Abstract: Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (ΔΨm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.

Abstract: Dysregulated neutrophil extracellular traps (NETs) formation contributes to the pathogenesis of anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV). Increasing evidence indicates that autophagy is involved in the process of NETs formation. In this study, we aimed to investigate whether ANCA could induce autophagy in the process of NETs formation. Autophagy was detected using live cell imaging, microtubule-associated protein light chain 3B (LC3B) accumulation and Western blotting. The results showed that autophagy vacuolization was detected in neutrophils treated with ANCA-positive IgG by live cell imaging. This effect was enhanced by rapamycin, the autophagy inducer, and weakened by 3-methyladenine (3-MA), the autophagy inhibitor. In line with these results, the autophagy marker, LC3B, showed a punctate distribution pattern in the neutrophils stimulated with ANCA-positive IgG. In the presence of rapamycin, LC3B accumulation was further increased; however, this effect was attenuated by 3-MA. Moreover, incubated with ANCA-positive IgG, the NETosis rate significantly increased compared with the unstimulated group. And, the rate significantly increased or decreased in the neutrophils pretreated with rapamycin or 3-MA, respectively, as compared with the cells incubated with ANCA-positive IgG. Overall, this study demonstrates that autophagy is induced by ANCA and promotes ANCA-induced NETs formation.

Abstract: Many nanomaterials are reported to disrupt lysosomal function and homeostasis, but how cells sense and then respond to nanomaterial-elicited lysosome stress is poorly understood. Nucleus translocation of transcription factor EB (TFEB) plays critical roles in lysosome biogenesis following lysosome stress induced by starvation. The authors previously reported massive cellular vacuolization, along with autophagy induction, in cells treated with rare earth oxide (REO) nanoparticles. Here, the authors identify these giant cellular vacuoles as abnormally enlarged and alkalinized endo/lysosomes whose formation is dependent on macropinocytosis. This vacuolization causes deactivation of mammalian target of rapamycin (mTOR), a TFEB-interacting kinase that resides on the lysosome membrane. Subsequently, TFEB is dephosphorylated at serine 142 and translocated into cell nucleus. This nucleus translocation of TFEB is observed only in vacuolated cells and it is critical for maintaining lysosome homeostasis after REO nanoparticle treatment, as knock-down of TFEB gene significantly compromises lysosome function and enhances cell death in nanoparticle-treated cells. Our results reveal that cellular vacuolization, which is commonly observed in cells treated with REOs and other nanomaterials, represents a condition of profound lysosome stress, and cells sense and respond to this stress by facilitating mTOR-dependent TFEB nucleus translocation in an effort to restore lysosome homeostasis.

Abstract: Our aim was to evaluate the ultrastructure of human metaphase II oocytes subjected to slow freezing and fixed after thawing at different intervals during post-thaw rehydration. Samples were studied by light and transmission electron microscopy. We found that vacuolization was present in all cryopreserved oocytes, reaching a maximum in the intermediate stage of rehydration. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates decreased following thawing, particularly in the first and intermediate stages of rehydration, whereas mitochondria-vesicle (MV) complexes augmented in the same stages. At the end of rehydration, vacuoles and MV complexes both diminished and M-SER aggregates increased again. Cortical granules (CGs) were scarce in all cryopreserved oocytes, gradually diminishing as rehydration progressed. This study also shows that such a membrane remodeling is mainly represented by a dynamic process of transition between M-SER aggregates and MV complexes, both able of transforming into each other. Vacuoles and CG membranes may take part in the membrane recycling mechanism.

