Acute Toxicity Assay Research Articles

Isolation and characterization of Bifidobacterium spp. From breast milk with different human milk oligosaccharides utilization and anti-inflammatory capacity

Breast milk is the best source of nutrition for infants. Human milk oligosaccharides (HMOs) and the corresponding HMOs-consuming Bifidobacterium positively influence infant health. This study aims to isolate and characterize Bifidobacterium from breast milk of healthy Chinese mothers, identifying the most efficacious strains for inclusion in simulated maternal milk formulas. Nine Bifidobacterium strains (two of B. breve and seven of B. infantis) were isolated, exhibiting a broad spectrum of probiotic potential. This included tolerance to simulated infant gastrointestinal conditions, notable adhesion, antibacterial, antioxidant activities, and HMOs utilization ability. Lacto-N-Tetraose (LNT) is preferred in early growth among Bifidobacterium isolates. B. breve showed a preference for LNT, whereas B. infantis showed a preference for fucosylated HMOs, and displayed reduced utilization of sialylated HMOs. They also exhibited robust safety profiles, including no hemolytic activity, an appropriate D/L lactate-producing ratio, and non-toxicity in an acute oral toxicity assay on mice. It is noteworthy that B. breve N-90, O-147, B. infantis O-161 and R-1 exhibited anti-inflammatory effects in LPS-induced RAW 264.7 cells. Specifically, a notable reduction in TNF-α levels was observed in pre-treatment, while a decrease in IL-1β and IL-6 levels in co-treatment. B. breve N-90 and B. infantis R-1 were identified finally as promising probiotic candidates. Their whole-genome sequencing analysis confirmed presence of functional genes associated with gastrointestinal colonization, antioxidation, and glycoside hydrolase activity on HMOs. The annotation for antibiotic resistance and virulence genes concurred with phenotypes, further validating the safety. Breast milk is a good source for Bifidobacteria isolation, while Bifidobacteria utilize HMOs in a strain-dependent manner. The two selected strains, B. breve N-90 and B. infantis R-1, are potential candidates for inclusion in simulated maternal milk formulas and deserved further in vivo investigation for their health-promoting effects.

Uncovering cocoa shell as a safe bioactive food ingredient: Nutritional and toxicological breakthroughs

This work aimed to analyze the chemical composition of the cocoa shell flour (CSF) and aqueous extract (CSE) and conduct acute and sub-chronic toxicity assays to ensure safety. CSF was composed of 59.1 % dietary fiber, primarily insoluble fiber (48.1 %). CSE consisted entirely of soluble fiber (72.8 %), mainly pectic polysaccharides. The amino acid profile revealed CSF's potential as a high-quality plant protein source, whereas CSE was rich in free amino acids, especially leucine, valine, and alanine (59.6–62.5 mg 100 g−1). Higher levels of macroelements, especially potassium, were found in CSE. Silicon was the most abundant microelement, and heavy metal levels were within acceptable limits for both cocoa shell matrices. This study confirmed the bioactive potential of CSF and CSE, enriched in phenolic compounds and methylxanthines, including theobromine, caffeine, gallic acid, (+)-catechin, as well as N-phenylpropenoyl-l-amino acids. The high theobromine content in both matrices, especially in CSE (2605.3 mg 100 g−1), did not harm the mice's health; acute and sub-chronic toxicity studies demonstrated the safety of oral administration of CSF and CSE in mice, showing no lethality or remarkable histopathological changes. This research supports the safe use of CSF and CSE, establishing these ingredients as suitable for incorporation into food products.

Unveiling the Nutritional Profile and Safety of Coffee Pulp as a First Step in Its Valorization Strategy.

