1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU Ecotoxic effects - Cellular and organisms levels - OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Toxicity at cellular level http://www.sszdra-karvina.cz/bunka/bi/03eu/obr/euk.gif Molecular mechanisms (effects on proteins, membranes, DNA) manifest at cellular level 1212569_21823227.jpg logo_mu_cerne.gif Life trajectories of the cell Regular pathways of cell life 1) Cycling (cell cycle, proliferation) 2) Due to limited proliferation à senescence or or terminal differentiation or cell death (controlled) – apoptosis Homeostasis assured through careful check of key processes, i.e. Cell membrane integrity Aerobic respiration (mitochondria) Proteosynthesis (ribozomes) DNA integrity …. Effects on these processes à toxicity https://www.i-med.ac.at/imcbc/bc/bilder/picture3.jpg 1212569_21823227.jpg logo_mu_cerne.gif http://www.aibnsus.org/images/Cell.jpg 1212569_21823227.jpg logo_mu_cerne.gif IMPACTS and manifestation of toxicity at cell level Disruption of cell proliferation •Tumors, cancer •Immune system disruption (proliferation in many processes) Disruptions of differentiation •Important for early development (embryotoxicity, teratogenicity) •Tumors (cells often NOT differentiated) •Immune systém Disruptions of apoptosis •Tumors (cells escape apoptosis) •Effects on immune system –(TCDD induced activation of AhR à apoptosis in thymus à loss of functional immune reactions • 1212569_21823227.jpg logo_mu_cerne.gif Oxidative stress Important general mechanism of celluar toxicity 1212569_21823227.jpg logo_mu_cerne.gif Importance of redox (oxido-reduction) homeostasis •Redox homeostasis –natural homeostatic levels of prooxidants and antioxidants –keeping cell metabolism and signalling balanced – •Disruptions of homeostasis –à depletion of oxygen •Change in metabolism, acidosis in tissues, signalling (e.g. TUMORS) •Less studied – new field – REDOX SIGNALLING à overproduction of prooxidants = oxidative stress •GENERAL MECHANISM OF TOXICITY AND AGING • http://www.neurogenol.co.uk/images/oxidativestress.gif 1212569_21823227.jpg logo_mu_cerne.gif •Oxygen (O2) –principal molecule in living organisms •terminal acceptor of electones –highly reactive molecule •formation of reactive derivatives à ROS à toxicity • •Other reactive molecules and ROS sources •production in mitochondria (byproducts of metabolism) •oxidations in detoxification mediated via MFOs (CYPs) •Fenton-reaction (toxic metals) •Depletion of antioxidants … caused by presence of all kinds of reactive chemicals •Redox-cycling (quinones of xenobiotics) •and others Pro oxidants 1212569_21823227.jpg logo_mu_cerne.gif Key Reactive Oxygen Species (ROS) SOD = Superoxide dismutase 1212569_21823227.jpg logo_mu_cerne.gif Reactivity of ROS (short rate à instability = reactivity) 1212569_21823227.jpg logo_mu_cerne.gif http://www.qub.ac.uk/schools/SchoolofBiologicalSciences/People/DrAGalkin/Research/Image1,172487,en. jpg Mitochondria (= metabolism!) Unwanted (side effect) production os O2*- (superoxide) during ATP synthesis = during oxidative respiration 1212569_21823227.jpg logo_mu_cerne.gif Metals and impacts on redox homeostasis (* direct ROS production / * binding to proteins) http://www.cell.com/cms/attachment/600379/4726988/gr1.jpg , Cr + H2O2 (Fenton reaction) 1212569_21823227.jpg logo_mu_cerne.gif CYP450 as ROS source (example CYP2E1, MEOS – microsomal ethanol oxidising system) http://themedicalbiochemistrypage.org/images/cyp2e1-activities.jpg 1212569_21823227.jpg logo_mu_cerne.gif Irradiation as a source of ROS and oxidative damage (reminder – check lectures on toxicity towards DNA) http://image.slidesharecdn.com/radioactivity-120828105904-phpapp02/95/radioactivity-12-728.jpg?cb=1 346151619 ROS 1212569_21823227.jpg logo_mu_cerne.gif Oxidative damage to cellular components & biomarkers of oxidative damage 1212569_21823227.jpg logo_mu_cerne.gif Effects of oxidative stress … multiple e.g. acute coronary syndrome (ACS) à myocardial infarction http://www.geneactivatornrf2.org/wp-content/uploads/2013/03/oxidative_stress_diseases-600.gif 1212569_21823227.jpg logo_mu_cerne.gif The cellular effects further propate à level of the ORGANISM 1212569_21823227.jpg logo_mu_cerne.gif Acute lethal toxicity (fish) & relevant toxicity mechanisms Russom et al. Environmental Toxicology and Chemistry, Vol. 16, No. 5, pp. 948–967, 1997 1212569_21823227.jpg logo_mu_cerne.gif „Chronic“ mechanisms less explored