during clonal hematopoiesis by conditions with elevated type II interferon signaling. We have recently demonstrated that loss of functional Dnmt3a endows HSCs with a fitness advantage during chronic IFNg-driven inflammation. The increased fitness of Dnmt3a-mutant HSCs in these conditions can be partly attributed to the insensitivity to IFNg-primed apoptosis upon proliferative stress and delayed cell cycle activation from quiescence. However, it is unknown if these two mechanisms are related; if not, what is the underlying mechanism of inflammation-induced apoptotic resistance by Dnmt3a loss in HSCs? If the apoptotic resistance was merely due to delayed cell cycle activation from quiescence, proliferative activation should normalize the apoptotic levels of IFNg-exposed Dnmt3a-mutant HSCs. To test this, we induced HSCs into cell cycle by injecting 5-fluorouracil (5FU) into control (Vav-cre-; Dnmt3af/f) and mice lacking Dnmt3a in hematopoietic cells (3aKO, Vav-cre+; Dnmt3af/f). Kinetic analysis by BrdU labeling revealed that HSCs reached proliferation peak 8-days post-5FU treatment (>90% BrdU+). To capture the maximal number of proliferative HSCs, we injected PBS or recombinant mouse IFNg into 5FU-treated control and 3aKO mice on day 7 and 8. Phenotypic HSCs (Lin-cKit+EPCR+CD150+CD48-) were purified 2h after the second injection and analyzed for apoptosis. Cycling control HSCs were significantly more apoptotic upon IFNg exposure, while cell-cycle activated 3aKO HSCs remained insensitive to IFNg-primed apoptosis. Functional validation by colony formation assay showed IFNg-exposed cycling control HSCs formed significantly fewer colonies, while 3aKO HSCs gave rise to significantly more colonies regardless of exposure. These data suggest that Dnmt3a mutation may render HSCs resistant to IFNg-primed apoptosis via cell-cycle independent mechanisms. It has been documented that inflammation causes HSC apoptosis by inducing DNA damage through various mechanisms. Thus, the apoptotic resistance observed in Dnmt3a mutant HSCs could be due to 1) resistance to inflammation-induced DNA damage or 2) enhanced resolution of DNA damage. To test if Dnmt3a mutation renders HSCs resistant to IFNg-primed DNA damage, neutral comet assay was performed using PBS or IFNg-exposed quiescent HSCs. Interestingly, both genotypes showed increased DNA double-strand breaks (DSBs) upon IFNg exposure, suggesting that quiescent Dnmt3a-mutant HSCs are susceptible to IFNg-primed DSBs. DNA repair is one mechanism enabling DSB resolution, and is functionally active in proliferative HSCs. Thus, we performed neutral comet assay on cycling HSCs to quantify DSBs following PBS/IFNg exposure. Expectedly, IFNg significantly increased DSBs in cycling control but not in 3aKO HSCs, suggesting Dnmt3a loss may endow HSCs with augmented DNA repair. Similarly, when DNA damage was determined in control and Dnmt3a-knockout (3aKO) murine myeloblast cells (32D) 4h and 24h post-IFNg exposure, a comparable increase in DNA damage was observed 4h post-exposure in both genotypes. Interestingly, DNA damage levels were significantly reduced 24h later only in the 3aKO cells. This data paralleled HSC data, implying that Dnmt3a ablation facilitates augmented DNA repair. DSBs are repaired primarily via three universal yet distinct pathways, homologous recombination (HR), non-homologous end joining (NHEJ), and alternative NHEJ. To investigate how Dnmt3a loss facilitates enhanced DNA repair in response to IFNg exposure, we performed a DNA repair reporter assay in 32D genotypes 24h after IFNg exposure. The plasmid repair reporter assay was built on the detection of a green fluorescent protein (GFP) expression, which is otherwise disrupted by the insertion of I-SceI restriction enzyme recognition sites. After cutting with I-SceI, GFP can only be expressed after specific repair by one of the three pathways. Results showed Dnmt3a loss specifically enhanced the NHEJ pathways upon IFNg exposure, while other pathways were comparable between genotypes and treatments. In summary, our results demonstrated that Dnmt3a loss protects cell-cycle activated HSCs from inflammation-primed DSBs and apoptosis via augmented NHEJ as one mechanism. Future studies will investigate the molecular mechanisms underlying the enhanced NHEJ in Dnmt3a-mutant HSCs in response to chronic inflammation.