The coffee pulp, a significant by-product of coffee processing, is often discarded but has potential for recycling and high-value uses. This study aimed to investigate the chemical composition of two coffee pulp ingredients, a flour (CPF) and an aqueous extract (CPE), and conducted acute and sub-chronic toxicity assays to determine their safety. The proximate composition revealed the high fiber content of both ingredients; the CPF mainly contained insoluble fiber, while CPE consisted exclusively of soluble pectic polysaccharides. The CPF had higher concentrations of amino acids and a better balance of essential/non-essential amino acids, whereas the CPE exhibited higher concentrations of free amino acids, ensuring higher bioavailability. Both ingredients showed elevated mineral content, while heavy-metal concentrations remained within acceptable limits. This study established the bioactive potential of the CPF and the CPE, demonstrating the high content of caffeine and gallic, protocatechuic, and 4-caffeoylquinic acids. The toxicity studies revealed that the CPF and the CPE exhibited safety when orally administered to mice. Administered doses were non-toxic, as they did not induce lethality or adverse effects in the mice or produce significant histopathological or biochemical adverse changes. This study represents a first step in valorizing the CPF and the CPE as safe novel food ingredients with health benefits for functional and nutritional foods.

Sensitivity assessment of Biomphalaria glabrata (SAY, 1818) using reference substance sodium dodecyl sulfate for ecotoxicological analyzes.

Sodium dodecyl sulfate (SDS) is a surfactant used and recommended by regulatory agencies as a reference substance in ecotoxicological analyzes. In this work, acute toxicity assays were performed with adults and embryos of the freshwater snail Biomphalaria glabrata, an endemic organism with environmental and public health importance, to evaluate the effects of the surfactant and establish a sensitivity control chart. The organisms were exposed to SDS for 24 h to a range of concentrations, as well as a control group. Six assays were performed to establish the control chart for adults (with a median LC50 of 36.87 mg L-1) and differential sensitivity was observed at each embryonic stage (EC50 = blastulae 33.03, gastrulae 35.03, trochophore 39.71 and veliger 72.55 mg L-1). The following behavioral responses were observed in exposed adult snails: release of hemolymph and mucus, body outside the shell, and penile overexposure. Embryos at the blastulae and gastrulae stages were more sensitive, and teratogenic effects were accentuated in the trochophore stage. The difference in results obtained between adults and embryos underscore the importance of carrying out analyzes at different developmental stages. The serial assays established with SDS for B. glabrata demonstrated efficiency and constancy conditions in the assays with good laboratory practice standards. The wide distribution of Biomphalaria species in several countries, their easy maintenance and cultivation in the laboratory, in addition to ecological importance, make them economical alternatives for ecotoxicological assays.

Cryptic species complex shows population-dependent, rather than lineage-dependent tolerance to a neonicotinoid

Cryptic species are rarely considered in ecotoxicology, resulting in misleading outcomes when using a single morphospecies that encompasses multiple cryptic species. This oversight contributes to the lack of reproducibility in ecotoxicological experiments and promotes unreliable extrapolations. The important question of ecological differentiation and the sensitivity of cryptic species is rarely tackled, leaving a substantial knowledge gap regarding the vulnerability of individual cryptic species within species complexes. In times of agricultural intensification and the frequent use of pesticides, there is an urgent need for a better understanding of the vulnerability of species complexes and possible differences in adaptive processes. We used the cryptic species complex of the aquatic amphipod Gammarus roeselii, which comprises at least 13 genetic mtDNA lineages and spans from small-scale endemic lineages in Greece to a large-scale widely distributed lineage in central Europe. We exposed eleven populations belonging to four lineages to the neonicotinoid thiacloprid in an acute toxicity assay. We recorded various environmental variables in each habitat to assess the potential pre-exposure of the populations to contaminants. Our results showed that the populations differed up to 4-fold in their tolerances. The lineage identity had a rather minor influence, suggesting that the cryptic species complex G. roeselii does not differ significantly in tolerance to the neonicotinoid thiacloprid. However, the observed population differentiation implies that recent pre-exposure to thiacloprid (or similar substances) or general habitat contamination has triggered adaptive processes. Though, the extent to which these mechanisms are equally triggered in all lineages needs to be addressed in the future. Our study provides two key findings: Firstly, it shows that observed phylogenetic differences within the G. roeselii species complex did not reveal differences in thiacloprid tolerance. Second, it confirms that differentiation occurs at the population level, highlighting that susceptibility to toxicants is population-dependent. The population-specific differences were within the range of accepted intraspecific variability from a regulatory standpoint. From an evolutionary-ecological perspective, it remains intriguing to observe how persistent stresses will continue to influence tolerance and whether different populations are on distinct pathways of adaptation. Given that the potential selection process has only lasted a relatively short number of generations, it is crucial to monitor these populations in the future, as even brief exposure periods significantly impact evolutionary responses.

