Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Erythrocytes possess an unusual programmed cell death mechanism termed eryptosis, and several compounds have been previously claimed to induce eryptosis in vitro. Malaria parasites (genus Plasmodium) reside in erythrocytes during the pathogenic part of their life cycle, and the potential of several eryptosis inducers to act as antimalarials has been tested in recent years. However, the eryptosis-inducing capacity of these compounds varies significantly between eryptosis-focused studies and malaria investigations. Here, we investigated the reasons for these discrepancies, we developed a protocol to investigate eryptosis in malaria cultures and we re-evaluated the potential of eryptosis inducers as antimalarials. First, we showed that eryptosis read-out in vitro is dependent on culture conditions. Indeed, conditions that have consistently been used to study eryptosis do not support P. falciparum growth and prime erythrocytes for eryptosis. Next, we defined culture conditions that allow the detection of eryptosis while supporting P. falciparum survival. Finally, we selected six eryptosis-inducers based on their clinical use, molecular target and antimalarial activities, and re-evaluated their eryptosis inducing capacities and their potential as antimalarials. We demonstrate that none of these compounds affect the viability of naïve or P. falciparum-infected erythrocytes in vitro. Nevertheless, three of these compounds impair parasite development, although through a mechanism unrelated to eryptosis and yet to be elucidated. We conclude that careful consideration of experimental set up is key for the accurate assessment of the eryptosis-inducing potential of compounds and their evaluation as potential antimalarials.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

Humans are exposed daily to artificial and naturally occurring magnetic fields that originate from many different sources. We review recent studies that examine the biological effects of and medical applications involving electromagnetic fields, review the properties of static and pulsed electromagnetic fields that affect biological systems, describe the use of a pulsed electromagnetic field in combination with an anticancer agent as an example of a medical application that incorporates an electromagnetic field, and discuss the recently updated safety guidelines for static electromagnetic fields. The most notable modifications to the 2009 International Commission on Non-Ionizing Radiation Protection guidelines are the increased exposure limits, especially for those who work with or near electromagnetic fields (occupational exposure limits). The recommended increases in exposure were determined using recent scientific evidence obtained from animal and human studies. Several studies since the 1994 publication of the guidelines have examined the effects on humans after exposure to high static electromagnetic fields (up to 9.4 tesla), but additional research is needed to ascertain further the safety of strong electromagnetic fields.

The following genotoxic chemicals were tested in the in vitro micronucleus assay, at Covance Laboratories, Harrogate, UK in the Chinese hamster lung cell line CHL. Etoposide (a topoisomerase inhibitor), colchicine (an aneugen), mitomycin C (a DNA cross linking agent) and cyclophosphamide (an alkylating agent requiring metabolic activation) were treated with and without cytokinesis block (by addition of cytochalasin B). This work formed part of a collaborative evaluation of the toxicity measures recommended in the draft OECD Test Guideline 487 for the in vitro micronucleus test. The toxicity measures used, detecting both cytostasis and cell death, were relative population doubling, relative increase in cell counts and relative cell counts for treatments in the absence of cytokinesis block, and replication index or cytokinesis blocked proliferation index in the presence of cytokinesis block. All of the chemicals tested gave significant increases in the percentage of micronucleated cells with and without cytokinesis block at concentrations giving approximately 60% toxicity (cytostasis and cell death) or less by all of the toxicity measures used. The outcomes from this series of tests support the use of relative increase in cell counts and relative population doubling, as well as relative cell counts, as appropriate measures of cytotoxicity for the non-cytokinesis blocked in vitro micronucleus assay.

The known aneugens vinblastine and diethylstilboestrol (DES) were tested in the in vitro micronucleus assay, with and without cytokinesis block in Chinese hamster CHO cells, at the laboratories of Swansea University, Swansea, UK. These experiments were carried out to determine the suitability of the cell death and cytostasis measures used in the assay, as recommended in the draft OECD Test Guideline 487, 2007. Both compounds were positive in the assay without cytokinesis block at concentrations giving approximately 50% or less cell death and cytostasis, using relative population doublings and relative increase in cell counts. Moreover, both compounds were positive in the assay with cytokinesis block at concentrations giving approximately 50% cell death and cytostasis, using replicative index. Vinblastine was also positive for mitotic slippage, causing micronuclei in mononucleate cells with cytokinesis block. Relative population doublings and relative increase in cell counts were appropriate measures of cell death and cytostasis for the non-cytokinesis block in vitro micronucleus assay. In the cytokinesis blocked micronucleus assay, replicative index and cytokinesis block proliferation index were suitable cell death and cytostasis measures.

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

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A larger data base will be required to relate the extent, duration, and nature of chemically induced cell proliferation to carcinogenic potential and to establish the importance of this end point in chemical carcinogenesis. As that relationship becomes clearer, the information gained will become valuable in investigations of mechanisms of carcinogenesis, development of short-term tests for nongenotoxic carcinogens, selection of appropriate doses for cancer bioassays, and in the formulation of improved risk-assessment models. Because the quality of chemically induced cell replication data depends on the procedures and protocols followed for these labor-intensive studies, efforts among laboratories must be coordinated so that the work and the expenditure of substantial resources will yield a cohesive data base.