Usually not tested in ecotoxicity assays Slow manifestation and effects in ecosystems - Various effects: à growth inhibition (~ lower food uptake) à diseases such as carcinogenicity à teratogenicity and embryotoxicity, developmental toxicity àReproduction toxicity à à à Organ-specific types of toxicity à Imunotoxicity à Neurotoxicity à Nefrotoxicity etc. - - CHRONIC and DELAYED TOXICITY „Systemic“ effects 1212569_21823227.jpg logo_mu_cerne.gif •Organism level – important in ecotoxicology (see Bioassays) §Effects on structure §Effects on metabolism (maintenance) §Effects on regulation § àChanges in functions (e.g. Ethinylestradiol) àRepair, survival, growth àDeath (lethality) àProliferation = Reproduction Effects at different levels - ORGANISM 3 key apical endpoints (reflected e.g. in regulations) 1212569_21823227.jpg logo_mu_cerne.gif Metabolism Control, Interactions with environment Defence against pathogens predators … Defence against toxicants Energy hv food Losses heat faeces Life (maintenance) Growth to sexual maturity Reproduction Chemical stress Chemical stress + ... another stress (food scarcity) REMINDER: Energy & Life 1212569_21823227.jpg logo_mu_cerne.gif Example - GROWTH inhibition in fish Exposures to PAHs +/- UV (phototoxicity) Model fish = Japanese medaka Growth is proportional to food/feed consumption (measuring of food consumption answers how toxicant affects the growth) 1212569_21823227.jpg logo_mu_cerne.gif Example – ecotoxicity of cytostatic drugs and their metabolites (Zounková et al. 2010 Chemosphere 81:253-260) 5-Fluorouracil Cytarabin Gemcitabin Metabolity 1212569_21823227.jpg logo_mu_cerne.gif (Zounková et al. 2010 Chemosphere 81:253-260) Acute toxicity – D. magna Reproduction toxicity 5-FU Fig. 1. Ecotoxicity (concentration–response curves) of the studied cytostatic drugs and their metabolites. (A) Daphnia magna acute immobilization test. Fig. 2. Effects of 5-fluorouracil (5-FU) on the reproduction of Daphnia magna (numbers of offsprings) in the 21-d chronic test. Example – aquatic ecotoxicity of cytostatic drugs 1212569_21823227.jpg logo_mu_cerne.gif Zounkova, R., Z. Kliemesova, L. Nepejchalova, K. Hilscherova and L. Blaha (2011). "Complex Evaluation of Ecotoxicity and Genotoxicity of Antimicrobials Oxytetracycline and Flumequine Used in Aquaculture." Environmental Toxicology and Chemistry 30(5): 1184-1189. Example – aquatic ecotoxicity of cytostatic drugs Oxytetracyklin – Wikipedie Flumequine - Wikipedia 1212569_21823227.jpg logo_mu_cerne.gif Zounkova, R., Z. Klimesova, L. Nepejchalova, K. Hilscherova and L. Blaha (2011). "Complex Evaluation of Ecotoxicity and Genotoxicity of Antimicrobials Oxytetracycline and Flumequine Used in Aquaculture." Environmental Toxicology and Chemistry 30(5): 1184-1189.0 Flumequine OTC 1212569_21823227.jpg logo_mu_cerne.gif Carcinogenicity 1212569_21823227.jpg logo_mu_cerne.gif E3 E3 1212569_21823227.jpg logo_mu_cerne.gif Reproduction toxicity, developmental toxicity, embryotoxicity and teratogenicity 1212569_21823227.jpg logo_mu_cerne.gif Reproduction and development are closely related https://sites.google.com/site/adesignframeworkforevolution/design-patterns-in-evolution/developpmet mouse.jpg Reprod Reprod 1212569_21823227.jpg logo_mu_cerne.gif Embryotoxicity = general term – toxicity to embryo Teratogenicity = morphological developmental effects Malformations, missing organs etc. - well characterized in aquatic vertebrates -ecotoxicity tests - Danio rerio, Xenopus laevis DEVELOPMENTAL TOXICITY 1212569_21823227.jpg logo_mu_cerne.gif Fig2C Fig2B Teratogenicity effects Kunisuke01 Examples of teratogens - organochlorine compounds (DDT, DDE) - new types of pesticides ATRAZIN - PCBs and compounds with dioxin-like mechanims - toxic metals - natural toxins (e.g. From cyanobacteria) Embryos of frogs X. laevis Controls exposure to cyanotoxins Japanese medaka teratogenicity of PCBs 1212569_21823227.jpg logo_mu_cerne.gif Dvořáková, D., K. Dvořáková, L. Bláha, B. Maršálek and Z. Knotková (2002). "Effects of cyanobacterial biomass and purified microcystins on malformations in Xenopus laevis: teratogenesis assay (FETAX)." Environmental Toxicology 17(6): 547-555. Toxic cyanobacteria and microcystin dynamics in a tropical reservoir: assessing the influence of environmental variables | Environmental Science and Pollution Research 1212569_21823227.jpg logo_mu_cerne.gif Fig. 1. Mortality in the 96-h FETAX test after exposure to purified