In silico, in vitro, and in vivo acute and sub-acute toxicity profiling of whole plant methanol extract of Equisetum diffusum D. Don from the sub-Himalayan West Bengal, India, having ethnobotanical uses

BackgroundEquisetum diffusum D. Don commonly known as ‘Himalayan horsetail’, has been traditionally used in the treatment of back pain, bone fracture and dislocation, and arthritis by various tribal communities of India. Our previous study confirmed the anti-inflammatory efficacy of the plant through in silico, in vitro, and in vivo model studies. Therefore, the current research is focused on safety dose evaluation for the first-time of the whole-plant methanol extract (EDME) of E. diffusum through appropriate in silico, in vitro, and in vivo approaches.MethodThe whole plant, along with its rhizomes, was collected, and the methanol extract was prepared. The in silico ADMET study was performed to predict the pharmacokinetics profile and toxicity of all the identified phyto-compounds of EDME previously screened by GC–MS study. In vitro cytotoxicity study of EDME was performed using two cell lines: kidney (HEK293) and liver (Huh7) cell lines. The in vivo toxicity study of EDME was validated by the acute toxicity (OECD 423, 2002) and sub-acute toxicity assays (OECD 407, 2008) in the Wistar Albino rat model.ResultsThe in silico ADMET study of all 47 bioactives predicted good pharmacokinetic and low toxicity profiles. In vitro cytotoxicity showed higher IC50 values of EDME viz., 672 ± 15.7 μg/mL and 1698 ± 6.54 μg/mL for both kidney (HEK293) and liver (Huh7) cell lines, respectively, which were considered as low-toxic. Based on acute oral toxicity, the LD50 value of the extract was considered “non-toxic” up to a feeding range of 2000 mg/kg of body weight. The regular consumption of the extract for an extended period (28 days) was also qualified as safe based on the body and organ weight, hematological, biochemical, and histoarchitecture results in the sub-acute toxicity assay.ConclusionThe detailed in silico, in vitro, in vivo (acute and sub-acute oral toxicity) studies gave us a new insight to the safety dose evaluation of Equisetum diffusum, which may serve as a reliable documentation for undertaking the experimental validation of the ethnobotanical uses of the plant which would help in the field of drug development for the treatment of inflammation related complications.

Novel collagen gradient membranes with multiphasic structures: Preparation, characterization, and biocompatibility

Scaffolds with multiphasic structures are considered to be superior for guided tissue regeneration. Two types of tilapia skin collagen gradient membranes (stepped gradient and linear gradient) with multiphasic structures were prepared by controlling the collagen concentrations and the freezing rates. The results revealed that collagen gradient membranes were more capable of guiding tissue regeneration compared to homogeneous membranes. These two gradient membranes featured a dense outer layer and a loose inner layer, with good mechanical properties as indicated by tensile strengths of more than 250 Kpa and porosities exceeding 85 %. Additionally, these membranes also showed good hydrophilicity and water absorption, with an inner layer contact angle of less than 91° and a water absorption ratio greater than 40 times. Furthermore, the multiphasic scaffolds were proved to be biocompatible by the acute toxicity assay, the intradermal irritation test and so on. Gradient membranes could effectively promote the adhesion and proliferation of fibroblasts and osteoblasts, through elevating the TGF-β/Smad signaling pathway by TGF-β and Smads, and activating the Wnt/β-catenin signaling pathway by LRP5 and β-catenin, similar to homogenous membranes. Therefore, collagen gradient membranes from tilapia skin show important application value in guiding tissue regeneration.

