Mn/S diatomic sites in C3N4 to enhance O2 activation for photocatalytic elimination of emerging pollutants.

Oxygen activation leading to the generation of reactive oxygen species (ROS) is essential for photocatalytic environmental remediation. The limited efficiency of O2 adsorption and reductive activation significantly limits the production of ROS when employing C3N4 for the degradation of emerging pollutants. Doping with metal single atoms may lead to unsatisfactory efficiency, due to the recombination of photogenerated electron-hole pairs. Here, Mn and S single atoms were introduced into C3N4, resulting in the excellent photocatalytic performances. Mn/S-C3N4 achieved 100% removal of bisphenol A, with a rate constant 11 times that of pristine C3N4. According to the experimental results and theoretical simulations, S-atoms restrict holes, facilitating the photo-generated carriers' separation. Single-atom Mn acts as the O2 adsorption site, enhancing the adsorption and activation of O2, resulting the generation of ROS. This study presents a novel approach for developing highly effective photocatalysts that follows a new mechanism to eliminate organic pollutants from water.

Nutrient vitamins enabled metabolic regulation of ferroptosis via reactive oxygen species biology.

Vitamins are dietary components necessary for cellular metabolic balance, especially redox homeostasis; deficient or excessive supply may give rise to symptoms of psychiatric disorders. Exploring the nutritional and metabolic pathways of vitamins could contribute to uncovering the underlying pathogenesis of ferroptosis-associated diseases. This mini-review aims to provide insights into vitamins closely linked to the regulation of ferroptosis from the perspective of cellular reactive oxygen species biology. The mainstream reprogramming mechanisms of ferroptosis are overviewed, focusing on unique biological processes of iron metabolism, lipid metabolism, and amino acid metabolism. Moreover, recent breakthroughs in therapeutic interventions targeting ferroptosis via fully utilizing vitamin-based pharmacological tools were overviewed, covering vitamins (B, C, E, and K). Finally, mechanism insight related to vitamin-associated nutrient signaling was provided, highlighting the pharmacological benefits of metabolically reprogramming ferroptosis-associated diseases.

Primaquine-5,6-orthoquinone is directly hemolytic to older G6PD deficient RBCs in a humanized mouse model.

Primaquine and tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species (ROS) generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinone-imine cause robust redox cycling in red blood cells (RBCs), but are so labile as to not be detected in blood or urine. Rather, the 5,8-quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PDMed-) that recapitulates the human biology of RBC age dependent enzyme decay, as well as an isogenic matched control mouse with human non-deficient G6PD hG6PDND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PDMed- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. Significance Statement These findings demonstrate that 5,6-POQ, which has been suggested to be an inert waste product of active primaquine metabolites, directly induces ROS that lead specifically to removal of older G6PDd RBCs from circulation. As 5,6-POQ is relatively stable compared to other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. In addition, a new model of G6PDd is used to show that young G6PDd RBCs are resistant to primaquine induced hemolysis.

Fisetin disrupts mitochondrial homeostasis via superoxide dismutase 2 acetylation in pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PDAC) is one of the most lethal malignant tumors with an urgent need for precision medicine strategies. The present study seeks to assess the antitumor effects of fisetin, and characterize its impact on PDAC. Multi-omic approaches include proteomic, transcriptomic, and metabolomic analyses. Further validation includes the assessment of mitochondria-derived reactive oxygen species (mtROS), mitochondrial membrane potential, as well as ATP generation. Molecular docking, immunoprecipitation, and proximity ligation assay were used to detect the interactions among fiseitn, superoxide dismutase 2 (SOD2), and sirtuin 2 (SIRT2). We showed that fisetin disrupted mitochondrial homeostasis and induced SOD2 acetylation in PDAC. Further, we produced site mutants to determine that fisetin-induced mtROS were dependent on SOD2 acetylation. Fisetin inhibited SIRT2 expression, thus blocking SOD2 deacetylation. SIRT2 overexpression could impede fisetin-induced SOD2 acetylation. Additionally, untargeted metabolomic analysis revealed an acceleration of folate metabolism with fisetin. Collectively, our findings suggest that fisetin disrupts mitochondrial homeostasis, eliciting an important cancer-suppressive role; thus, fisetin may serve as a promising therapeutic for PDAC.

