Thiazolidine derivatives: In vitro toxicity assessment against promastigote and amastigote forms of Leishmania infantum and ultrastructural study.

Neglected diseases, such as Leishmaniasis, constitute a group of communicable diseases that occur mainly in tropical countries. Considered a public health problem with limited treatment. Therefore, there is a need for new therapies. In this sense, our proposal was to evaluate in vitro two series of thiazolidine compounds (7a-7e and 8a-8e) against Leishmania infantum. We performed in vitro evaluations through macrophage cytotoxicity assays (J774) and nitric oxide production, activity against promastigotes and amastigotes, as well as ultrastructural analyzes in promastigotes. In the evaluation of cytotoxicity, the thiazolidine compounds presented CC50 values between 8.52 and 126.83 µM. Regarding the evaluation against the promastigote forms, the IC50 values ranged between 0.42 and 142.43 µM. Compound 7a was the most promising, as it had the lowest IC50. The parasites treated with compound 7a showed several changes, such as cell body shrinkage, shortening and loss of the flagellum, intense mitochondrial edema and cytoplasmic vacuolization, leading the parasite to cell inviability. In assays against the amastigote forms, the compound showed a low IC50 (0.65 µM). These results indicate that compound 7a was efficient for both evolutionary forms of the parasite. In silico studies suggest that the compound has good oral bioavailability. These results show that compound 7a is a potential drug candidate for the treatment of Leishmaniasis.

Transovarial toxicity matters: lethal and sublethal effects of hexythiazox on the two-spotted spider mite (Acari: Tetranychidae).

The effects of hexythiazox on life-history traits and demographic parameters of Tetranychus urticae Koch (Acari: Tetranychidae) were evaluated using the age-stage two-sex life table (in fecundity-based and fertility-based variants), with emphasis on its transovarial toxicity. Hexythiazox was applied when T. urticae females were either in the preovipositional period or in the first day of oviposition. In the F0 generation bioassay, treatments with concentrations of 50, 12.5 and 3.125 mg/l significantly reduced the longevity of females and their fecundity. These effects were mostly the result of mortality of treated females (18-23%) over the 24-h exposure period. Even though the net reproductive rate (R0) decreased significantly, the intrinsic rate of increase (r), finite rate of increase (λ) and doubling time (D) were not significantly different from the control. The strongest transovarial toxic effect occurred within the first 4 days following treatment, when 52-89% of the eggs laid by treated females (96% in control) hatched. Fertility was significantly reduced by concentrations of 50, 12.5, 3.125, 0.781 and 0.195 mg/l. These concentrations caused significant reductions in R0 (34-54%), r (12-24%) and λ (3-5%), whereas D was extended for 0.4-0.7 days. In the F1 generation bioassay, 50, 12.5, 3.125, 0.781, 0.049 and 0.012 mg/l caused significant reductions in R0 (34-92%), r (10-68%) and λ (3-17%), whereas extending D for 0.3-5.6 days. These effects were mostly the consequence of transovarial toxicity. Application of the fecundity-based life table underestimated population-level effects of hexythiazox on T. urticae.

A Role for Plant KASH Proteins in Regulating Stomatal Dynamics.

Stomatal movement, which regulates gas exchange in plants, is controlled by a variety of environmental factors, including biotic and abiotic stresses. The stress hormone abscisic acid (ABA) initiates a signaling cascade, which leads to increased H2O2 and Ca2+ levels and F-actin reorganization, but the mechanism of, and connection between, these events is unclear. SINE1, an outer nuclear envelope component of a plant Linker of Nucleoskeleton and Cytoskeleton complex, associates with F-actin and is, along with its putative paralog SINE2, expressed in guard cells. Here, we have determined that Arabidopsis (Arabidopsis thaliana) SINE1 and SINE2 play an important role in stomatal opening and closing. Loss of SINE1 or SINE2 results in ABA hyposensitivity and impaired stomatal dynamics but does not affect stomatal closure induced by the bacterial elicitor flg22. The ABA-induced stomatal closure phenotype is, in part, attributed to impairments in Ca2+ and F-actin regulation. Together, the data suggest that SINE1 and SINE2 act downstream of ABA but upstream of Ca2+ and F-actin. While there is a large degree of functional overlap between the two proteins, there are also critical differences. Our study makes an unanticipated connection between stomatal regulation and nuclear envelope-associated proteins, and adds two new players to the increasingly complex system of guard cell regulation.

