Analysis of programmed cell death (PCD) and cell cycle in ecotoxicology

Ecotoxic impact of chemicals is often analysed using quantitative molecular and biochemical markers such as enzymes involved in detoxification as well as elimination of reactive oxygen species (ROS). However, so far very little attention has been devoted to environmental toxicants-induced programmed cell death as well as perturbations mitochondrial functions (mitotoxicity) and cell cycle.

The toxicants can damage the cells in diverse organs as a result of specific or non-specific (e.g. generation of reactive oxygen species etc) effects. Pollutant-induced cell death can be also a result of DNA damage if the toxicants have genotoxic properties and the damage induced exceeds the ability of cells to perform DNA repair processes. In all cases excessive cell death and mitochondrial dysfunctions can lead to patho-physiologies and functional deficiencies.

Aquatic organisms with ex utero development are particularly vulnerable to cell damage during embryogenesis. Perturbations in PCD as well as cell cycle can have a tremendous impact on survival and teratological phenotypes impacting them at later stages in life cycle.

Markers for many diverse modes of caspase-dependent cell death have been particularly well developed in aquatic vertebrate models and can be analysed using techniques ranging from molecular techniques, microscopy and flow cytometry as well as imaging cytometry.

 

Xenobiotic-induced disturbances in cell proliferation and programmed cell death.

Intertwined molecular signalling network that involves reactive oxygen species generation and sub-cellular oxidative damage. Some pollutants can also induce direct damage to organelles such as mitochondria and endoplasmic reticulum. Genotoxic contaminants inducing DNA damage will initiate a DNA damage response. In all of the above examples irreparable changes exceeding cellular capacity to repair them will lead to cell cycle arrest and subsequent activation of programmed and/or accidental cell death.

A cross-talk between xenobiotic induced mitochondrial damage, ROS generation, oxidative damage and induction of different modes of cell death. Note that low level oxidative damage despite survival and cell repairs can lead to subsequent oncogenic events.

The biology of cell death

Particular interest in biology of cell death comes from the discovery of multitude complex regulatory circuits that control the cellular demise.

Historically cells were known die in two distinct morphologically and biochemically processes such as apoptosis and necrosis often referred to as programmed and accidental cell death (necrosis), respectively.

Both were initially identified based on characteristic changes in cell morphology. Figures below outline major morphological and molecular changes occurring during apoptosis versus accidental cell death (herein termed necrosis).

Alterations in those complex cellular parameters become a basis for development of a variety of markers for fluorescence microscopy and flow cytometry.

Despite subsequent introduction of numerous molecular assays, the morphological changes, detected by light and electron microscopy, still remain the ‘‘gold standard’’ to differentiate these two distinct modes of cell death.

Excessive cell death can lead to many developmental as well functional patho-physiologies.

An example of mitochondrial pathway of caspase-dependent apoptosis commonly triggered by chemical damage is shown above. 

Morphological hallmarks of apoptosis and accidental cell death (necrosis). Note that some features of apoptosis may not be present and depend heavily on a particular cell type, stimuli, and cellular microenvironment.

Biochemical hallmarks of apoptosis and accidental cell death (necrosis). Note that some features of apoptosis may not be present and depend heavily on a particular cell type, stimuli, and cellular microenvironment.

Mitochondria as sentinels of xenobiotic-induced cell damage

Apart from functions in bioenergetics the mitochondrial networks stand at the nexus of sensing and integrating diverse incoming stress signals, and mitochondrial disturbances often occur long before any marked morphological symptoms of cell death.

In recent years multiple mechanisms have been revealed that explain mitochondrial function in apoptosis, including release of apoptogenic proteins into the cytosol upon mitochondrial outer membrane permeabilization (MOMP), loss of mitochondrial physiological processes indispensable for cell survival and generation of reactive oxygen species (ROS).

The MOMP is a fundamental event leading to a release of holocytochrome c (cyt c) and an array of cell death modulating small proteins such as AIF, EndoG, Omi/HtrA2, Smac/DIABLO, Smac b, normally enclosed in the intermembrane space of the organell. Dissipation of mitochondrial inner transmembrane potential is frequently associated with MOMP.

Interestingly, as described by our laboratory and others, dissipation of mitochondrial inner transmembrane potential may not be an ultimate point of no return for cell commitment to die.

The detection of mitochondrial inner transmembrane potential loss is a sensitive marker of early apoptotic events. Procedures are based on lipofilic cationic probes that are readily taken up by live cells and accumulate in mitochondria according to the Nernst equation. The extent of their uptake, as measured by intensity of cellular fluorescence, is proportional to mitochondrial inner transmembrane potential.

Majority of sensitive probes are easily applicable for multiparameter detection with other apoptotic markers including caspase activation by fluorescently labelled inhibitors of caspases (FLICA), phosphatidylserine (PS) exposure by Annexin V and plasma membrane permeabilization by propidium iodide (PI) or YO-PRO 1.

Our laboratory has pioneered a plethora of cytometric and imaging teniques and bioassays for the detection of apoptosis as well as mitochondrial function.