Hexyltrimethylammonium ion enhances potential copper-chelating properties of ammonium thiomolybdate in an in vivo zebrafish model

Ammonium and hexyltrimethylammonium thiomolybdates (ATM and ATM-C6) and thiotungstates (ATT and ATT-C6) were synthesized. Their toxicity was evaluated using both in vitro and in vivo approaches via the zebrafish embryo acute toxicity assay (ZFET), while the copper-thiometallate interaction was studied using cyclic voltammetry, as well as in an in vivo assay. Cyclic voltammetry suggests that all thiometallates form complexes with copper in a 2:1 Cu:thiometallate ratio. Both in vitro and in vivo assays demonstrated low toxicity in BALB/3T3 cells and in zebrafish embryos, with high IC50 and LC50 values. Furthermore, the hexyltrimethylammonium ion played a crucial role in enhancing viability and reducing toxicity during prolonged treatments for ATM and ATT. In particular, the ZEFT assay uncovered the accumulation of ATM in zebrafish yolk, averted by the incorporation of the hexyltrimethylammonium ion. Notably, the copper-thiometallate interaction assay highlighted the improved viability of embryos when cultured in CuCl2 and ATM-C6, even at high CuCl2 concentrations. The hatching assay further confirmed that copper-ATM-C6 interaction mitigates inhibitory effects induced by thiomolybdates and CuCl2 when administered individually. These results suggest that the incorporation of the hexyltrimethylammonium ion in ATM increase its ability to interact with copper and its potential application as a copper chelator.

Assessment of acute toxicity, genotoxicity, and anti-inflammatory activity of SteLL, a lectin from Schinus terebinthifolia Raddi. Leaves, in mice

Ethnopharmacology relevanceSchinus terebinthifolia Raddi (Anacardiaceae), known as Brazilian pepper tree, stands out as a medicinal plant widely used in traditional medicine. The leaves are popularly used as anti-inflammatory agent and to relieve inflammatory conditions such as bronchitis, ulcers, and wounds, for example. Aim of the studyThe present study evaluated the acute toxicity, genotoxicity, and anti-inflammatory activity of S. terebinthifolia leaf lectin (SteLL) in mice (Mus musculus). Materials and methodsIn the acute toxicity assay, the animals were treated intraperitoneally (i.p.) or orally (per os) with a single dose of 100 mg/kg. Genotoxicity was assessed by the comet and micronucleus assays. Carrageenan-induced peritonitis and paw edema models were used to evaluate the anti-inflammatory effects of SteLL (1, 5 and 10 mg/kg, i.p.). ResultsNo animal died and no signs of intoxication or histopathological damage were observed in the acute toxicity assay. Genotoxic effect was not detected. In peritonitis assay, SteLL reduced in 56–69% leukocyte migration to the peritoneal cavity; neutrophil count decreased by 25–32%, while mononuclear cell count increased by 67–74%. SteLL promoted a notable reduction of paw edema after 4 h (61.1–63.4%). Morphometric analysis showed that SteLL also decreased the thickness of epidermal edema (30.2–40.7%). Furthermore, SteLL decreased MPO activity, plasma leakage, NO release, and modulated cytokines in both peritoneal fluid and paw homogenate. ConclusionSteLL did not induce acute toxicity or genotoxicity in mice and stands out as a promising candidate in the development of new phytopharmaceuticals with anti-inflammatory action.

Discovery of α-hexyl cinnamaldehyde and its derivatives as novel larvicides against Aedes albopictus