Abstract: Chemotherapy is an important treatment modality for colon cancer, and concurrent chemoradiation therapy (CCRT) is the preferred treatment route for patients with stage II and III rectal cancer. We examined whether DangguiBuxue Tang (DBT), a traditional Chinese herbal extract, sensitizes colorectal cancer cells to anticancer treatments. The polysaccharide-depleted fraction of DBT (DBT-PD) contains greater amounts of astragaloside IV (312.626 µg/g) and ferulic acid (1.404 µg/g) than does the original formula. Treatment of the murine colon carcinoma cell line (CT26) with DBT-PD inhibits growth, whereas treatment with comparable amounts of purified astragaloside IV and ferulic acid showed no significant effect. Concurrent treatment with DBT-PD increases the growth inhibitory effect of 5-fluorouracil up to 4.39-fold. DBT-PD enhances the effect of radiation therapy (RT) with a sensitizer enhancement ratio (SER) of up to 1.3. It also increases the therapeutic effect of CCRT on CT26 cells. Cells treated with DBP-PD showed ultrastructural changes characteristic of autophagy, including multiple cytoplasmic vacuoles with double-layered membranes, vacuoles containing remnants of degraded organelles, marked swelling and vacuolization of mitochondria, and autolysosome-like vacuoles. We conclude that DBT-PD induces autophagy-associated cell death in CT26 cells, and may have potential as a chemotherapy or radiotherapy sensitizer in colorectal cancer treatment.

Abstract: Lead is a toxic heavy metal that adversely affects nervous tissues; it often occurs as an environmental pollutant. We investigated histological changes in the cerebral cortex, hippocampus and cerebellum of adult albino mice following exposure to lead acetate. We also studied the possible ameliorative effect of the chelating agent, L-cysteine, on lead-induced neurotoxicity. We divided albino mice into six groups: 1) vehicle-only control, 2) L-cysteine control, 3 and 4) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, and 5 and 6) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, followed by 50 mg/kg L-cysteine for 7 days. Lead acetate administration caused disorganization of cell layers, neuronal loss and degeneration, and neuropil vacuolization. Brain sections from lead-intoxicated mice treated with L-cysteine showed fewer pathological changes; the neuropil showed less vacuolization and the neurons appeared less damaged. L-cysteine at the dose we used only marginally alleviated lead-induced toxicity.

Abstract: Simian virus 40 (SV40), a polyomavirus that has served as an important model to understand many aspects of biology, induces dramatic cytoplasmic vacuolization late during productive infection of monkey host cells. Although this activity led to the discovery of the virus in 1960, the mechanism of vacuolization is still not known. Pentamers of the major SV40 capsid protein VP1 bind to the ganglioside GM1, which serves as the cellular receptor for the virus. In this report, we show that binding of VP1 to cell surface GM1 plays a key role in SV40 infection-induced vacuolization. We previously showed that SV40 VP1 mutants defective for GM1 binding fail to induce vacuolization, even though they replicate efficiently. Here, we show that interfering with GM1-VP1 binding by knockdown of GM1 after infection is established abrogates vacuolization by wild-type SV40. Vacuole formation during permissive infection requires efficient virus release, and conditioned medium harvested late during SV40 infection rapidly induces vacuoles in a VP1- and GM1-dependent fashion. Furthermore, vacuolization can also be induced by a nonreplicating SV40 pseudovirus in a GM1-dependent manner, and a mutation in BK pseudovirus VP1 that generates GM1 binding confers vacuole-inducing activity. Vacuolization can also be triggered by purified pentamers of wild-type SV40 VP1, but not by GM1 binding-defective pentamers or by intracellular expression of VP1. These results demonstrate that SV40 infection-induced vacuolization is caused by the binding of released progeny viruses to GM1, thereby identifying the molecular trigger for the activity that led to the discovery of SV40. IMPORTANCE The DNA tumor virus SV40 was discovered more than a half century ago as a contaminant of poliovirus vaccine stocks, because it caused dramatic cytoplasmic vacuolization of permissive host cells. Although SV40 played a historically important role in the development of molecular and cellular biology, restriction mapping, molecular cloning, and whole-genome sequencing, the basis of this vacuolization phenotype was unknown. Here, we show that SV40-induced vacuolization is triggered by the binding of the major viral capsid protein, VP1, to a cell surface ganglioside receptor, GM1. No other viral proteins or virus replication is required for vacuole formation. Other polyomaviruses utilize different ganglioside receptors, but they do not induce vacuolization. This work identifies the molecular trigger for the phenotype that led to the discovery of this important virus and provides the first molecular insight into an unusual and enigmatic cytopathic effect due to virus infection.