A rapid cytometric bioassay that measures dissipation of mitochondrial transmembrane potential in living cells as a marker of mitochondrial damage.

Analysis by staining with a lipofilic cationic probe tetramethylrhodamine methyl ester (TMRM). Cells were either untreated (Ctrl) or treated with cycloheximide (CHX) to induce apoptosis and supravitally loaded with TMRM as described.

Cells with collapsed mitochondrial transmembrane potential have decreased intensity of orange TMRM fluorescence (mito loss gate).

An advanced cytometric bioassay enabling to perform rapid multiparameter labelling of mitochondrial function and DNA content in living cells using tetramethylrhodamine methyl ester (TMRM) and stoichiometric DNA probe DRAQ5 probes, respectively.

The bivariate dot plots represent the staining with the status of incorporation of TMRM in active, energised mitochondria in relation to DNA content (DRAQ5) with G1, S and G2/M phases of the cell cycle gated for quantitative analysis. A typical run used a sample of 5000 cells.

Many ways to die: beyond apoptosis and necrosis

Last decade has led to the characterization of many alternative cell demise modes beyond apoptosis and necrosis.

Those including among others caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necroptosis, necrosis-like PCD, and mitotic catastrophe.

Autophagy for instance is an intracellular bulk degradation system for long-lived proteins and whole organelles. Emerging evidence suggests that while autophagy may enhance survival of cells exposed to nutrient deprivation, hypoxia or certain xenobiotics, it may also contribute to cell death when induced above an acceptable for cellular homeostasis threshold.

Although still a matter of debate, these non-canonical pathways appear to have wide reaching connotations in pathogenesis and treatment of human diseases but have been so far completely unexplored in ecotoxicology. Moreover, they present an increasingly complex network of molecular cross-talks reflecting in a wide diversity of cellular phenotypes.

These discoveries raised also an ongoing debate aiming at the classification of cell death programs. It must be acknowledged that the general term apoptosis, commonly exploited in many research articles, tends sometimes to misinterpret the actual mechanisms underlying molecular suicide.

 

Current concepts on the complexity of cell demise modes.

Last decade has led to the characterisation of many alternative cell demise including among others caspase-independent apoptosis-like programmed cell death (PCD), autophagy, necroptosis, necrosis-like PCD, and mitotic catastrophe

Analysis of programmed cell death

In view of recent discoveries of alternative cell death pathways it has been, postulated to restrict the term apoptosis to only the traditional cascade featuring all canonical ‘‘hallmarks of apoptotic cell death,’’ such as (i) activation of caspases as an absolute marker of cell death ; (ii) high degree of compaction of chromatin; (iii) activation of endonucleases(s) causing internucleosomal DNA cleavage and extensive DNA fragmentation; (iv) appearance of distinctive cellular morphology with preservation of organelles, (v) cell shrinkage, (vi) plasma membrane blebbing, and (vii) nuclear fragmentation followed by formation of apoptotic bodies..

Markers for many diverse modes of caspase-dependent cell death have been particularly well developed in aquatic vertebrate models and can be analysed using techniques ranging from molecular techniques, microscopy and flow cytometry as well as imaging cytometry.

Our laboratory employs diverse methods that allow for implementation of apoptotic assays on live suspension, adherent cells, in situ using transgenic models and innovative fluorescent probes as well as fixed specimens such as cell and tissue samples.

We specialise in multi parameter flow and imaging bioassays to detect programmed cell death, cell proliferation and analyse changes in cell cycle such as for instance cell cycle arrest. The major advantages of cytometry include the possibility of multiparameter measurements (correlation of different cellular events at a time), single cell analysis (avoidance of bulk analysis), and rapid analysis of cell populations. Cytometry overcomes a frequent problem of traditional bulk techniques such as fluorimetry, spectrophotometry, or gel techniques (e.g., Western blot, WB). These are based on analysis of a total cell population that averages the results from every given cell.

Moreover, by virtue of multiparameter analysis, cytometry allows correlative studies between many cell attributes based on both light scatter and fluorescence measurements. For example, when cellular DNA content, the parameter that reports the cell cycle position, is one of the measured attributes, an expression of other measured attribute(s) can be then directly related to the cell cycle position.

This advantageous when investigating mechanisms of toxicant action because change in expression of particular cell constituents, or co-expression of different events, if correlated within the same cell, may yield clues regarding a possible cause–effect relationship between the detected events.

Novel technologies such as cell imaging in flow and laser scanning cytometry (LSC) deliver even more sophisticated features that combine superior statistical power of cytometric analysis coupled with in situ and in vivo imaging capabilities.

Multi-parameter analysis of caspase 3 activation in relation to the cell cycle profile (DNA content) using flow cytometry.