Utility of outdoor central site monitoring in assessing exposure of school children to ultrafine particles

Epidemiological studies investigating the association between daily particle exposure and health effects are frequently based on a single monitoring site located in an urban background. Using a central site in epidemiological time-series studies has been established based on the premises of low spatial variability of particles within the areas of interest and hence the adequacy of the central sites to monitor the exposure. This is true to a large extent in relation to larger particles (PM2.5, PM10) that are typically monitored and regulated. However, the distribution of ultrafine particles (UFP), which in cities predominantly originate from traffic, is heterogeneous. With increasing pressure to improve the epidemiology of UFP, an important question to ask is, whether central site monitoring is representative of community exposure to this size fraction of particulate matter; addressing this question is the aim of this paper. To achieve this aim, we measured personal exposure to UFP, expressed as particle number concentration (PNC), using Philips Aerasense Nanotracers (NT) carried by the participants of the study, and condensation particle counters (CPC) or scanning mobility particle sizers (SMPS) at central fixed-site monitoring stations. The measurements were conducted at three locations in Brisbane (Australia), Cassino (Italy) and Accra (Ghana). We then used paired t-tests to compare the average personal and average fixed-site PNC measured over the same 24-h, and hourly, periods. We found that, at all three locations, the 24-h average fixed-site PNC was no different to the personal PNC, when averaged over the study period and all the participants. However, the corresponding hourly averages were significantly different at certain times of the day. These were generally times spent commuting and during cooking and eating at home. Our analysis of the data obtained in Brisbane, showed that maximum personal exposure occurred in the home microenvironment during morning breakfast and evening dinner time. The main source of PNC for personal exposure was from the home-microenvironment. We conclude that the 24-h average PNC from the central-site can be used to estimate the 24-h average personal exposure for a community. However, the hourly average PNC from the central site cannot consistently be used to estimate hourly average personal exposure, mainly because they are affected by very different sources.

Dynamics and mechanisms of bioaccumulation and elimination of nonylphenol in zebrafish

Nonylphenol (NP) has been widely concerned for its endocrine disrupting effects. In this study, we investigated the accumulation and elimination of NP for the whole body and trunk of zebrafish (Danio rerio). The results show that the LC50 values of NP in zebrafish ranged from 474 μg·L−1 (24-h exposure) to 238 μg·L−1 (96-h exposure). Meanwhile, the NP concentrations in zebrafish during the depuration stage fitted the first-order kinetic model well, and the depuration rate constant (K2) was reduced from 0.412 d−1 to 0.2827 d−1 with higher NP. The half-life (t1/2) of NP was 1.75–2.45 d in the whole fish and 0.56–0.86 d in the trunk under low to high NP, respectively. Both the accumulation and elimination of NP in trunk were faster than those in whole fish, indicating the preferential transfer from viscera to muscle and rapidly diffusion in reverse. The bioconcentration factors (BCFSS) of NP were 104–112 L·kg−1 in whole body and 76–104 L·kg−1 in trunk, respectively, suggesting that the muscle was a major position for NP storage.

Six marine thecate Heterocapsa (Dinophyceae) from Malaysia, including the description of three novel species and their cytotoxicity potential

Thirty-four strains of Heterocapsa were established from Malaysian waters and their morphologies were examined by light, scanning, and transmission electron microscopy. Three species, H. bohaiensis, H. huensis, and H. rotundata, and three new species, H. borneoensis sp. nov., H. limii sp. nov., and H. iwatakii sp. nov. were described in this study. The three species were differentiated morphologically by unique characteristics of cell size, shape, displacement of the cingulum, shape and position of nucleus, the number and position of pyrenoids, and body scale ultrastructure. The species delimitations were robustly supported by the molecular data. A light-microscopy-based key to species of Heterocapsa is established, with two major groups, i.e., species with a single pyrenoid, and species with multiple pyrenoids. Bioassays were conducted by exposing Artemia nauplii to Heterocapsa densities of 1–5 × 105 cells mL−1, and treatments exposed to H. borneoensis showed naupliar mortality, while no naupliar death was observed in the treatments exposed to cells of H. bohaiensis, H. huensis, H. limii, and H. iwatakii. Naupliar death was observed during the initial 24 h for both tested H. borneoensis strains, and mortality rates increased up to 50% after 72-h exposure. This study documented for the first time the diversity and cytotoxic potency of Heterocapsa species from Malaysian waters.

Effects of the natural antimicrobial peptide aureocin A53 on cells of Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis in the excised teat model.