Formulation of Garlic Essential Oil-assisted Silver Nanoparticles and Mechanistic Evaluation of their Antimicrobial Activity against a Spectrum of Pathogenic Microorganisms.

BACKGROUND: The synthesis of nanoparticles using the principle of green chemistry has achieved huge potential in nanomedicine. Here, we report the synthesis of silver nanoparticles (Ag- NPs) employing garlic essential oil (GEO) due to wide applications of GEO in the biomedical and pharmaceutical industry. OBJECTIVE: This study aimed to synthesise garlic essential oil-assisted silver nanoparticles and present their antimicrobial and antibiofilm activities with mechanistic assessment. METHOD: Initially, the formulation of AgNPs was confirmed using different optical techniques, such as XRD, FT-IR, DLS, zeta potential, SEM, and EDX analysis, which confirmed the formulation of well-dispersed, stable, and spherical AgNPs. The antimicrobial and antibiofilm activity of GEO-assisted AgNPs was evaluated against a spectrum of pathogenic microorganisms, such as Gram-positive (S. aureus and B. subtilis) and Gram-negative (E. coli and P. aeruginosa) bacteria. RESULTS: The AgNPs exhibited remarkable antimicrobial and anti-biofilm activity against all tested strains. The mechanism behind the antimicrobial activity of AgNPs was explored by estimating the amount of reactive oxygen species (ROS) generated due to the interaction of AgNP with bacterial cells and observing the morphological changes of bacteria upon AgNP interaction. CONCLUSION: The findings of this study concluded that ROS generation due to the interaction of AgNPs with bacterial cells put stress on bacterial membranes, altering the morphology of bacteria, exhibiting remarkable antimicrobial activity, and preventing biofilm formation.

Temperature Is a Key Factor Governing the Toxic Impact of Ultra-Violet Radiation-Emitting Nail Dryers When Used on Human Skin Cells.

The skin is the largest organ in the body and the only one to come into contact with solar UV radiation (UVR). UVA (320-400 nm) is a significant contributor to UV-related skin damage. The UVA spectrum makes up over 95% of solar-UV energy reaching the earth's surface causing the majority of the visible signs of skin photoaging. Many consumer products also emit UVA, including nail dryers. There have been sporadic reports suggesting that these units may be contributing to skin cancer incidence. This notion was recently bolstered by a finding that nail dryer-irradiated mammalian skin cells develop a mutational signature consistent with UVA exposure. This report was surprising considering the comparatively low level of UVA to which the skin is exposed during nail treatments. In this research, we investigated how UVA-emitting devices caused cytotoxic/genotoxic impact after only low levels of UVA exposure. Our data showed that levels of UVA in the unit are highly variable and location dependent. We confirm previous reports that using prolonged exposure protocols could induce significant levels of DNA damage. It was also determined that UV-induced DNA damage only partially correlated with the level of UVA fluency. On investigation, we found that the unit had a rapid increase in internal temperature when in use. Exposing human cells to these elevated temperatures acted synergistically with UVA to magnify the cytotoxic and genotoxic impact of UV irradiation.

Insight into the role of TXNRD2 in steroidogenesis through a novel homozygous TXNRD2 splice variant.