Cells were grown in the presence of a pan-kinase inhibitor staurosporine (1 nM, 10 nM) and a xanthene H-type dimmer caspase substrate dye PhiPhiLux. After 24 h of culture, cells were counterstained stained with DRAQ5 probe for 20 min and immediately analyzed on the microflow cytometer. To perform cell cycle analysis DRAQ5 fluorescent signals were amplified using linear mode whereas PhiPhiLux signals using logarithmic mode. The cell subpopulation incorporating PhiPhiLux probe (representing activated caspases) is shown above the threshold marked by the dashed line.

Flow cytometric analysis of apoptosis in planarian primary cells using Annexin V and 7-AAD fluorescent probes.

Dissociation of planarians into single cells provide also opportunity for multiparameter analysis of apoptosis and cell cycle using flow cytometry. Such assays can be particularly useful in studies of toxicant mode of action as well as screening of genotoxic chemicals

Cell proliferation and apoptosis markers in a planarian model Schmidtea mediterranea and Dugesia japonica enable rapid in situ cellular toxicology studies.

Convenient whole-mount immunohistochemistry protocols as well as an array of monoclonal antibodies and in situ hybridization probes enables mechanistic studies of the impact of toxicant exposures on the cell adaptive mechanisms, cytotoxicity, perturbations in the cell cycle as well as genotoxic responses.

References

Bownik A and Wlodkowic D 2021 Applications of advanced neuro-behavioural analysis strategies in aquatic ecotoxicology, Science of The Total Environment, 772, 145577DOI

Walpitagama M, Carve M, Douek AM, Trestrail C, Bai Y, Kaslin J, Wlodkowic D 2019 Additives migrating from 3D-printed plastic induce developmental toxicity and neuro-behavioural alterations in early life zebrafish (Danio rerio), Aquatic Toxicology, 213, 105227DOI

Wlodkowic D, Akagi J, Dobrucki J, Errington R, Smith PJ, Takeda K, Darzynkiewicz Z 2013 Kinetic Viability Assays Using DRAQ7 Probe, Current Protocols in Cytometry, 65(1), 9.41.1-9.41.8DOI

Akagi J, Kordon M, Zhao H, Matuszek A, Dobrucki J, Errington R, Smith PJ, Takeda K, Darzynkiewicz Z, Wlodkowic D 2013 Real-Time Cell Viability Assays Using a New Anthracycline Derivative DRAQ7, Cytometry Part A 􏰆 83A: 227􏰇234DOI

Akagi J, Takeda K, Fujimura Y, Matuszek A, Khoshmanesh K, Wlodkowic D 2013 Microflow cytometry in studies of programmed tumor cell death, Sensors and Actuators B: Chemical, 189, 11-20DOI

Skommer J, Akagi J, Takeda K, Fujimura Y, Khoshmanesh K, Wlodkowic D 2013 Multiparameter Lab-on-a-Chip flow cytometry of the cell cycle, Biosensors and Bioelectronics, 42, 586-591DOI

Wlodkowic D, Faley S, Darzynkiewicz Z, Cooper JM 2011 Real-Time Cytotoxicity Assays, Cancer Cell Culture Part of the Methods in Molecular Biology book series (MIMB, volume 731), 285-291DOI

Darzynkiewicz Z, Traganos T, Zhao H, Halicka D, Skommer J, Wlodkowic D 2011 Analysis of Individual Molecular Events of DNA Damage Response by Flow- and Image-Assisted Cytometry, Methods in Cell Biology
103, 115-147DOI

Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z 2011 Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death, Methods in Cell Biology, 103, 55-98DOI

Skommer J, Darzynkiewicz Z, Wlodkowic D 2010 Cell death goes LIVE: Technological advances in real-time tracking of cell death, Cell Cycle, 9:12, 2330-234DOI

Wlodkowic D, Skommer J, Darzynkiewicz Z 2010 Cytometry in cell necrobiology revisited. Recent advances and new vistas, Cytometry Part A, 77A(7), 591-606DOI

Zhao H, Oczos J, Janowski P, Trembecka D, Dobrucki J, Darzynkiewicz Z, Wlodkowic D 2010 Rationale for the real‐time and dynamic cell death assays using propidium iodides, Cytometry Part A, 77A(4), 399-405DOI

Wlodkowic D, Skommer J, Faley S, Darzynkiewicz Z, Cooper JM 2009 Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry, Experimental Cell Research, 315(10), 1706-1714DOI

Wlodkowic D, Skommer J, Darzynkiewicz Z 2009 Flow Cytometry-Based Apoptosis Detection, Apoptosis Part of the Methods in Molecular Biology book series (MIMB, volume 559), 19-32DOI

Wlodkowic D, Skommer J, Darzynkiewicz Z 2008 SYTO probes in the cytometry of tumor cell death, Cytometry Part A, 73A(6), 496-507DOI

Wlodkowic D, Skommer J, Hillier C, Darzynkiewicz Z 2008 Multiparameter detection of apoptosis using red‐excitable SYTO probes, Cytometry Part A, 73A(6), 563-569DOI

Skommer J, Wlodkowic D, Deptala A 2007 Larger than life: Mitochondria and the Bcl-2 family, Leukemia Research, 31(3), 277-286DOI