Aureocin A53 is an N-formylated antimicrobial peptide (AMP) produced by Staphylococcus aureus. Aureocin A53 has a broad spectrum of antimicrobial activity against human and animal pathogens. In the present study, its antagonistic activity was investigated towards 30 strains of S. aureus and 30 strains of Streptococcus spp. isolated from bovine mastitis cases in Brazil. Bovine mastitis is a disease that causes a major economic impact worldwide. Aureocin A53 inhibited the growth of all 60 strains tested, including multidrug-resistant streptococcal isolates and strains of S. aureus belonging to different pulsotypes. This AMP proved to be bactericidal against the six target strains randomly selected among staphylococci and streptococci, also exhibiting a lytic mode of action against the staphylococcal cells. Furthermore, it was determined that 2,048 AU/mL of the AMP were required to inhibit 99.99% of the cell growth of the strain less sensitive to aureocin A53. Aureocin A53 was not toxic to bovine mammary gland epithelial cells after a 24-h exposure and maintained its antimicrobial activity when tested in the excised-teat model against strains of S. aureus and Streptococcus agalactiae, the species responsible for most intramammary infections, not only in Brazil but in other countries as well. Therefore, the use of aureocin A53 in the development of new pharmacological products for the prophylaxis and/or treatment of bovine mastitis was considered promising.

Abstract 9419: Empagliflozin Modulates Cardiac Na Homeostasis via Late I Na and I NCX

Introduction: The effects of empagliflozin on excitation-contraction coupling (ECC) have not been fully elucidated. We previously showed that empagliflozin directly reduces Ca 2+ /calmodulin kinase II (CaMKII) activity in human and murine heart failure, thereby improving contraction. Upon acute exposure, however, only subsarcolemmal [Na + ] is decreased, while [Na + ] cytosolic and [Ca 2+ ] cytosolic are only decreased after 24 h empagliflozin. Hypothesis: We hypothesize that empagliflozin reduces cardiac late Na current (late I Na ) via a reduction in CaMKII activity. We further hypothesize that Na + /Ca 2+ -exchanger (NCX) is acutely masking effects on cardiac [Na + ], thereby delaying downstream effects on cardiac ECC. Methods: Informed consent was obtained according to the Declaration of Helsinki. Human/murine cardiomyocytes (CM) were isolated and incubated with 1 μM empagliflozin for 30 min or 24 h. Some CM were treated with H 2 O 2 or ATX-II to induce late I Na . Whole-cell patch clamp was used to measure late I Na or I NCX . Epifluorescence with SBFI was used to obtain cardiac [Na + ]. Some CM were treated with NCX inhibitor KB-R7943 (5 μM). Results and Conclusions: Late I Na induced by H 2 O 2 was acutely inhibited in human and murine CM by empagliflozin, similar to CaMKII inhibition with AIP (2 μM). In CM lacking CaMKII or the CaMKII phosphosite S571 on Na V 1.5, no effect of empagliflozin on late I Na was present (also not under ATX-II stimulation). As shown before, subsarcolemmal [Na + ] estimated via patch clamp (Nernst equation) was already reduced after 30min (T30 min ). However, empagliflozin did not acutely reduce cytosolic [Na + ] at T30 min , but potently did so after 24h exposure (SBFI measurement). Acute inhibition of NCX with KB-R7943 showed no effect on cardiac Na + , but when performed in addition to empagliflozin, potently reduced [Na + ] already at T30 min . Matching this, NCX forward mode (patch clamp) was acutely induced by empagliflozin at T30 min , thus resulting in Na + influx that initially masks the reduction in [Na + ] resulting from a reduction in late I Na . This may explain why Na + /Ca 2+ homeostasis is only affected after 24h empagliflozin exposure. Our results may explain discrepant results on ECC by different groups on acute vs. prolonged empagliflozin exposure.

In vitro toxicity assessment of polyethylene terephthalate and polyvinyl chloride microplastics using three cell lines from rainbow trout (Oncorhynchus mykiss)

The RTgill-W1 (gill), RTG-2 (gonad), and RTL-W1 (liver) cell lines derived from a freshwater fish rainbow trout (Oncorhynchus mykiss), were used to assess the toxicity of polyethylene terephthalate (PET) and two forms of polyvinyl chloride (PVC). Two size fractions (25-μm and 90-μm particles) were tested for all materials. The highest tested concentration was 1 mg/ml, corresponding to from 70 000 ± 9000 to 620 000 ± 57 000 particles/ml for 25-μm particles and from 2300 ± 100 to 11 000 ± 1000 particles/ml for 90-μm particles (depending on the material). Toxicity differences between commercial PVC dry blend powder and secondary microplastics created from a processed PVC were newly described. After a 24-h exposure, the cells were analyzed for changes in viability, 7-ethoxyresorufin-O-deethylase (EROD) activity, and reactive oxygen species (ROS) generation. In addition to the microplastic suspensions, leachates and particles remaining after leaching resuspended in fresh exposure medium were tested. The particles were subjected to leaching for 1, 8, and 15 days. The PVC dry blend (25 μm and 90 μm) and processed PVC (25 μm) increased ROS generation, to which leached chemicals appeared to be the major contributor. PVC dry blend caused substantially higher ROS induction than processed PVC, showing that the former is not suitable for toxicity testing, as it can produce different results from those of secondary PVC. The 90-μm PVC dry blend increased ROS generation only after prolonged leaching. PET did not induce any changes in ROS generation, and none of the tested polymers had any effect on viability or EROD activity. The importance of choosing realistic extraction procedures for microplastic toxicity experiments was emphasized. Conducting long-term experiments is crucial to detect possible environmentally relevant effects. In conclusion, the tested materials showed no acute toxicity to the cell lines.