Assessing different thiazolidine and thiazole based compounds as antileishmanial scaffolds.

The compounds from eight different thiazolidine and thiazole series were assessed as potential antileishmanial scaffolds. They were tested for antileishmanial activity against promastigotes of Leishmania major using in vitro primary screen and dose response assays. The compounds from six thiazolidine and thiazole series were identified as the hits with antileishmanial activity against L. major. However, the analyses of structure-activity relations (SARs) showed that the interpretable SARs were obtained only for phenyl-indole hybrids (compounds C1, C2, C3 and C5) as the most effective compounds against L. major promastigotes (IC50 < 10 µM) with low toxicity to human fibroblasts. For the scaffold of these compounds, the most significant SAR patterns were: free N3 position of thiazolidinone core, absence of big fragments at the C5 position of thiazolidinone core and presence of halogen atoms or nitro group in the phenyl ring of phenyl-indole fragment. As previous studies showed that these compounds also have activity against the two Trypanosoma species, Trypanosoma brucei and Trypanosoma gambiense, their scaffold could be associated with a broader antiparasitic activity.

Synthesis of metronidazole based thiazolidinone analogs as promising antiamoebic agents.

Metronidazole and its derivatives are widely used for the treatment of amoebiasis. However, metronidazole is considered as the standard drug but it has many side effects. The present study describes the synthesis of a series of metronidazole based thiazolidinone analogs via Knoevenagel condensation of 4-[2-(2-methyl-5-nitro-1H-imidazole-1-yl)ethoxy]benzaldehyde 1 with various thiazolidinone derivatives 2-14 to get the new scaffold (15-27) having better activity and lesser toxicity. Six compounds have shown better efficacy and lesser cytotoxicity than the standard drug metronidazole towards HM1: IMSS strain of Entamoeba histolytica. These compounds may combat the problem of drug resistance and might be effective in identifying potential alternatives for future drug discovery against EhOASS.

2-(2-Methoxyphenyl)-3-((Piperidin-1-yl)ethyl)thiazolidin-4-One-Loaded Polymeric Nanocapsules: In Vitro Antiglioma Activity and In Vivo Toxicity Evaluation.

Among gliomas types, glioblastoma is considered the most malignant and the worst form of primary brain tumor. It is characterized by high infiltration rate and great angiogenic capacity. The presence of an inflammatory microenvironment contributes to chemo/radioresistance, resulting in poor prognosis for patients. Recent data show that thiazolidinones have a wide range of pharmacological properties, including anti-inflammatory and antiglioma activities. Nanocapsules of biodegradable polymers become an alternative to cancer treatment since they provide targeted drug delivery and could overcome blood-brain barrier. Therefore, here we investigated the in vitro antiglioma activity and the potential in vivo toxicity of 2- (2-methoxyphenyl) -3- ((piperidin-1-yl) ethyl) thiazolidin-4-one-loaded polymeric nanocapsules (4L-N). Nanocapsules were prepared and characterized in terms of particle size, polydispersity index, zeta potential, pH, molecule content and encapsulation efficiency. Treatment with 4L-N selectively decreased human U138MG and rat C6 cell lines viability and proliferation, being even more efficient than the free-form molecule (4L). In addition, 4L-N did not promote toxicity to primary astrocytes. We further demonstrated that the treatment with sub-therapeutic dose of 4L-N did not alter weight, neither resulted in mortality, toxicity or peripheral damage to Wistar rats. Finally, 4L as well as 4L-N did not alter makers of oxidative damage, such as TBARS levels and total sulfhydryl content, and did not change antioxidant enzymes SOD and CAT activity in liver and brain of treated rats. Taken together, these data indicate that the nanoencapsulation of 4L has potentiated its antiglioma effect and does not cause in vivo toxicity.

Design, synthesis, molecular docking and biological evaluation of thiophen-2-iminothiazolidine derivatives for use against Trypanosoma cruzi.