Mosquitoes are the vectors of numerous diseases such as malaria, Dengue and Zika. Controlling mosquito vectors is essential for preventing and managing diseases transmitted by mosquitoes. Larvicides target larvae within their breeding habitats before they mature into adult mosquitoes and disperse. In this study, using α-hexyl cinnamaldehyde (HCA) as a lead compound, three series of derivatives comprising a total of 78 compounds were designed and synthesized. The larvicidal activities of these compounds against Aedes albopictus were evaluated, resulting in the identification of ten promising compounds. Among them, furan-3-ylmethyl cinnamate (compound 2v) and (E)-allyl 3-(4-chlorophenyl)acrylate (compound 4c) exhibited the lowest LC50 values (8.0 ppm and 7.2 ppm respectively). In a 96-h acute toxicity assay conducted on zebrafish, 2v showed no or very low toxicity with an LC50 higher than 100 ppm. To elucidate their mode of action, morphological and histopathologic analyses were performed, and the effects of the compounds on the acetylcholinesterase (AChE) and phosphatase activities were evaluated. Results suggested that the active HCA derivatives induce injury to intestinal epithelial cells by inhibiting phosphatase activity, ultimately resulting in the mortality of mosquito larvae.

Discovery of novel benzimidazole derivatives as selective and reversible monoamine oxidase B inhibitors for Parkinson's disease treatment

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. The development of novel scaffolds for human monoamine oxidase B (hMAO-B) inhibitors with reversible properties represents an important strategy to improve the efficacy and safety for PD treatment. In the current work, we have devised and assessed two innovative derivative series serving as hMAO-B inhibitors. These series have utilized benzimidazole as a scaffold and strategically incorporated a primary amide group, which is recognized as a pivotal pharmacophore in subsequent activity screening and reversible mode of action. Among these compounds, 16d has emerged as the most potent hMAO-B inhibitor with an IC50 value of 67.3 nM, comparable to safinamide (IC50 = 42.6 nM) in vitro. Besides, 16d demonstrated good selectivity towards hMAO-B isoenzyme with a selectivity index over 387. Importantly, in line with the design purpose, 16d inhibited hMAO-B in a competitive and reversible manner (Ki = 82.50 nM). Moreover, 16d exhibited a good safety profile in both cellular and acute toxicity assays in mice. It also displayed ideal pharmacokinetic properties and blood-brain barrier permeability in vivo, essential prerequisites for central nervous system medicines. In the MPTP-induced PD mouse model, 16d significantly alleviated the motor impairment, especially muscle relaxation and motor coordination. Therefore, 16d, serving as a lead compound, holds instructive significance for subsequent investigations regarding its application in the treatment of PD.

Ecological risk evaluation of spent engine oil pollution using earthworm and microbial bioassays

The study aimed to assess the ecotoxicological risk associated with the indiscriminate disposal of spent engine oil on terrestrial ecosystem using earthworm and microbial assays. Soil samples were collected from a depth of 0-20 cm and subjected to standard analytical protocols for analysis. Earthworms (assessed by mortality rate) and microorganisms (evaluated for inhibitory effects) covered a wide range of short-term lethal and sub-lethal endpoints used for risk characterization, analyzed through Probit analysis. The result of acute toxicity assay revealed that microbial absorbance rated depended on the dose and type of organism, ranking in the order: Acinetobacter > Enterobacter > Bacillus species >Pseudomonas. Aas oil concentratoin increase, mortality among earthworm was observed. Risk Quotient (RQ) values for Zea mays, Vigna unguiculata, Glycine max and earthworm varied from low to very high risk based on estimated Predicted No Effect Concentration (PNEC) values. Microorganisms exhibited differing level of biotolerance to spent engine oil exposure as indicated by respective risk quotient values. Exposure to spent engine oil posed minimal risk to Pseudomonas sp., Bacillus sp., and Acinetobacter sp., with RQ values below unity (< 1). Conversely, Enterobacter sp. Showed a high risk with values above unity. Earthworms play a pivotal role in agriculture due to their numerous economic benefots. Soil microorganisms are essential for maintening soil quality by performing vital processes. The antimicrobial properties of spent engine oil on soil may distort microbial activities, potentially inhibiting their growth and leading to alterations in ecological functionality of the soil.

Phytochemical Profile, Antioxidant, Enzyme Inhibition, Acute Toxicity, In Silico Molecular Docking and Dynamic Analysis of Apis Mellifera Propolis as Antidiabetic Supplement.