Aerosolized aqueous dust extracts collected near a drying lake trigger acute neutrophilic pulmonary inflammation reminiscent of microbial innate immune ligands

BackgroundA high incidence of asthma is prevalent among residents near the Salton Sea, a large inland terminal lake in southern California. This arid region has high levels of ambient particulate matter (PM); yet while high PM levels are often associated with asthma in many environments, it is possible that the rapidly retreating lake, and exposed playa or lakebed, may contribute components with a specific role in promoting asthma symptoms. ObjectivesOur hypothesis is that asthma may be higher in residents closest to the Salton Sea due to chronic exposures to playa dust. Playa emissions may be concentrating dissolved material from the lake, with microbial components capable of inducing pulmonary innate immune responses. To test this hypothesis, we used a mouse model of aerosol exposures to assess the effects of playa dust. MethodsFrom dust collected around the Salton Sea region, aqueous extracts were used to generate aerosols, which were injected into an environmental chamber for mouse exposure studies. We compared the effects of exposure to Salton Sea aerosols, as well as to known immunostimulatory reference materials. Acute 48-h and chronic 7-day exposures were compared, with lungs analyzed for inflammatory cell recruitment and gene expression. ResultsDust from sites nearest to the Salton Sea triggered lung neutrophil inflammation that was stronger at 48-h but reduced at 7-days. This acute inflammatory profile and kinetics resembled the response to innate immune ligands LTA and LPS while distinct from the classic allergic response to Alternaria. ConclusionLung inflammatory responses to Salton Sea dusts are similar to acute innate immune responses, raising the possibility that microbial components are entrained in the dust, promoting inflammation. This effect highlights the health risks at drying terminal lakes from inflammatory components in dust emissions from exposed lakebed.

ODP272 ROUNDUP IS AN IMPORTANT DISRUPTOR THAT CAUSES PROLIFERATIVE EFFECTS ON NORMAL AND ON CANCER THYROID CELLS

Abstract Glyphosate has long be suspected to promote endocrine disruptive and even carcinogenic effects, but little is known about its effect at different concentrations on normal and on cancer thyroid cells. Since the endocrine system responds to low chemical concentrations and abnormal cells may have different sensitivity, it is essential to determine the effects of this compound at different levels, including the Acceptable Occupational Exposure Level (AOEL) and the Acceptable Daily Intake (IDA) doses. We used two human thyroid-derived cell lines strains, Nthy-ori 3-1 (from thyroid normal follicular cells) and TPC-1 (from papillary carcinoma) to test 15 different concentrations of Roundup® Original DI, ranging from 6.5 µg/L to 6.500 µg/L, including AOEL and IDA according to the Technical Note Process No. 25351. 056754/2013-17 of the National Health Surveillance Agency (ANVISA) in Brazil. Trypan blue assay and Cell Counting Kit – 8 (CCK-8) were employed to evaluate cell viability and cytotoxicity. Exposures were made for 24 and 48 hours in technical and biological triplicates. We observed a decrease in the number of viable cells at the 24h exposure compared to 48h in both strains. Exposure of Nthy-ori 3-1 and TPC-1 cells for 24h at the AOEL concentration (160µg/L) caused death of 52% and 58% of the cells, respectively, whereas IDA (830µg/L) resulted in 43% e 58% cell death. These data evidence an important disruptive effect of Roundup Original DI® at the acceptable concentrations. After 48h of exposure, AOEL and IDA concentrations caused a lower number of Nthy-ori 3-1 (19% e 15%) and TPC-1 (29% e 23%) dead cells, respectively, suggesting both thyroid strains have compensatory mechanisms that protect them from longer exposures to the herbicide. The CCK-8 assay, on the contrary, showed a proliferative effect on small concentrations: 6.5 µg/L increased the number of viable Nthy-ori 3-1 and TPC-1 cells to 113% and 118%, respectively, at 24h, and this stimulatory effect persisted after 48h of exposure. In contrast, AOEL and IDA concentrations produced a reduction in the number of both strains cells, likewise observed with the Trypan blue assay. Our data demonstrate that thyroid cells exposed to Roundup Original DI® present a non-monotone dose-response curve, which is a well-known characteristic of an endocrine disruptor. The proliferative effect produced on thyroid cells, especially TPC-1 mutant cells, by very low concentrations of this glyphosate-based herbicide widely used around the world, suggests that it may play a role in the increasing incidence rate of thyroid nodules and/or cancer that have been observed worldwide. Presentation: No date and time listed