In this study, we designed and synthesized a series of thiophen-2-iminothiazolidine derivatives from thiophen-2-thioureic with good anti-Trypanosoma cruzi activity. Several of the final compounds displayed remarkable trypanocidal activity. The ability of the new compounds to inhibit the activity of the enzyme cruzain, the major cysteine protease of T. cruzi, was also explored. The compounds 3b, 4b, 8b and 8c were the most active derivatives against amastigote form, with significant IC50 values between 9.7 and 6.03µM. The 8c derivative showed the highest potency against cruzain (IC50=2.4µM). Molecular docking study showed that this compound can interact with subsites S1 and S2 simultaneously, and the negative values for the theoretical energy binding (Eb=-7.39kcal·mol(-1)) indicates interaction (via dipole-dipole) between the hybridized sulfur sp(3) atom at the thiazolidine ring and Gly66. Finally, the results suggest that the thiophen-2-iminothiazolidines synthesized are important lead compounds for the continuing battle against Chagas disease.

Synthesis, Biological Activity, and Docking Study of Novel Isatin Coupled Thiazolidin-4-one Derivatives as Anticonvulsants.

A series of 2-(substituted-phenyl)-3-(2-oxoindolin-3-ylidene)amino)-thiazolidin-4-one derivatives were designed and synthesized under microwave irradiation, using an eco-friendly, efficient, microwave-assisted synthetic protocol that involves cyclocondensation of 3-substituted benzylidine-hydrazono-indolin-2-one 3a-j with thioglycolic acid in dimethyl formamide (DMF) as solvent and anhydrous zinc chloride as a catalyst, keeping in view the structural requirement of the pharmacophore. The intermediate compounds 3a-j were obtained by condensation of the hydrazone of indoline-2,3-dione with aromatic aldehydes. The synthesized derivatives were evaluated for CNS depressant activity and anticonvulsant activity in mice using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (sc-PTZ) induced seizure tests. All the derivatives showed good CNS depressant activity and showed protection in the MES test, indicative of their ability to inhibit the seizure spread. A histopathological study was performed to evaluate liver toxicity caused by the synthesized compounds. The compounds were nontoxic. A computational study was performed, in which log P values were calculated experimentally. Virtual screening was performed by molecular docking of the designed compounds into the ATP binding sites of the NMDA and AMPA receptors, to predict if these compounds have analogous binding modes.

Ultrasound-assisted synthesis, anticonvulsant activity, and docking study of indole-appended thiazolidin-4-ones.

Two series of novel indolyl thiazolidin-4-one derivatives 4a-j and 5a-j were obtained by an ecofriendly synthetic protocol by treating a mixture of Schiff's bases (0.01 mol) with thioglycolic acid or thiolactic acid (0.01 mol) and anhydrous zinc chloride in catalytic amount in DMF as solvent under ultrasound irradiation, using an ultrasound synthesizer with a synthetic solid probe. The structures of the synthesized compounds were confirmed by IR, (1) H NMR, (13) C NMR, MS, and elemental analysis. The anticonvulsant activity and neurotoxicity of the newly synthesized compounds were established by MES and sc-PTZ model and by rotarod test, respectively, in vivo using mouse models. The actophotometer was used for the screening of behavioral activity. The compounds exhibited promising anticonvulsant activity; especially, the compounds showed maximum protection in the MES model at a dose of 100 mg/kg. Further, docking studies of the synthesized compounds were performed against the sodium channel receptor and showed good binding interactions with the receptor. A computational study was carried out to highlight the pharmacophore distance mapping, log p determination, and pharmacokinetic parameters.

An efficient sonochemical synthesis of novel Schiff's bases, thiazolidine, and pyrazolidine incorporating 1,8-naphthyridine moiety and their cytotoxic activity against HePG2 cell lines.

Novel Schiff's bases 4a-e, 5a, 5b, and 6, thiazolidine 7a-d, and pyrazolidine 8 have been synthesized using the versatile synthon 4-hydroxy-2,7-dimethyl-1,8-naphthyridine 1. Reactions carried out under ultrasound irradiation showed higher rates and yields than those done under silent conditions. The newly synthesized compounds were evaluated for HepG2 cell growth inhibition. The results obtained revealed that the tested compounds possess inhibitory effect on the growth of HepG2 liver cancer cells. The results were compared to doxorubicin as a reference drug (IC50: 0.04). Compounds 4a and 7b showed the highest inhibition activity against the HepG2 cell line (IC50: 0.047 and 0.041 µM, resp.) among all the tested compounds.

Computational Modeling Oriented Substructure Splicing Application in the Identification of Thiazolidine Derivatives as Potential Low Honeybee Toxic Neonicotinoids.