OBJECTIVE: Adrenal cortisol production occurs through a biosynthetic pathway which depend on NADH and NADPH for energy supply. The mitochondrial respiratory chain and the reactive oxygen species (ROS) detoxification system are therefore important for steroidogenesis. Mitochondrial dysfunction leading to oxidative stress has been implicated in the pathogenesis of several adrenal conditions. Nonetheless, only very few patients with variants in one gene of the ROS detoxification system, Thioredoxin Reductase 2 (TXNRD2), have been described with variable phenotypes. DESIGN: Clinical, genetic, structural, and functional characterization of a novel, biallelic TXNRD2 splice variant. METHODS: On human biomaterial, we performed whole exome sequencing to identify and RNA analysis to characterize the specific TXNRD2 splice variant. Amino acid conservation analysis and protein structure modeling were performed in silico. Using patient's fibroblast-derived human induced pluripotent stem cells, we generated adrenal-like cells (iALC) to study the impact of wild-type (WT) and mutant TXNRD2 on adrenal steroidogenesis and ROS production. RESULTS: The patient had a complex phenotype of primary adrenal insufficiency (PAI), combined with genital, ophthalmological, and neurological features. He carried a homozygous splice variant c.1348-1G > T in TXNRD2 which leads to a shorter protein lacking the C-terminus and thereby affecting homodimerization and flavin adenine dinucleotide binding. Patient-derived iALC showed a loss of cortisol production with overall diminished adrenal steroidogenesis, while ROS production was significantly increased. CONCLUSION: Lack of TXNRD2 activity for mitochondrial ROS detoxification affects adrenal steroidogenesis and predominantly cortisol production.

Variants of human DECTIN-1 rs16910526 are linked to differential reactive oxygen species production and susceptibility to tuberculosis.

BACKGROUND: Dectin-1 is a transmembrane receptor that plays a pivotal role in recognising fungi and Mycobacterium tuberculosis (Mtb). A specific variant, DECTIN-1 rs16910526, results in a truncated receptor that disrupts membrane expression and ligand binding and is clinically associated with recurrent cutaneous mycoses. Previous research has clarified the role of Dectin-1 in boosting immune defenses against mycobacteria by enhancing reactive oxygen species (ROS) production in neutrophils (PMNs). Here, we investigated the association between the rs16910526 variant and Dectin-1 expression in PMNs, as well as intracellular ROS production in response to Mtb. Furthermore, we explored the potential link between the rs16910526 gene variant and TB outcomes in Argentina. METHODS: DNA was extracted from blood samples obtained from a cohort of 178 TB patients and healthy subjects (HS) in Argentina. PCR amplification and sequencing were performed to identify the rs16910526 variant. Flow cytometry was utilised to assess Dectin-1 expression on the PMN plasma membrane and to measure intracellular ROS levels, as indicated by the oxidation of DHR123 in response to the Mtb antigen. RESULTS: PMNs carrying the rs16910526 variant exhibited diminished Dectin-1 expression and ROS production in response to Mtb (p < 0.0001). In a case‒control study, the rs16910526 variant had an allelic frequency of 0.112 in TB patients and 0.051 in HS. Notably, 10 out of 88 HS and 18 out of 62 TB patients harboured the variant (odds ratio [OR]: 2.55 [95% CI 1.1-5.9, p = 0.03]), indicating a potential association with TB disease. Furthermore, TB patients with the rs16910526 variant exhibited a delayed sputum smear conversion time (p < 0.004) and 100% positivity for acid-fast bacilli smears (p < 0.00001). CONCLUSION: Our study identified a significant association between the SNP variant rs16910526 in the DECTIN-1 gene and Dectin-1 expression in the PMN, leading to altered ROS production. The higher frequency of this variant in TB patients compared to HS suggests a possible link with susceptibility to TB disease in Argentina.

Hypersensitivity to man-made electromagnetic fields (EHS) correlates with immune responsivity to oxidative stress: a case report.