Sex-specific Feeding Behavior of Adult House Flies, Musca domestica L. (Diptera: Muscidae).

House flies, Musca domestica L., (Diptera: Muscidae) mechanically vector diverse disease-causing microorganisms while foraging for food in agricultural and urban habitats. Although flies are diverse feeders, nutrient composition of food is important for both fly longevity and reproduction, especially for anautogenous females who require protein for egg production. We investigated whether fly sex and/or mating status influenced their preference for foods with varying macronutrient composition. Presumably mated or unmated male and female flies were separated by sex and offered four food, each in 10% solution offered on cotton wicks: sugar (carbohydrate-rich), fat-free milk (protein-rich, moderate carbohydrate), egg-yolk (protein and lipid-rich), and water (no macronutrients). Foods were colored with nontoxic dyes, which were rotated between replicates. After 4h exposure, flies were dissected to determine the type of food(s) ingested. The interaction of house fly sex and food type significantly influenced food preference, where females preferred milk (protein and carbohydrate-rich food), and males preferred mainly sugar (carbohydrate-rich). Furthermore, 32.8% of females and 10.6% of males foraged on multiple foods. While interaction of sex and mating status had no effect on food preference, milk preference was significantly higher in presumably mated than unmated females. We also tested whether food color influenced fly feeding preference, and found that color was most significant when flies were offered one food type, but negligible when multiple food types were present. This study suggests that bait-based fly control strategies should consider sex-specific preferences for various food attractants if aiming to target and control both male and female house flies.

Residual efficacy of the neonicotinoid insecticide clothianidin against pyrethroid-resistant Aedes aegypti.

Here we report the residual efficacy of the neonicotinoid insecticide clothianidin against pyrethroid-resistant Aedes aegypti. We first conducted a range-finding evaluation of clothianidin on three different substrates (wall, wood, cloth) using three doses (100, 300 and 600 mg a.i.m-2 ) and conducting World Health Organization (WHO) cone bioassays to assess acute (24 h) and delayed (up to 7 days) mortality. In experimental houses located in Merida (Mexico) and using free-flying pyrethroid-resistant Ae. aegypti females, we quantified the acute and delayed mortality after a 24-h exposure to the targeted indoor residual spraying (TIRS) of two clothianidin doses (100 and 300 mg a.i.m-2 ). Range-finding studies with WHO cones showed low (<50%) acute mortality for all surfaces, doses and times post spraying. Delayed mortality was higher, with average values above or close to the 60% mark (and 95% confidence interval estimates crossing 80% for the 600 mg a.i.m-2 dose). In experimental houses, a similar low acute mortality was quantified (range of mortality across 12 months was 2-44% for 100 mg a.i.m-2 and 8-61% for 300 mg a.i/m2 ). However, delayed mortality showed a strong effect of clothianidin on free-flying Ae. aegypti, with values above 80% up to 7months post-TIRS. Novel residual insecticide molecules have a promising outlook for Ae. aegypti control and can contribute to the expansion and adoption of TIRS in urban areas. clothianidin can contribute to the control of resistant Ae. aegypti and provide residual control for up to 7 months after application. © 2022 Society of Chemical Industry.

Acute exposure of early-life stage zebrafish (Danio rerio) to Deepwater Horizon crude oil impairs glomerular filtration and renal fluid clearance capacity.