This study aims to identify the phytochemical profile of Apis mellifera propolis and explore the potential of its anti-diabetic activity through inhibition of α-amylase (α-AE), α-glucosidase(α-GE), as well as novel antidiabetic compounds of propolis. Apis mellifera propolis extract (AMPE) exhibited elevated polyphenol 33.26±0.17 (mg GAE/g) and flavonoid (15.45±0.13 mg RE/g). It also indicated moderate strong antioxidant activity (IC50 793.09±1.94 μg/ml). This study found that AMPE displayed promising α-AE and α-GE inhibition through in vitro study. Based on LC-MS/MS screening, 18 unique AMPE compounds were identified, with majorly belonging to anthraquinone and flavonoid compounds. Furthermore, in silico study determined that 8 compounds of AMPE exhibited strong binding to α-AE that specifically interacted with its catalytic residue of ASP197. Moreover, 2 compounds exhibit potential inhibition of α-GE, by interacting with crucial amino acids of ARG315, ASP352, and ASP69. Finally, we suggested that 2,7-Dihydroxy-1-(p-hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene and 3(3-(3,4-Dihydroxybenzyl)-7-hydroxychroman-4-one as novel inhibitors of α-AE and α-GE. Notably, these compounds were initially discovered from Apis mellifera propolis in this study. The molecular dynamic analysis confirmed their stable binding with both enzymes over 100 ns simulations. The in vivo acute toxicity assay reveals AMPE as a practically non-toxic product with an LD50 value of 16,050 mg/kg. Therefore, this propolis may serve as a promising natural product for diabetes mellitus treatment.

A group of emerging heterocyclic nitrogenous disinfection byproducts: Formation and cytotoxicity of halopyridinols in drinking water

Recently, a new group of halopyridinol disinfection byproducts (DBPs) was reported in drinking water. The in vivo developmental and acute toxicity assays have shown that they were more toxic than a few commonly known aliphatic DBPs such as bromoform and iodoacetic acid. However, many pyridinol DBPs with the same main structures but different halogen substitutions were still unknown due to complicated water quality conditions and various disinfection methods applied in drinking water treatment plants. Studies on their transformation mechanisms in drinking water disinfection were quite limited. In this study, comprehensive detection and identification of halopyridinols were conducted, and five new halopyridinols were first reported, including 2-chloro-3-pyridinol, 2,6-dichloro-3-pyridinol, 2-bromo-5-chloro-3-pyridinol, 2,4,6-trichloro-3-pyridinol and 2,5,6-trichloro-3-pyridinol. Formation conditions and mechanisms of the halopyridinols were explored, and results showed that chlorination promoted their formation compared with chloramination. Halopyridinols were intermediate DBPs that could undergo further transformation/degradation with increasing contact time, disinfectant dose, bromide concentration, and pH. The in vitro cytotoxicity of the halopyridinols was evaluated using human hepatocellular carcinoma cells. Results showed that the cytotoxicity of 3,5,6-trichloro-2-pyridinol was the highest (EC50 = 474.3 μM), which was 13.0 and 1.6 times higher than that of 2-bromo-3-pyridinol (EC50 = 6214.5 μM) and tribromomethane (EC50 = 753.6 μM), respectively.

Comprehensive analysis of cyanobacterial secondary metabolites distribution and toxicity in urban water bodies

This study addresses the increasing concern regarding cyanotoxin contamination of water bodies, highlighting the diversity of these toxins and their potential health implications. Cyanobacteria, which are prevalent in aquatic environments, produce toxic metabolites, raising concerns regarding human exposure and associated health risks, including a potential increase in cancer risk. Although existing research has primarily focused on well-known cyanotoxins, recent technological advancements have revealed numerous unknown cyanotoxins, necessitating a comprehensive assessment of multiple toxin categories. To enhance the cyanotoxin databases, we optimized the CyanoMetDB cyanobacterial secondary metabolites database by incorporating secondary fragmentation patterns using the Mass Frontier fragmentation data prediction software. Water samples from diverse locations in Shanghai were analyzed using high-resolution mass spectrometry. Subsequently, the toxicity of cyanobacterial metabolites in the water samples was examined through acute toxicity assays using the crustacean Thamnocephalus platyurus. After 24 h of exposure, the semi-lethal concentrations (LC50) of the water samples ranged from 0.31 mg L−1 to 1.78 mg L−1 (MC-LR equivalent concentration). Our findings revealed a critical correlation between the overall concentration of cyanobacterial metabolites and toxicity. The robust framework and insights of this study underscore the need for an inclusive approach to water quality management, emphasizing continuous efforts to refine detection methods and comprehend the broader ecological impact of cyanobacterial blooms on aquatic ecosystems.