Abstract: Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10 minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50 μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100 μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

Abstract: // Reo Nozaki 1 , Toru Kono 1, 2 , Hiroki Bochimoto 3 , Tsuyoshi Watanabe 3 , Kaori Oketani 1 , Yuichi Sakamaki 1 , Naoto Okubo 1 , Koji Nakagawa 1 , Hiroshi Takeda 1 1 Pathophysiology and Therapeutics, Hokkaido University Faculty of Pharmaceutical Sciences, Sapporo, Hokkaido, Japan 2 Center for Clinical and Biomedical Research, Sapporo Higashi-Tokushukai Hospital, Sapporo, Hokkaido, Japan 3 Department of Microscopic Anatomy and Cell Biology, Asahikawa Medical University, Asahikawa, Hokkaido, Japan Correspondence to: Toru Kono, email: kono@toru-kono.com Keywords: autophagy, autophagic cell death, vacuolization, colon cancer, zanthoxylum fruit Received: February 09, 2016 Accepted: September 02, 2016 Published: September 10, 2016 ABSTRACT Zanthoxylum fruit, obtained from the Japanese pepper plant ( Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Abstract: The present study was aimed to investigate the antioxidant, biochemical, and histological effects of alpha lipoic acid (ALA) on polychlorinated biphenyl (PCB)-induced testicular toxicity in male rats. The rats were divided into five groups: In the control group, the rats were not administered any chemicals for 30 days. In the sham group, the rats were administered corn oil for 30 days. In the PCB group, the rats were administered with Aroclor 1254 for 30 days. In the ALA group, the rats were treated with ALA for 30 days. In the ALA+PCB group, the rats were treated with ALA 24 hours before Aroclor 1254 was administered for 30 days. The total oxidant status (TOS) level in the serum and testis, number of apoptotic cells, vacuolization at the basal membrane, immature spermatids in the tubular lumen, heme oxygenase-1 (HO-1) staining density, and abnormal spermatozoa were significantly increased in the PCB group. Moreover, in the PCB group, the seminiferous tubule diameter (STD) was decreased in stage VII-VIII and XII-XIV tubules. The TOS level in the serum and testis, vacuolization at the basal membrane, immature spermatids in the tubular lumen, and apoptosis were significantly decreased in the ALA+PCB groups. These findings suggested that ALA has a protective role against PCB-induced testicular toxicity.

Abstract: Penicillin G oversecretion by Penicillium chrysogenum PQ-96 is associated with a strictly adjusted cellular organization of the mature and senescent mycelial cells. Abundant vacuolar phagy and extended cellular vacuolization combined with vacuolar budding resulting in the formation of vacuolar vesicles that fuse with the cell membrane are the most important characteristic features of those cells. We suggest as follows: if the peroxisomes are integrated into vacuoles, the penicillin G formed in peroxisomes might be transferred to vacuoles and later secreted out of the cells by an exocytosis process. The peroxisomal cells of the mycelium are privileged in penicillin G secretion.

Abstract: Due to the negative effects of pesticide usage in environment, i.e., decline in amphibian populations, this study was designed to examine histopathologic changes following carbaryl exposure for 96 h in the digestive tract of variable toad, Bufotes variabilis. After exposure to single doses of carbaryl (low dose 50 μg/g, medium dose 100 μg/g, and high dose: 200 μg/g), the toads were euthanized and dissected and digestive tract samples were quickly removed. Histopathology revealed esophageal vacuolization and necrosis in esophageal cells. Hemorrhage was also observed in the esophagus. In the stomach, necrosis in the glandular epithelium, congested blood vessels, edema among gastric glands, dilated tips of the mucosal epithelial layer, gastric glandular atrophy, and hemorrhage were found. In the intestine, edema within villi, hemorrhage, inflammation, vacuolization, and necrosis in epithelial cells of villi were detected. This study clearly showed that carbaryl caused adverse effects on the digestive tract of B. variabilis in all dose groups.