With the aim of identifying novel neonicotinoid insecticides with low bee toxicity, a series of compounds bearing thiazolidine moiety, which has been shown to be low bee toxic, were rationally designed through substructure splicing strategy and evaluated insecticidal activities. The optimal compounds A24 and A29 exhibited LC50 values of 30.01 and 17.08 mg/L against Aphis craccivora, respectively. Electrophysiological studies performed on Xenopus oocytes indicated that compound A29 acted on insect nAChR, with EC50 value of 50.11 µM. Docking binding mode analysis demonstrated that A29 bound to Lymnaea stagnalis acetylcholine binding protein through H-bonds with the residues of D_Arg55, D_Leu102, and D_Val114. Quantum mechanics calculation showed that A29 had a higher highest occupied molecular orbit (HOMO) energy and lower vertical ionization potential (IP) value compared to the high bee toxic imidacloprid, showing potentially low bee toxicity. Bee toxicity predictive model also indicated that A29 was nontoxic to honeybees. Our present work identified an innovative insecticidal scaffold and might facilitate the further exploration of low bee toxic neonicotinoid insecticides.

Differential binding of latrunculins to G-actin: a molecular dynamics study.

Latrunculins are unique macrolides containing a thiazolidinone moiety. Latrunculin A (1), latrunculin B (2), 16-epi-latrunculin B (3), and latrunculin T (4) were isolated from the Red Sea sponge Negombata magnifica. In the present study, after testing compounds 2-4 for cytotoxic activity, they were docked into the crystal structure of G-actin and subjected to binding energy calculation and a 20 ns MD simulation. The modeling study shows that latrunculins binding depends on both hydrophobic interaction of the macrocycle as well as H bonding of the thiazolidinone ring with Asp157 and Thr186. It was noticed that epimerization at C16 of latrunculin B was well tolerated as it could form an alternative H bonding network. However, opening of the macrocyclic ring deteriorates the actin binding due to reduced hydrophobicity. MD simulation showed that latrunculin B (2) possesses a more significant stabilizing effect on G-actin than latrunculin T (4) and could efficiently hinder the flattening transition of G-actin into F-actin. These findings could explain, at the molecular level, the impact of epimerization and macrolide ring-opening on latrunculins activity, an issue that has not been addressed before. Also, the study gives insights into the mechanism of cytotoxicity of diverse latrunculins and provides direction for future lead optimization studies.

Synthesis and characterization of celecoxib derivatives as possible anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV agents.

A series of novel N-(3-substituted aryl/alkyl-4-oxo-1,3-thiazolidin-2-ylidene)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamides 2a-e were synthesized by the addition of ethyl a-bromoacetate and anhydrous sodium acetate in dry ethanol to N-(substituted aryl/alkylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoro-methyl)-1H-pyrazol-1-yl]benzene sulfonamides 1a-e, which were synthesized by the reaction of alkyl/aryl isothiocyanates with celecoxib. The structures of the isolated products were determined by spectral methods and their anti-inflammatory, analgesic, antioxidant, anticancer and anti-HCV NS5B RNA-dependent RNA polymerase (RdRp) activities evaluated. The compounds were also tested for gastric toxicity and selected compound 1a was screened for its anticancer activity against 60 human tumor cell lines. These investigations revealed that compound 1a exhibited anti-inflammatory and analgesic activities and further did not cause tissue damage in liver, kidney, colon and brain compared to untreated controls or celecoxib. Compounds 1c and 1d displayed modest inhibition of HCV NS5B RdRp activity. In conclusion, N-(ethylcarbamothioyl)-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (1a) may have the potential to be developed into a therapeutic agent.

Permeability and toxicity characteristics of L-cysteine and 2-methyl-thiazolidine-4-carboxylic acid in Caco-2 cells.