There is increasing evidence that exposure to weak electromagnetic fields (EMFs) generated by modern telecommunications or household appliances has physiological consequences, including reports of electromagnetic field hypersensitivity (EHS) leading to adverse health effects. Although symptoms can be serious, no underlying mechanism for EHS is known and there is no general cure or effective therapy. Here, we present the case study of a self-reported EHS patient whose symptoms include severe headaches, generalized fatigue, cardiac arrhythmia, attention and memory deficit, and generalized systemic pain within minutes of exposure to telecommunications (Wifi, cellular phones), high tension lines and electronic devices. Tests for cerebral, cardiovascular, and other physiological anomalies proved negative, as did serological tests for inflammation, allergies, infections, auto-immune conditions, and hormonal imbalance. However, further investigation revealed deficits in cellular anti-oxidants and increased radical scavenging enzymes, indicative of systemic oxidative stress. Significantly, there was a large increase in circulating antibodies for oxidized Low-Density Lipoprotein (LDLox), byproducts of oxidative stress accumulating in membranes of vascular cells. Because a known primary effect of EMF exposure is to increase the concentration of cellular oxidants, we propose that pathology in this patient may be causally related to a resulting increase in LDLox synthesis. This in turn could trigger an exaggerated auto-immune response consistent with EHS symptoms. This case report thereby provides a testable mechanistic framework for EHS pathology with therapeutic implications for this debilitating and poorly understood condition.

Emerging nanozyme therapy incorporated into dental materials for diverse oral pathologies.

OBJECTIVE: Nanozyme materials combine the advantages of natural enzymes and artificial catalysis, and have been widely applied in new technologies for dental materials and oral disease treatment. Based on the role of reactive oxygen species (ROS) and oxidative stress pathways in the occurrence and therapy of oral diseases, a comprehensive review was conducted on the methods and mechanisms of nanozymes and their dental materials in treating different oral diseases. METHODS: This review is based on literature surveys from PubMed and Web of Science databases, as well as reviews of relevant researches and publications on nanozymes in the therapy of oral diseases and oral tumors in international peer-reviewed journals. RESULTS: Given the unique function of nanozymes in the generation and elimination of ROS, they play an important role in the occurrence, development, and treatment of different oral diseases. The application of nanozymes in dental materials and oral disease treatment was introduced, including the latest advances in their use for dental caries, pulpitis, jaw osteomyelitis, periodontitis, oral mucosal diseases, temporomandibular joint disorders, and oral tumors. Future approaches were also summarized and proposed based on the characteristics of these diseases. SIGNIFICANCE: This review will guide biomedical researchers and oral clinicians to understand the mechanisms and applications of nanozymes in the therapy of oral diseases, promoting further development in the field of dental materials within the oral medication. It is anticipated that more suitable therapeutic agents or dental materials encapsulating nanozymes, specifically designed for the oral environment and simpler for clinical utilization, will emerge in the forthcoming future.

Calcium leakage involved in nematotoxic effects of the Conidiobolus obscurus CytCo protein on the pine wood nematode, Bursaphelenchus xylophilus.

BACKGROUND: The pine wood nematode Bursaphelenchus xylophilus, a severe invasive species, is responsible for causing widespread pine wilt disease. The CytCo protein, a pore-forming toxin derived from Conidiobolus obscurus, exhibits nematotoxicity towards B. xylophilus. RESULTS: Our present study reveals the expression variation of a range of gene products in B. xylophilus that respond to the effects of CytCo using the isobaric tags for relative and absolute quantification proteomics technology. Functional enrichment analysis indicates that many differentially expressed proteins are linked to calcium signaling system, proteasome, energy production and conversion, and the determination of adult lifespan. It suggests that the dysregulation of calcium homeostasis, energy metabolism, and apoptosis contribute to the CytCo nematotoxicity. Using the calcium ion (Ca2+)-indicator calcein, we detected changes in Ca2+ levels in B. xylophilus, with a significantly increase in fluorescence in the nematode's intestine and pseudocoelom following CytCo treatments. Meanwhile, the apoptosis and reactive oxygen species (ROS) assays showed an enhancement of fluorescence in B. xylophilus cells, with increased CytCo concentrations. CONCLUSION: The protein toxin CytCo triggers Ca2+ leakage, disrupts Ca2+ balance in B. xylophilus, and induces apoptosis and ROS outburst, thereby intensifying its nematotoxic effects. This finding facilitates our understanding of the modes of action of nematotoxic proteins, and contributes to the development of innovative nematode control strategies. © 2024 Society of Chemical Industry.