Abstract: This study was aimed to evaluate the efficacy of treatment modalities for minimizing salivary gland damage caused by radiotherapy. Forty rats were divided into five groups. Group 1 had no irradiation or any treatment. Group 2 underwent only 15 Gy single dose radiotherapy. N-acetylcysteine, dexamethasone, hyperbaric oxygen treatment were given, respectively to the group 3, 4 and 5 for 5 days. 15 Gy single dose radiotherapy was applied to the group 3, 4 and 5 on the second day. Pyknosis, lysis, and vacuolization were examined in ductal cells and pyknosis, lysis, vacuolization, inflammation and collective duct damage in acinar cells. Dexamethasone and hyperbaric oxygen did not prove to have a positive effect on acinar and ductal cell. N-acetylcysteine-applied group had statistically significantly lower amount of damage. We determined that the decrease of ductal and acinar cell damage in parotid glands of N-acetylcysteine-applied rats was more distinct and statistically.

Abstract: Background: Bisphenol A is a xenoestrogen, synthesized in large quantities for the production of polymers (polycarbonates, epoxy resins) and thermal paper, and is widely used in products of everyday use (packaging, containers and bottles). Data concerning the occurrence of BPA in food, water and indoor environments as well as its appearance in tissues and body fluids of the human body are available in the literature. Male accessory sex glands are also vulnerable to environmental endocrine disruptors with adverse effects in adulthood. The developing prostate gland is particularly sensitive to estrogens and high-dose exposures during a critical developmental window results in intraepithelial prostatic neoplasia (PIN) in adult rodent models. Bisphenol A is also an endocrine disruptor. High levels of BPA exposure correlate with increased risk of mammary gland, brain and prostate cancers and have adverse effects on the tissues of the prostate and seminal vesicles. Objectives: The aim of this study was to investigate the effects of BPA doses on the histological structure and ultrastructure of prostate and seminal vesicle glands. Methods: Forty male Wistar rats were randomly divided into four groups (n = 10): A control group and three treatment groups, receiving BPA at doses of 10, 50 and 100 µg/kg. Bisphenol A was administrated intraperitoneally for 15 days. As the prostate and seminal vesicle glands were dissected on the day 16, the structure and ultrastructure histological parameters were analyzed, using light and electron microscopes. Results: The main pathological finding observed in the prostrate and seminal vesicle glands, using light microscope, revealed congestion in the connective tissue and vacuolization in the secretory units of epithelial cells and epithelium rupture at 50 and 100 µg/kg doses of BPA. Ultrastructure study showed vacuolization of mitochondria, condensation of nuclei and disappearance of nucleus in the prostate tissue. Pathological changes showed the vacuolization and dilation of endoplamic reticulum (ER) and secretory glands and disappearance of microvilli. They also showed an increase in collagen fibers around the cells in the seminal vesicle tissue in the treatment group that received 100 µg/kg BW BPA. Conclusions: This study revealed that BPA has adverse effects on the reproductive system in male rats. The results also revealed that BPA has destructive effects on the prostate and seminal vesicle glands.

Abstract: ized by a signet ring aspect. ‘RCM signet ring-like cells’ correspond to a complete annular bright ring with a polar reinforcement, surrounding a dark center. This aspect brings to mind the eccentric pyknotic nucleus encompassed by a vacuolized cytoplasm (appearing as a dark center) seen on the histopathological examination of a skin biopsy performed immediately after RCM on the very same area in 4 cases ( fig. 1 g, h, 2 c, f) – in 1 case, the biopsy had been performed before RCM ( fig. 1 i). We observed a higher number of signet ring-like cells with RCM images compared to the histopathological sections. This could be related to the en face horizontal view given by RCM. Keratinocyte necrosis and apoptotic keratinocytes are considered to be responsible for the vacuolar degeneration of the DEJ seen in interface dermatitis [4] . Intraepidermal necrotic keratinocytes have been described with RCM in lichen planus [2] and lupus erythematosus [3] as mildly bright, polygonal structures larger than surrounding keratinocytes. However, the cellular changes we observed at the DEJ, and which we correlated to basal cell vacuolization, have not been described in these previous pilot studies. We believe it is worth emphasizing this morphological alteration, which could represent an additional clue for the RCM diagnosis of interface dermatitis and which deserves to be confirmed by complementary studies.

