1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz BIOMARKERS AND TOXICITY MECHANISMS 08 –Toxicity mechanisms at cell level OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif INTRACELLULAR signals as target to toxicants http://site.motifolio.com/images/Intracellular-signaling-pathways-5111156.png 1212569_21823227.jpg logo_mu_cerne.gif •- Regulation controlled by complex signalling • - "network" of general pathways • - similar in all cells / different cell-specific effects • •- Consequences of signalling disruption • - unwanted changes in „homeostatic“ rates among proliferation / differentiation / apoptosis –à cell transformation (carcinogenicity) –à embryotoxicity –à immunotoxicity –à reproduction toxicity – .... and other chronic types of toxicity Intracellular signal transduction: target of toxicants 1212569_21823227.jpg logo_mu_cerne.gif Signal transduction - principles •Two major intracellular signalling processes – protein-(de)phosphorylation • ProteinKinases - PKs, ProteinPhosphatases - PPases • - secondary messengers • cAMP / IP3, PIP2, DAG, Ca2+, AA • •Three major types of signalling –1: Membrane receptors - G-proteins / kinases à activation of protein kinase A (PKA): major messenger: cAMP – –2: Membrane receptors à activation of membrane lipases à and later proteinkinase C IP3, PIP2, DAG, Ca2+, AA – –3: Cytoplasmic (nuclear) receptors (discussed in detail in other sections) – 1212569_21823227.jpg logo_mu_cerne.gif •Membrane receptors acting as ProteinKinases G-proteins & G-protein coupled receptors - GPCRs • C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif •Signalling mechanism 1 •à Activation of adenylate cyclase à cAMP à PKA C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif ßPKA is central to a number of signalling events and following effects ßIncluding modulation of “MAPKs” 1212569_21823227.jpg logo_mu_cerne.gif Mitogen Activated Protein Kinases (MAPKs) & dependent effects C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif •Signalling mechanism 2 •Activation of Phospholipase C à release of PIPs à DAG à PKC / arachidonic acid + IP3 à activation of Ca2+ signalling C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Obrázky\1.jpg Different “types” of signalling crosstalk à networks 1212569_21823227.jpg logo_mu_cerne.gif •Cholera toxin •CT acts as adenylate cyclase enzyme àincreasing cAMP levels àTOXICITY • Disruption of intracellular signaling - EXAMPLES 1212569_21823227.jpg logo_mu_cerne.gif •Example: Lipopolysaccharides (LPS) from cell walls • à hyperactivation of intracellular signals à immunotoxicity • 1212569_21823227.jpg logo_mu_cerne.gif DOI: 10.1021/acs.est.5b02049 Environ. Sci. Technol. 2015, 49, 12457−12464 1212569_21823227.jpg logo_mu_cerne.gif Cell and its basic functions and life trajectories •Metabolism •Proliferation (cell division) – cell cycle •Diferentiation •Senescence •Cell death -Apoptosis -Necroptosis -Necrosis https://www.i-med.ac.at/imcbc/bc/bilder/picture3.jpg Influence of toxicity mechanisms on cellular life trajectories •Various toxicity mechanisms / modes of action –i.e. those discussed previously •PROTEINS – enzyme inhibitions, protein damage/oxidation •DNA damage •MEMBRANE disruption –as well as others •including mainly INTRACELLULAR signalling disruptions • •… affect the cell fate, and propagate to systemic effects: …examples… –Disruption of metabolism •Acute à (cell) death (CO, CN- à effects on mitochondria/haeme) •Chronic à various diseases (e.g. diabetes) –Effects on proliferation (cell division) – cell cycle •Tumor growth, carcinogenesis, effects on immune system / haemopoiesis –Diferentiation •Developmental toxicity, embryotoxicity, teratogenicity, immune system effects –Senescence (Usually not adverse or toxic) –Cell death •NECROSES (e.g. after irradiation) •APOPTOSES (bone marrow – haemopoetic effects; effects on tumors) 1212569_21823227.jpg logo_mu_cerne.gif CELL CYCLE and its careful CONTROL - importance •GENERAL –Control of genetic material and information (including reparation) –Proper distribution of genetic material into daughter cells – •EARLY DEVELOPMENT –Regulation of development, embryo- and organogenesis – •ADULTHOOD –Reconstruction and renewal of adult tissues –Control of proliferation / tumor growth Cell cycle regulation and control •Factors controlling proper cell cycle •Extracellular signals (hormones, neurotrasmitters…) •Intracellular „stress sensors“ and signals •p53 protein among others •Correct sequence of individual events (phases) •Error-free events • •Controlling principles –General: •Phosphorylation (kinases) / dephosphorylation (phosphatases) of proteins à discussed further –Such as … for cell cycle: •cyclines and CDK (cycline-dependent kinases) • • • Výsledek obrázku pro cdk cell cycle 1212569_21823227.jpg logo_mu_cerne.gif Role and functions of p53 Výsledek obrázku pro p53 phosphorylation * Phosphorylation of p53 * acetylation (A), methylation (M) * and ubiquitylation (Ub) of p53 * as well as „mutations“ (SNPs) of p53 à Control and affect key cellular processes http://a.static-abcam.com/CmsMedia/Media/p53-acetylation-methylation-and-ubiquitylation-sites.jpg 1212569_21823227.jpg logo_mu_cerne.gif Example - p53 in control of intracellular stress / such as DNA damage p53-diagram 1212569_21823227.jpg logo_mu_cerne.gif Cell deaths Necrosis •Pathology •Membrane damage •Cell „explosion“/ lysis •Chromatin disintegration • •à immune reaction (inflammation) •„scars“ formation Apoptosis • •Physiological •Suicidal process (internal •Carefully controlled •DNA fragmentation •Membrane „blebbing“ •Apoptotic bodies à fagocytosis • • Further cell death variants also recognized (different cell fate and control) •Necroptosis •Ferroptosis 1212569_21823227.jpg logo_mu_cerne.gif http://www.aibnsus.org/images/Cell.jpg Výsledek obrázku pro apoptosis control