Photoactivated rose bengal mitigates a fibrotic phenotype and improves cutaneous wound healing in full-thickness injuries.

Healing of deep cutaneous wounds often results in detrimental sequelae, including painful and debilitating scars. Current therapies for full-thickness injuries that target specific phases of wound healing have moderate success; however, full resolution remains incomplete and negative consequences persist if skin homeostasis is not achieved. Photoactivated molecules can modulate cellular responses by generating reactive oxygen species and may provide a novel therapeutic option to improve wound healing. In the current study, we investigated the effects of Rose bengal (RB) dye in a preclinical model of full-thickness cutaneous injury. Monochromatic green light activates RB to generate ROS in the presence of oxygen, subsequently crosslinking collagen fibrils. In in vitro studies, we show that photoactivated RB is well tolerated by epidermal keratinocytes and dermal fibroblasts and can mitigate fibrotic signalling by downregulating collagen production. In a murine model of full-thickness injury, topically-applied and photoactivated RB closed wounds faster than control and vehicle treatments and showed significantly improved wound healing outcomes, including enhanced early granulation, better collagen organisation and increased vascularity in the presence of protracted tissue ROS. These data support an overall improved cutaneous wound healing profile after RB phototherapy and warrant further investigations into this versatile molecule.

Fusarium graminearum effector FgEC1 targets wheat TaGF14b protein to suppress TaRBOHD-mediated ROS production and promote infection.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat globally. However, the molecular mechanisms underlying the interactions between F. graminearum and wheat remain unclear. Here, we identified a secreted effector protein, FgEC1, that is induced during wheat infection and is required for F. graminearum virulence. FgEC1 suppressed flg22- and chitin-induced callose deposition and reactive oxygen species (ROS) burst in Nicotiana benthamiana. FgEC1 directly interacts with TaGF14b, which is upregulated in wheat heads during F. graminearum infection. Overexpression of TaGF14b increases FHB resistance in wheat without compromising yield. TaGF14b interacts with NADPH oxidase respiratory burst oxidase homolog D (TaRBOHD) and protects it against degradation by the 26S proteasome. FgEC1 inhibited the interaction of TaGF14b with TaRBOHD and promoted TaRBOHD degradation, thereby reducing TaRBOHD-mediated ROS production. Our findings reveal a novel pathogenic mechanism in which a fungal pathogen acts via an effector to reduce TaRBOHD-mediated ROS production.

Role of ROS and autophagy in the pathological process of atherosclerosis.

Activation of autophagy and production of reactive oxygen species occur at various stages of atherosclerosis. To clarify the role and mechanism of autophagy and reactive oxygen species in atherosclerosis is of great significance to the prevention and treatment of atherosclerosis. Recent studies have shown that basal autophagy plays an important role in protecting cells from oxidative stress, reducing apoptosis and enhancing atherosclerotic plaque stability. Autophagy deficiency and excessive accumulation of reactive oxygen species can impair the function of endothelial cells, macrophages and smooth muscle cells, trigger autophagic cell death, and lead to instability and even rupture of plaques. However, the main signaling pathways regulating autophagy, the molecular mechanisms of autophagy and reactive oxygen species interaction, how they are initiated and distributed in plaques, and how they affect atherosclerosis progression, remain to be clarified. At present, there is no autophagy inducer used to treat atherosclerosis clinically. Therefore, it is urgent to clarify the mechanism of autophagy and find new targets for autophagy. Antioxidant agents generally have defects such as low reactive oxygen species scavenging efficiency and high cytotoxicity. Highly potent autophagy inducers and reactive oxygen species scavengers still need to be further developed and validated to provide more possibilities for innovative treatments for atherosclerosis.

Momordica cochinchinensis extract alleviates oxidative stress and skin damage caused by fine particulate matter.