Abstract: Aim To compare the effects of thiamine pyrophosphate (TPP) and thiamine (TM) in oxidative optic neuropathy in rats induced by ethambutol. Methods The animals were divided into four groups: a control group (CG), an ethambutol control (ETC) group, TM plus ethambutol group (TMG), and TPP plus ethambutol group (TPPG). One hour after intraperitoneal administration of TM 20 mg/kg to the TMG group and TPP 20 mg/kg to TPPG group, 30 mg/kg ethambutol was given via gavage to all the groups but the CG. This procedure was repeated once daily for 90d. After that period, all rats were exposed to high levels of anaesthesia in order to investigate the gene expression of malondialdehyde and glutathione in removed optic nerve tissue and histopathologically to examine these tissues. Results Malondialdehyde gene expression significantly increased, whereas glutathione gene expression significantly decreased in the ETC group compared to the CG. TM could not prevent the increase of malondialdehyde gene expression and the decrease of glutathione, while TPP significantly could suppress. Histopathologically, significant vacuolization in the optic nerve, single-cell necrosis in the glial cells, and a decrease in oligodendrocytes were observed in the ETC group. Vacuolization in the optic nerve, a decrease in oligodendrocytes and single-cell necrosis were found in the TMG group, while no pathological finding was observed in the TPPG group except for mild vacuolization. Conclusion TPP protects the optic nerve against the ethambutol-induced toxicity but TM does not. TPP can be beneficial in prophilaxis of optic neuropathy in ethambutol therapy.

Abstract: The objective of study is to study the peculiarities of morphological changes in different subdivisions of the intralobular duct of the submandibular gland (SMG) in rats in case of experimental diabetes mellitus (DM). The study included sexually mature male Wistar rats. Experimental DM was induced by streptozotocin. Electron microscopic study of subdivisions of the intralobular duct of the SMG was carried out on the 14th, 28th, 42nd, 56th, and 70th days of the experiment. In early stages of experimental DM the intercalated ducts are characterized by a relatively unchanged structure, and in late stages vacuolization of the cytoplasm of their epithelial cells is observed. Since the 14th day vacuolization of mitochondria is observed in epithelial cells of the granular ducts being the most pronounced on the 28th day and not apparent over the subsequent periods. The degree of filling with granules reduces till 56th day, however, it increases sligthly on the last day of the experiment. On the 28th-70th days vacuolization of the cytoplasm is observed in epithelial cells of the striated ducts. In addition, on the 14th day the mitochondrial matrix of these cells condenses; over the next periods it becomes enlightened and mitochondrial cristae are clearly visualized and disorganized. CONCLUSION In the intralobular duct of the SMG in experimental DM dystrophic changes of different intensity occur in the granular and striated ducts on the 14th day and in the intercalated ducts only since the 42nd day of the experiment.

Abstract: The nucleus is the center of vital activity and is the brain of any cell. This stands true in case of physiological and pathological cells. Nucleus is the most active and prominent cellular organelle which shows many forms, has varied shapes and sizes, depicting the activity of the cell. Morphology of the nuclear shape and presentation can get altered because of changes in nuclear lamina or it can also be because of the forces exerted by the cytoplasm.[1] Change in nuclear shape might affect the function of a cell, though it is not very clear. Two hypothesis are given for this: The first states that nuclear shape change might give it a beneficial plasticity whereas the second states that the changed shape results in chromatin reorganization which might affect gene expression.[1] Common forms of nucleus physiologically, are open, closed and semi‐open.[1] A variety of nuclear shapes, sizes and presentation are associated with numerous pathologies. The cleaved nucleus, owl eye nucleus, Orphan Annie nucleus and buckled nucleus are a few to name. An interesting and uncommon change in the nucleus is nuclear vacuoles and vacuolization.