Acetaldehyde is a known mutagenic substance and has been classified as a group-one carcinogen by the WHO. It is possible to bind acetaldehyde locally in the gastrointestinal (GI) tract with the semi-essential amino acid l-cysteine, which reacts covalently with acetaldehyde and forms compound 2-methyl-thiozolidine-4-carboxylic acid (MTCA). The Caco-2 cell line was used to determine the permeation of l-cysteine and MTCA, as well as the possible cell toxicity of both substances. Neither of the substances permeated through the Caco-2 cells at the concentrations used in this study, and only the highest concentration of MTCA affected the viability of the cells in the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) test. These results showed that when l-cysteine is administered in formulations releasing it locally in the lower parts of GI tract, it is not absorbed but can react with acetaldehyde, and that neither l-cysteine nor MTCA is harmful to the cells when present locally in the upper parts of GI tract. This study also shows that MTCA is sensitive at a lower pH of 5.5. Since stable MTCA is desired in different parts of the GI tract, this observation raises concern over the influence of lower pH on l-cysteine-containing product ability to bind and eliminate carcinogenic acetaldehyde.

Microfluidics-based assessment of cell deformability.

Mechanical properties of cells have been shown to have a significant role in disease, as in many instances cell stiffness changes when a cell is no longer healthy. We present a high-throughput microfluidics-based approach that exploits the connection between travel time of a cell through a narrow passage and cell stiffness. The system resolves both cell travel time and relative cell diameter while retaining information on the cell level. We show that stiffer cells have longer transit times than less stiff ones and that cell size significantly influences travel times. Experiments with untreated HeLa cells and cells made compliant with latrunculin A and cytochalasin B further demonstrate that travel time is influenced by cell stiffness, with the compliant cells having faster transit time.

Effects of spindle poisons in peripheral human lymphocytes by the in vitro cytokinesis-block micronucleus assay.

The search for micronuclei (MN) in binucleated cells is not always the best choice to recognize microtubule-perturbing agents, as they give rise to (micronucleated) mononucleated cells, mainly via mitotic slippage. We therefore treated peripheral lymphocytes with vincristine (VCR), nocodazole (NOC) and colcemid (COL): (i) to quantify the formation of MN in mononucleated cells and the occurrence of abnormal mitoses (c-anaphases, endoreduplicated or tetraploid metaphases); (ii) to investigate the role of cytokinesis inhibition in determining or modulating the cytogenetic effects induced by the spindle poisons (we used either cytochalasin B (cyt B) or latrunculin A, a cytokinesis inhibitor that acts differently as compared with cyt B); (iii) to assess the ploidy of cells bearing MN by fluorescence in situ hybridisation (FISH) analysis; and (iv) to evaluate the levels of the mitotic arrest deficient (MAD2) protein, that blocks the cell at the metaphase-anaphase transition, by immunoblotting. We observed the induction of numerous abnormal mitoses and tetraploid interphase nuclei, as well as of MN in mononucleated cells, a high percentage of which had a diploid complement. We also found that the effects were generally not dose but chemical dependent, where NOC was proven to be more effective than COL and VCR in inducing overall MN formation and, specifically, diploid micronucleated lymphocytes. Aneugens damaged cells to a greater extent in the presence of cytokinesis inhibitors rather than in their absence. MAD2 protein was expressed in controls to an extent reflecting the amount of lymphocytes which were initially in the G2/M transition phase. The same trend was seen in aneugen-treated cells where MAD2 levels decreased with increasing spindle poison concentration. Here, we demonstrate that micronucleated mononucleated cells and aberrant mitoses can be considered useful markers of exposure to aneugens-like spindle poisons causing preferentially, but not exclusively, mitotic slippage. Assessment of MAD2 levels can be used to confirm the cell-damaging activity of the compounds.

Proteomic response of wheat embryos to fosthiazate stress in a protected vegetable soil.

A proteomic analysis of wheat defense response induced by the widely used organophosphorus nematicide fosthiazate is reported. Seed germination and two-dimensional gel electrophoresis (2-DE) experiments were performed using a Chinese wheat cultivar, Zhenmai No. 5. Root and shoot elongation decreased but thiobarbituric acid reactive substances (TBARS) content in embryos increased with increasing pesticide concentration. More than 1000 protein spots were reproducibly detected in each silver-stained gel. Thirty-seven protein spots with at least 2-fold changes were identified using MALDI-TOF MS/MS analysis. Of these, 24 spots were up-regulated and 13 were down-regulated. Proteins identified included some well-known classical stress responsive proteins under abiotic or biotic stresses as well as some unusual responsive proteins. Ten responsive proteins were reported for the first time at the proteomic level, including fatty acyl CoA reductase, dihydrodipicolinate synthase, DEAD-box ATPase-RNA-helicase, fimbriata-like protein, waxy B1, rust resistance kinase Lr10, putative In2.1 protein, retinoblastoma-related protein 1, pollen allergen-like protein and S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase. The proteins identified were involved in several processes such as metabolism, defense/detoxification, cell structure/cell growth, signal transduction/transcription, photosynthesis and energy. Seven candidate proteins were further analyzed at the mRNA level by RT-PCR to compare transcript and protein accumulation patterns, revealing that not all the genes were correlated well with the protein level. Identification of these responsive proteins may provide new insight into the molecular basis of the fosthiazate-stress response in the early developmental stages of plants and may be useful in stress monitoring or stress-tolerant crop breeding for environmentally friendly agricultural production.