The pronephros (early-stage kidney) is an important osmoregulatory organ, and the onset of its function occurs relatively early in some teleost fishes. As such, any defects in kidney development and function are likely associated with a decreased ability to osmoregulate. Previous work has shown that early-life stage (ELS) zebrafish (Danio rerio) acutely exposed to Deepwater Horizon (DWH) crude oil exhibit transcriptional changes in key genes involved in pronephros development and function, as well as pronephric morphological defects and whole-animal osmoregulatory impairment. The objective of this study was to examine the acute effects of crude oil exposure during zebrafish ELS on pronephros function by assessing its fluid clearance capacity and glomerular filtration integrity. Following a 72-h exposure to control conditions, 20% or 40% dilutions of high-energy water-accommodated fractions (HEWAF) of DWH crude oil, zebrafish were injected into the common cardinal vein either with fluorescein-labeled (FITC) 70-kDa dextran to assess glomerular filtration integrity or with FITC-inulin to assess pronephric clearance capacity. Fluorescence was quantified after the injections at predetermined time intervals by fluorescence microscopy. The results demonstrated a diminished pronephric fluid clearance capacity and failed glomerular perfusion when larvae were exposed to 40% HEWAF dilutions, whereas only a reduced glomerular filtration selectivity was observed in zebrafish previously exposed to the 20% HEWAF dilution.

Influence of microplastics on the toxicity of chlorpyrifos and mercury on the marine microalgae Rhodomonas lens

The growing use of plastics, including microplastics (MPs), has enhanced their potential release into aquatic environments, where microalgae represent the basis of food webs. Due to their physicochemical properties, MPs may act as carriers of organic and inorganic pollutants. The present study aimed to determine the toxicity of polyethylene MPs (plain and oxidized) and the model pollutants chlorpyrifos (CPF) and mercury (Hg) on the red microalgae Rhodomonas lens, to contribute to the understanding of the effects of MPs and associated pollutants on marine ecosystems, including the role of MPs as vectors of potentially harmful pollutants to marine food webs. R. lens cultures were exposed to MPs (1–1000 μg/L; 25–24,750 particles/mL), CPF (1–4900 μg/L), Hg (1–500 μg/L), and to CPF- and Hg-loaded MPs, for 96 h. Average specific growth rate (ASGR, day−1), cellular viability and pigment concentration (chlorophyll a, c2 and carotenoids) were measured at 48 and 96 h. No significant effects were observed on the growth pattern of the microalgae after 96-h exposure to plain and oxidized MPs. However, a significant increase in cell concentration was detected after 48-h exposure to plain MPs. A decrease of the ASGR was noticed after exposure to CPF, Hg and to CPF/Hg-loaded MPs, whereas viability was affected by exposure to MPs, CPF and Hg, alone and in combination. Chlorophyll a and c2 significantly decreased when microalgae were exposed to plain MPs and CPF, while both pigments significantly increased when exposed to CPF-loaded MPs. Similarly, chlorophyll and carotenoids content significantly decreased after exposure to Hg, whereas a significant increase in chlorophyll a was observed after 48-h exposure to Hg-loaded MPs, at the higher tested concentration. Overall, the presence of MPs modulates the toxicity of Hg and CPF to these microalgae, decreasing the toxic effects on R. lens, probably due to a lower bioavailability of the contaminants.

Fluoride Exposure Provokes Mitochondria-Mediated Apoptosis and Increases Mitophagy in Osteocytes via Increasing ROS Production.

Fluoride is a persistent environmental pollutant, and its excessive intake causes skeletal and dental fluorosis. However, few studies focused on the effects of fluoride on osteocytes, making up over 95% of all bone cells. This study aimed to investigate the effect of fluoride on osteocytes in vitro, as well as explore the underlying mechanisms. CCK-8, LDH assay, fluorescent probes, flow cytometry, and western blotting were performed to examine cell viability, apoptosis, mitochondria changes, reactive oxygen species (ROS) and mitochondrial ROS (mtROS), and protein expressions. Results showed that sodium fluoride (NaF) exposure (4, 8mmol/L) for 24h inhibited the cell viability of osteocytes MLO-Y4 and promoted G0/G1 phase arrest and increased cell apoptosis. NaF treatment remarkably caused mitochondria damage, loss of MMP, ATP decrease, Cyto c release, and Bax/Bcl-2 ratio increase and elevated the activity of caspase-9 and caspase-3. Furthermore, NaF significantly upregulated the expressions of LC-3II, PINK1, and Parkin and increased autophagy flux and the accumulation of acidic vacuoles, while the p62 level was downregulated. In addition, NaF exposure triggered the production of intracellular ROS and mtROS and increased malondialdehyde (MDA); but superoxide dismutase (SOD) activity and glutathione (GSH) content were decreased. The scavenger N-acetyl-L-cysteine (NAC) significantly reversed NaF-induced apoptosis and mitophagy, suggesting that ROS is responsible for the mitochondrial-mediated apoptosis and mitophagy induced by NaF exposure. These findings provide in vitro evidence that apoptosis and mitophagy are cellular mechanisms for the toxic effect of fluoride on osteocytes, thereby suggesting the potential role of osteocytes in skeletal and dental fluorosis.