Abstract: Mercury (Hg) is a highly hazardous pollutant widely used in industrial, pharmaceutical and agricultural fields. Mercury is found in the environment in several forms, elemental, inorganic (iHg) and organic, all of which are toxic. Considering that the liver is the organ primarily involved in the regulation of metabolic pathways, homeostasis and detoxification we investigated the morphological and ultrastructural effects in Danio rerio liver after 96 h exposure to two low HgCl2 concentrations (7.7 and 38.5 μg/L). We showed that a short-term exposure to very low concentrations of iHg severely affects liver morphology and ultrastructure. The main effects recorded in this work were: cytoplasm vacuolization, decrease in both lipid droplets and glycogen granules, increase in number of mitochondria, increase of rough endoplasmic reticulum and pyknotic nuclei. Pathological alterations observed were dose dependent. Trough immunohistochemistry, in situ hybridization and real-time PCR analysis, the induction of metallothionein (MT) under stressor conditions was also evaluated. Some of observed alterations could be considered as a general response of tissue to heavy metals, whereas others (such as increased number of mitochondria and increase of RER) may be considered as an adaptive response to mercury.

Abstract: Introduction Amblyomin‐X is a recombinant protein, which cause apoptosis of tumor cells, in a selective way, beside tumor regression, in in vivo tumor models. Due to the interesting effects, this protein is being developed as an antitumor drug, and toxicity studies in animals are required by regulatory agencies. Amblyomin‐X selectively induces apoptosis in tumor cells and was able to promote a reduction in tumor size in a melanoma model. Aim The aim of this study was to perform preclinical assays to evaluate the toxicity of Amblyomin‐X in mice after acute (200 mg/kg) and chronic (5 and 50 mg/kg per day) intravenous administration. During the experimental period, indications of toxicity, body weight variations and water and food consumption were evaluated. No mortality was observed following treatment with Amblyomin‐X. Results Amblyomin‐X is well tolerated by mice, both male and female, that were treated with a high concentration of the drug (200 times higher than the effective dose therapy) and therefore is not be expected to have any acute or long‐term adverse effects in humans. The therapeutic concentration of Amblyomin‐X, which is less than 5 mg/kg, causes tumor reduction in mouse models and is pharmacologically safe. In general, Amblyomin‐X did not cause clinical alterations, and treated animals exhibited no changes in hematological and biochemical parameters. The macroscopic and histologic analysis of organs demonstrated that the protein increased the white pulp of the spleen, caused vacuolization in hepatocytes and induced intestinal lesions, which can be considered toxic effects of Amblyomin‐X, at concentrations of 50 mg/kg to 200 mg/kg. However, these effects do not appear to alter the function of the organ or homeostasis. Conclusion After this study, we can conclude that the liver changes that were observed in animals treated with high doses of Amblyomin‐X are changes that can take place without jeopardizing the integrity of the organ, which is responsible for the most important metabolic processes in the body. Careful interpretation and extrapolation of the data obtained from these models are essential to reduce the current clinical costs of new anticancer therapeutic agents for humans. This study provides data regarding important safety parameters that enable the implementation of further experiments aimed at developing this molecule as an anti‐tumor drug. Support or Funding Information Support by FAPESP

Abstract: Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of nonapoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methyl-1<i>H</i>-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis.

Abstract: Autophagy degrades cellular components during senescence, starvation, and stress. High-temperature (HT) stress can inhibit microsporogenesis, but the involvement of autophagy in HT injury is unknown. Here we show that Arabidopsis autophagy-defective (atg) mutants are hypersensitive to HT stress during microsporogenesis but not during seedling growth. Fertility was normal at 23 °C, but sporophytic male sterility occurred at 30 °C. At 30 °C, wild-type developing anthers showed increased vacuolization in tapetum and lipophagy in microspores. The atg5-1 mutant did not show these autophagic phenomena, but instead showed irregularly enlarged vacuoles and subsequent shrinkage, and failure of the tapetum to degenerate completely. HT specifically upregulated ATG8 in the developing anther, but not in seedlings, and reduced MYB80 signaling in the anther, which is required for the regulation of tapetal programmed cell death to promote microspore maturation. Interestingly, inhibition of auxin activated the ATG8 signal in seedlings at both 23 and 30 °C. These results, combined with our previous observation of anther-specific auxin depletion caused by HT, suggest that autophagy mitigates HT injury to microsporogenesis.