New ibuprofen derivatives with thiazolidine-4-one scaffold with improved pharmaco-toxicological profile.

BACKGROUND: Aryl-propionic acid derivatives with ibuprofen as representative drug are very important for therapy, being recommended especially for anti-inflammatory and analgesic effects. On other hand 1,3-thiazolidine-4-one scaffold is an important heterocycle, which is associated with different biological effects such as anti-inflammatory and analgesic, antioxidant, antiviral, antiproliferative, antimicrobial etc. The present study aimed to evaluated the toxicity degree and the anti-inflammatory and analgesic effects of new 1,3-thiazolidine-4-one derivatives of ibuprofen. METHODS: For evaluation the toxicity degree, cell viability assay using MTT method and acute toxicity assay on rats were applied. The carrageenan-induced paw-edema in rat was used for evaluation of the anti-inflammatory effect while for analgesic effect the tail-flick test, as thermal nociception in rats and the writhing assay, as visceral pain in mice, were used. RESULTS: The toxicological screening, in terms of cytotoxicity and toxicity degree on mice, revealed that the ibuprofen derivatives (4a-n) are non-cytotoxic at 2 µg/ml. In addition, ibuprofen derivatives reduced carrageenan-induced paw edema in rats, for most of them the maximum effect was recorded at 4 h after administration which means they have medium action latency, similar to that of ibuprofen. Moreover, for compound 4d the effect was higher than that of ibuprofen, even after 24 h of administration. The analgesic effect evaluation highlighted that 4 h showed increased pain inhibition in reference to ibuprofen in thermal (tail-flick assay) and visceral (writhing assay) nociception models. CONCLUSIONS: The study revealed for ibuprofen derivatives, noted as 4 m, 4 k, 4e, 4d, a good anti-inflammatory and analgesic effect and also a safer profile compared with ibuprofen. These findings could suggest the promising potential use of them in the treatment of inflammatory pain conditions.

Bioymifi, a novel mimetic of TNF-related apoptosis-induced ligand (TRAIL), stimulates eryptosis.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a cytokine that initiates apoptosis upon binding to death receptor 5 (DR5) on cancer cells. Small molecule TRAIL mimetics have therefore been investigated as promising chemotherapeutic agents. Since anemia of chemotherapy is common, our goal is to investigate the hemolytic and eryptotic properties of novel DR5 agonist bioymifi (BMF) and identify the underlying molecular mechanisms. Whole blood (WB) was stimulated with 100 µM of BMF, whereas red blood cells (RBCs) were treated with 10-100 µM of BMF for 24 h at 37 °C. WB was analyzed for RBC, leukocyte, and platelet indices, while RBCs were examined for hemolysis by light absorbance of free hemoglobin, membrane scrambling by Annexin V-FITC, calcium by Fluo4/AM, cellular morphology by light scatter, and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) using flow cytometry. Caspase inhibitor Z-VAD-FMK, p38 inhibitor SB203580, casein kinase 1α inhibitor D4476, receptor-interacting protein 1 inhibitor necrostatin-2, reduced glutathione, or cyclooxygenase (COX) inhibitor aspirin were added accordingly. BMF exerted dose-responsive, calcium-independent hemolysis, reduced RBC hemoglobin, significantly increased Annexin V-, Fluo4-, and DCF-positive cells, along with a dual effect on forward and side light scatter. Notably, the cytotoxic potential of BMF was significantly mitigated upon pharmacological inhibition of p38. Furthermore, BMF exhibited selective toxicity to eosinophils and significantly diminished reticulocyte hemoglobin content. Altogether, these novel findings highlight the adverse outcomes of BMF exposure on RBC physiology and provide the first toxicological assessment of BMF as an antitumor agent.