Toxicity and Phytochemical Analysis of Five Medicinal Plants

Recent studies have shown that long-term uses of herbs have been associated with a rise in morbidity and mortality rates. While most researches are focused on bioactivity investigations, the toxicity of many plants has not been reported. There is a paucity of data on the potential toxicity of the following plants: Harungana madagascariensis (HM), Pterocarpus osun (PO), Phoenix dactylifera (PD), Annona muricata (AM), and Rutidea parviflora (RP). To evaluate the toxicity of the above-mentioned plants; two tests were employed namely: The Brine shrimp lethality test (BSLT) and the Allium cepa test. A correlation between the oral acute toxicity assay in mice and the LC50 obtained from BSLT has been established. Allium cepa test measures the potential genotoxic effects of plant extracts exerted on the root meristem of A. cepa (onions). Plant extracts were administered in concentrations ranging from 100 to 2500 µg/ml to the A. cepa for 72 h to obtain their Mitotic Indices (MI) and EC50. Results of the MI at 2500 µg/ml for HM, PO, PD, AM, and RP were 3.75, 4.96, 5.96, 6.10, and 6.71 while 281.81, 398.11, 501.19, 630.96, and 707.9 µg/ml were obtained as the respective EC50 values. Furthermore, 10-1000 mcg/ml concentrations were administered in the BSLT and the obtained LC50 values were 116.3, 250, 581.5, 581.5, and 750 µg/ml. The toxicity result demonstrated that the five plants were moderately toxic, with RP exhibiting minimal toxicity values and thus potentially having a good safety profile. The phytochemical screening of these plants revealed the presence of some pharmacologically important classes of compounds that are abundant. Several bioactive and toxic compounds were identified in the GC-MS analysis for some of the plants.

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.

Toxicity of butylone and its enantiomers to Daphnia magna and its degradation/toxicity potential using advanced oxidation technologies

Butylone (BTL) is a chiral synthetic cathinone available as a racemate and reported as contaminant in wastewater effluents. However, there are no studies on its impact on ecosystems and possible enantioselectivity in ecotoxicity. This work aimed to evaluate: (i) the possible ecotoxicity of BTL as racemate or its isolated (R)- and (S)- enantiomers using Daphnia magna; and (ii) the efficiency of advanced oxidation technologies (AOTs) in the removal of BTL and reduction of toxic effects caused by wastewaters. Enantiomers of BTL were obtained by liquid chromatography (LC) using a chiral semi-preparative column. Enantiomeric purity of each enantiomer was > 97 %. For toxicity assessment, a 9-day sub-chronic assay was performed with the racemate (at 0.10, 1.0 or 10 μg L−1) or each enantiomer (at 0.10 or 1.0 μg L−1). Changes in morphophysiological, behavioural, biochemical and reproductive endpoints were observed, which were dependent on the form of the substance and life stage of the organism (juvenile or adult). Removal rates of BTL in spiked wastewater (10 μg L−1) treated with different AOTs (ultraviolet, UV; ozonation, O3; and UV/O3) were similar and lower than 29 %. The 48 h D. magna acute toxicity assays demonstrated a reduction in the toxicity of the treated spiked effluents, but no differences were found amongst AOTs treatments. These results warn for the contamination and negative impact of BTL on ecosystems and highlight the need for efficient removal processes.