Momordica cochinchinensis (MC), commonly known as gac fruit, is a tropical fruit rich in antioxidants and bioactive compounds. This research aimed to elucidate the effect of MC on apoptosis induced by fine particulate matter with a diameter of less than 10 µm (< PM10) in epidermal keratinocyte HaCaT cells. We found that PM10 significantly diminish the viability of HaCaT cells through cytotoxic mechanisms. However, the treatment with MC at a concentration of 10 µg/mL notably restored the cellular viability decreased by PM10. MC reduced the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) by mainly preventing the generation of reactive oxygen species (ROS) in HaCaT cells subjected to PM10. Furthermore, MC exhibited a regulatory effect on the expression of genes associated with apoptosis, including B-Cell Lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), and cleaved caspase-3 by inhibiting the activation of the transcription factor nuclear factor-kappa B (NF-κB). These findings demonstrate that MC aids in neutralizing the apoptotic signaling pathway of free radicals produced by environmental pollutants such as PM10, which have the potential to damage skin cells and accelerate the aging process.

Hyposalinity elicits physiological responses and alters intestinal microbiota in Korean rockfish Sebastes schlegelii.

Global warming significantly impacts aquatic ecosystems, with changes in the salt environment negatively affecting the physiological responses of fish. We investigated the impact of hyposalinity on the physiological responses and intestinal microbiota of Sebastes schlegelii under the context of increased freshwater influx due to climate change. We focused on the osmoregulatory capacity, oxidative stress responses, and alterations in the intestinal microbiome of S. schlegelii under low-salinity conditions. Our findings revealed compromised osmoregulatory capacity in S. schlegelii under low-salinity conditions, accompanied by the activation of oxidative stress responses, indicating physiological adaptations to cope with environmental stress. Specifically, changes in Na+/K+-ATPase (NKA) activity in gill tissues were associated with decreased osmoregulatory capacity. Furthermore, the analysis of the intestinal microbiome led to significant changes in microbial diversity. Exposure to low-salinity environments led to dysbiosis, with notable decreases in the relative abundance of Gammaproteobacteria at the class level and specific genera such as Enterovibrio, and Photobacterium. Conversely, Bacilli classes, along with genera like Mycoplasma, exhibited increased proportions in fish exposed to low-salinity conditions. These findings underscore the potential impact of environmental salinity changes on the adaptive capacity of fish species, particularly in the context of aquaculture. Moreover, they highlight the importance of considering both physiological and microbial responses in understanding the resilience of aquatic organisms to environmental stress. Additionally, they highlight the importance of intestinal microbiota analyses in understanding the immune system and disease management in fish.

Recent developments in the design of functional derivatives of edaravone and exploration of their antioxidant activities.

Edaravone, a pyrazalone derivative, is an antioxidant and free radical scavenger used to treat oxidative stress-related diseases. It is a proven drug to mitigate conditions prevailing to oxidative stress by inhibiting lipid peroxidation, reducing inflammation, and thereby preventing endothelial cell death. In recent years, considerable interest has been given by researchers in the derivatization of edaravone by adding varieties of substituents of versatile steric and functional properties to improve its antioxidant and pharmacological activity. This review accounts all the important methods developed for the derivatization of edaravone and the impacts of the structural modifications on the antioxidant activity of the motif.

You can't always get as much iron as you want: how rice plants deal with excess of an essential nutrient.

Iron (Fe) is an essential nutrient for almost all organisms. However, free Fe within cells can lead to damage to macromolecules and oxidative stress, making Fe concentrations tightly controlled. In plants, Fe deficiency is a common problem, especially in well-aerated, calcareous soils. Rice (Oryza sativa L.) is commonly cultivated in waterlogged soils, which are hypoxic and can cause Fe reduction from Fe3+ to Fe2+, especially in low pH acidic soils, leading to high Fe availability and accumulation. Therefore, Fe excess decreases rice growth and productivity. Despite the widespread occurrence of Fe excess toxicity, we still know little about the genetic basis of how rice plants respond to Fe overload and what genes are involved in variation when comparing genotypes with different tolerance levels. Here, we review the current knowledge about physiological and molecular data on Fe excess in rice, providing a comprehensive summary of the field.

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells.

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.