The single and combined effects of mercury and polystyrene plastic beads on antioxidant-related systems in the brackish water flea: toxicological interaction depending on mercury species and plastic bead size

Plastics are considered as a major threat to marine environments owing their high usage, persistence, and negative effects on aquatic organisms. Although they often exist as mixtures in combination with other pollutants (e.g., mercury (Hg)) in aquatic ecosystems, the combined effects of plastics and ambient pollutants remain unclear. Therefore, in the present study, we investigated the toxicological interactions between Hg and plastics using two Hg species (HgCl2 and MeHgCl) and different-sized polystyrene (PS) beads (diameter: 0.05, 0.5, and 6-μm) in the brackish water flea Diaphanosoma celebensis. The single and combined effects of Hg and PS beads on mortality were investigated, and changes in the antioxidant system and lipid peroxidation were further analyzed. After 48-h exposure to single Hg, HgCl2 induced a higher mortality rate than MeHgCl. The combined exposure test showed that 0.05-μm PS beads can enhance the toxicity of both the Hg species. The expression of GST-mu, glutathione S-transferease (GST) activity and malondialdehyde (MDA) content increased significantly after exposure to Hg alone (HgCl2 or MeHgCl) exposure. Combined exposure with PS beads modulated the effects of Hg on the antioxidant system depending on bead size and the Hg species. In particular, the 0.05-μm beads significantly increased the expression level of GST-mu, GST activity and MDA content, regardless of Hg species. These findings suggest that toxicological interactions between Hg and PS beads depend on the type of Hg species and the size of PS beads; nano-sized 0.05-μm PS beads can induce synergistic toxicity with Hg.

Statins Alleviate Tumor Hypoxia in Prostate Cancer Models by Decreasing Oxygen Consumption: An Opportunity for Radiosensitization?

Background: Because statins were found to decrease the oxygen consumption rate (OCR) of a variety of normal cells, our hypothesis was that statins may also decrease the OCR of cancer cells, alleviate tumor hypoxia and radiosensitize tumors. Methods: OCR was assessed using the Seahorse XF96 technology and EPR respirometry in PC-3 prostate cancer cells. Mitochondrial superoxide production was measured by EPR with mitoTEMPO-H as a sensing probe. Tumor pO2 was measured in vivo using low-frequency EPR oximetry to define the optimal window of reoxygenation, the time at which tumors were irradiated with a single 6 Gy dose with a Cesium-137 irradiator. Results: 24-h exposure to simvastatin and fluvastatin significantly decreased the OCR of PC-3 cancer cells. An increase in mitochondrial superoxide levels was also observed after fluvastatin exposure. The PC-3 prostate cancer model was found highly hypoxic at the basal level. When mice were treated with simvastatin or fluvastatin (daily injection of 20 mg/kg), tumor oxygenation increased 48 and 72 h after initiation of the treatment. However, despite reoxygenation, simvastatin did not sensitize the PC-3 tumor model to RT. Conclusions: exposure to statins affect tumor metabolism and tumor oxygenation, however, with limited impact on tumor growth with or without irradiation.

Sublethal effects of oregano essential oil and its major compound carvacrol on biological parameters of Aedes albopictus (Diptera: Culicidae)

Mosquito management programs rely basically on the use of conventional synthetic larvicides. However, frequent applications and misuse of some synthetic insecticides have led to problems related to mosquito resistance development, harmful effects on human health and unacceptable environmental effects on non-target organisms. Recently, a growing number of phytochemicals has been tested as more eco-friendly larvicides against various mosquito species, exerting high efficacy with multiple modes of action. In the laboratory, we investigated for the first time the sublethal effects of oregano oil and its major compound carvacrol, against Aedes albopictus (Asian tiger mosquito), a mosquito of great medical importance. We determined the effects of short term (24h) exposure of 3rd- 4th larvae to LC50 concentrations on survival and development of survived larvae until adulthood, as well as on fecundity, fertility, longevity and wing length of emerged adults. Only half of 24h survived larvae from oregano oil and carvacrol treatment finally reached adulthood. Abnormal shapes of dead larvae and pupae, and failed adult emergence were also observed, indicating a potential growth inhibitory activity of the tested materials. No particular effects from exposure to larvicidal LC50 concentrations were recorded on life cycle parameters of successfully emerged adults. These findings suggest the tested oregano oil and carvacrol as sufficiently effective larvicides against Ae. albopictus at lower than the acutely toxic concentrations, promoting a more eco-friendly and less costly profile for these biopesticides.

Eco-toxicological effect of a commercial dye Rhodamine B on freshwater microalgae Chlorella vulgaris.