Acute and Subchronic Toxicological Study of the Cocktail Extract from Curcuma xanthorrhiza Roxb, Phyllanthus niruri L. and Morinda citrifolia L.

Curcuma xanthorrhiza Roxb, Phyllanthus niruri L., and Morinda citrifolia L. are Indonesian medicinal herbs used empirically as traditional therapeutics for maintaining health. The cocktail extract of these three plants (CECPM) had been developed and demonstrated immunostimulant activity in rats. This study aimed to evaluate the acute and subchronic toxicity of CECPM in vivo. The acute toxicity assay was conducted by orally administering a range dose of CECPM (313, 625, 1250, 2500, or 5000 mg/kg body weight (bw) on female mice once and then evaluating the toxic symptom every day for 14 days later. The chronic toxicity test was carried out by giving various doses of CECPM (600, 800, and 1000 mg/kg·bw) to female and male rats orally continuously for 90 consecutive days. The signs of toxicities were evaluated at the 90- and 28 days postadministration. The acute oral toxicity assays showed that there was no toxic syndrome and mortality found during the period of the experiment. The lethal dose level (LD50) of CECPM was more than 5000 g/kg, which was categorized as practically non-toxic. Meanwhile, in the sub-chronic toxicity study, some parameters tested at 90 days postadministration and after 28 days of withdrawal, such as the body weight, relative organ weight, food intake, hematological and biochemical blood parameters, and also histopathological examination of five primary tissues (heart, liver, kidney, spleen, and lung) revealed no abnormalities. There was no-observed adverse effect level (NOAEL) for the present study of CECPM 1000 mg/kg·bw of the rat. Therefore, it is concluded that the orally administered CECPM was relatively nontoxic during acute and subchronic toxicology studies.

A novel pan-PI3K inhibitor KTC1101 synergizes with anti-PD-1 therapy by targeting tumor suppression and immune activation

BackgroundPhosphoinositide 3-kinases (PI3Ks) are critical regulators of diverse cellular functions and have emerged as promising targets in cancer therapy. Despite significant progress, existing PI3K inhibitors encounter various challenges such as suboptimal bioavailability, potential off-target effects, restricted therapeutic indices, and cancer-acquired resistance. Hence, novel inhibitors that overcome some of these challenges are needed. Here, we describe the characterization of KTC1101, a novel pan-PI3K inhibitor that simultaneously targets tumor cell proliferation and the tumor microenvironment. Our studies demonstrate that KTC1101 significantly increases the anti-PD-1 efficacy in multiple pre-clinical mouse models.MethodsKTC1101 was synthesized and characterized employing chemical synthesis, molecular modeling, Nuclear Magnetic Resonance (NMR), and mass spectrometry. Its target specificity was confirmed through the kinase assay, JFCR39 COMPARE analysis, and RNA-Seq analysis. Metabolic stability was verified via liver microsome and plasma assays, pharmacokinetics determined by LC–MS/MS, and safety profile established through acute toxicity assays to determine the LD50. The antiproliferative effects of KTC1101 were evaluated in a panel of cancer cell lines and further validated in diverse BALB/c nude mouse xenograft, NSG mouse xenograft and syngeneic mouse models. The KTC1101 treatment effect on the immune response was assessed through comprehensive RNA-Seq, flow cytometry, and immunohistochemistry, with molecular pathways investigated via Western blot, ELISA, and qRT-PCR.ResultsKTC1101 demonstrated strong inhibition of cancer cell growth in vitro and significantly impeded tumor progression in vivo. It effectively modulated the Tumor Microenvironment (TME), characterized by increased infiltration of CD8+ T cells and innate immune cells. An intermittent dosing regimen of KTC1101 enhanced these effects. Notably, KTC1101 synergized with anti-PD-1 therapy, significantly boosting antitumor immunity and extending survival in preclinical models.ConclusionKTC1101's dual mechanism of action—directly inhibiting tumor cell growth and dynamically enhancing the immune response— represents a significant advancement in cancer treatment strategies. These findings support incorporating KTC1101 into future oncologic regimens to improve the efficacy of immunotherapy combinations.