In this study, the acute toxicity effects of a fluorescent xanthene dye, Rhodamine B (RhB), widely used in textile, paper, and leather industries was investigated on a freshwater microalgae Chlorella vulgaris. The acute toxicity of RhB on C. vulgaris was determined by examining the growth, cell morphology, pigment production, protein content, and the activities of oxidative stress enzymes. Based on the results of the toxicity study of 24-96h, the median inhibitory concentration (IC50) values ranged from 69.94 to 31.29mgL-1. The growth of C. vulgaris was conspicuously inhibited by RhB exposure, and the cell surfaces appeared to be seriously shrunk in SEM analysis. The growth of C. vulgaris was hindered after exposure to graded concentrations (10-50mgL-1) of RhB. A significant reduction in growth rate, pigment synthesis (chlorophyll a, chlorophyll b, and carotenoid), and protein content was recorded in a dose-dependent manner. After 96h exposure of C. vulgaris to 50mgL-1 RhB, chlorophyll a, chlorophyll b, carotenoids, and protein contents were reduced by 71.59, 74.90, 65.84, and 74.20%, respectively. The activities of the antioxidant enzymes peroxidase (POD), and catalase (CAT) also increased markedly in the presence of RhB. A notable effect was observed on oxidative enzymes catalase and peroxidase, indicating that oxidative stress may be the primary factor in the inhibition of growth and pigment synthesis. Consequently, the experimental acute toxicity data were compared to the QSAR prediction made by the ECOSAR programme. Results showed that the experimental acute toxicity values were 67.74-fold lower than the ECOSAR predicted values. The study provides convincing evidence for the metabolic disruption in the ubiquitous microalgae C. vulgaris due to the RhB dye toxicity.

Determining Toxic Potencies of Water-Soluble Contaminants in Wastewater Influents and Effluent Using Gene Expression Profiling in C. elegans as a Bioanalytical Tool

With chemical analysis, it is impossible to qualify and quantify the toxic potency of especially hydrophilic bioactive contaminants. In this study, we applied the nematode C. elegans as a model organism for detecting the toxic potency of whole influent wastewater samples. Gene expression in the nematode was used as bioanalytical tool to reveal the presence, type and potency of molecular pathways induced by 24-h exposure to wastewater from a hospital (H), nursing home (N), community (C), and influent (I) and treated effluent (E) from a local wastewater treatment plant. Exposure to influent water significantly altered expression of 464 genes, while only two genes were differentially expressed in nematodes treated with effluent. This indicates a significant decrease in bioactive pollutant-load after wastewater treatment. Surface water receiving the effluent did not induce any genes in exposed nematodes. A subset of 209 genes was differentially expressed in all untreated wastewaters, including cytochromes P450 and C-type lectins related to the nematode’s xenobiotic metabolism and immune response, respectively. Different subsets of genes responded to particular waste streams making them candidates to fingerprint-specific wastewater sources. This study shows that gene expression profiling in C. elegans can be used for mechanism-based identification of hydrophilic bioactive compounds and fingerprinting of specific wastewaters. More comprehensive than with chemical analysis, it can demonstrate the effective overall removal of bioactive compounds through wastewater treatment. This bioanalytical tool can also be applied in the process of identification of the bioactive compounds via a process of toxicity identification evaluation.Graphical abstract

Nematicidal Potential of Sulla (Hedysarum coronarium L.) against the Root-Knot Nematode Meloidogyne incognita.

The content of nematicidal metabolites such as saponins, flavonoids and tannins in sulla (Hedysarum coronarium L.) suggests its potential nematicidal activity. In this study, the biocidal activity of 62.5–1000 μg mL−1 concentrations of flavonoid and tannin fractions from sulla was assessed in in vitro assays on the infective juveniles (J2) of the root-knot nematode (RKN) Meloidogyne incognita, while the suppressive effects of soil amendments with 10–40 g kg−1 soil rates of sulla biomass were investigated on potted tomato infested by M. incognita. The content of total nitrogen, carbon, flavonoids, tannins and saponins of sulla experimental material was also determined. After a 96-h exposure, more than 80% of the M. incognita J2 were killed even by a 125 µg mL−1 concentration of the flavonoid extract, while mortality peaked at 89% only at the 1000 µg mL−1 concentration of the tannin solution. Soil incorporation with sulla biomass significantly reduced the M. incognita densities both on tomato roots and in the soil, compared to either the non-treated control and chemical treatment with Fluopyram. The data confirmed the nematicidal potential of sulla, mainly due to its content of flavonoids and tannins, suggesting its suitability as green manure or a soil amendment for sustainable RKN management.