microcystin-LR (MLR) and the biomass of cyanobacterial water blooms: (A) Dose–response curves of purified MLR (scale in g/L on X axis), biomass containing natural microcystins (bloom dominated by Microcystis aeruginosa), and biomass with no detectable microcystins (bloom dominated by M. wesenbergii; scale milligrams of biomas d.w. per liter on X axis). Concentrations of purified MLR and the M. aeruginosa biomass are proportional (e.g., 12 mg of the biomass d.w. contained 10 g of MLR). (B) Toxic effects of externally added MLR (25–250 g/L) to the cyanobacterial biomass with no natural microcystins. Asterisks (**) indicate statistically significant difference from the effect of the biomass (300 g/L) with no MLR addition (Pearson’s chi-square, p 0.01). Bars represent means standard error of the mean of two independent experiments each performed in two parallels. 1212569_21823227.jpg logo_mu_cerne.gif Endocrine disruption •Interference of xenobiotics with normal functioning of hormonal system • •Known consequences •à Disruption of homeostasis, reproduction, development, and/or behavior (and other hormone-controlled processes), such as –Shift in sex ratio, defective sexual development –Low fecundity/fertility –Hypo-immunity, carcinogenesis –Developmental processes - malformations –etc. 1212569_21823227.jpg logo_mu_cerne.gif Endocrine disrupters in the environment? • • EDCs... •Persistent Organic Compounds (POPs and their metabolites) •steroid hormones and their derivatives from contraception pills •alkylphenols •organometallics (butyltins) •pharmaceuticals •Pesticides •+ number of unknowns … • Tributyl-tin alkylphenols 2,3,7,8-TCDD estradiol 1212569_21823227.jpg logo_mu_cerne.gif Effects of EDs in invertebrates (molluscs) One of the first EDC effects: = imposex •Development of male sexual characteristic in females •Effects of alkyltins (e.g. Tributyl tin) – anti-fouling agents A picture containing water, large, sitting, snow Description automatically generated BIOFOULING 1212569_21823227.jpg logo_mu_cerne.gif Female estrogens and contraception pills 1 Feminization Intersex Female eggs (oocytes) formed in male testes Reproduction disruption Decline in fish populations 1212569_21823227.jpg logo_mu_cerne.gif Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen. PNAS 104(21):8897-8901 Control lake lake with EE2 EE2 - 5 ng/L (!) 1212569_21823227.jpg logo_mu_cerne.gif Organ-specific ecotoxic effects 1212569_21823227.jpg logo_mu_cerne.gif Examples - Mortalities of seals, dolfins – morbillivirus infections / PCBs, PCDDs - Elevated skin lesions (fungi, bacteria) in fish from contaminated sites - Arsenic à direct toxicity to natural killer cells in immune system (responsible for removal of tumors à increased carcinogenicity) - Prenatal exposures to DIOXINS à complete „apoptosis“ (convolusion) of thymus à not immune system in offsprings (no T-cells) IMMUNOTOXIC EFFECTS OF ECOTOXICANTS 1212569_21823227.jpg logo_mu_cerne.gif 1] Acute toxicity - spasms, effects on CNS, suffocation, death 2] Chronic effects à effects on behaviour, learning etc.. Behavioral changes – critical for survival of individuals and populations - male-female attraction / reproduction, foraging, hiding from predators -Loss of synchronization in release of gametes (aquatic invertebrates and vertebrates) - Complex reproduction behaviour (birds and mammals) - Slower burrying of molluscs into sediments ß fast predation à lower fitness and lower reproduction success NEUROTOXIC EFFECTS (e.g. Insecticides) 1212569_21823227.jpg logo_mu_cerne.gif NEFROTOXICITY IN VULTURES •- Damaging effects of veterinary pharmaceuticals on vulture populations • - primary effect à kidney in vultures = nephrotoxicity http://img2.allvoices.com/thumbs/image/609/480/103574038-diclofenac-drug.jpg http://www.painstopanswers.com/images/diclofenac.jpg 1212569_21823227.jpg logo_mu_cerne.gif TOXIC EFFECTS TO PRODUCERS (plants, algae) Unique process of PHOTOSYNTHESIS Target to many herbicidies – e.g. Diuron (DCMU) and Paraquat 1212569_21823227.jpg logo_mu_cerne.gif figure4 figure4 figure4 figure4b figure4 Acute effects in producers Example: Effects of metals on chlorophyll-a content in algae Damage to photosynthetic pigments cell and plant death 1212569_21823227.jpg logo_mu_cerne.gif Example – ecotoxicity of cytostatic drugs and their metabolites (Zounková et al. 2010 Chemosphere 81:253-260) 5-Fluorouracil Cytarabin Gemcitabin Metabolity 1212569_21823227.jpg logo_mu_cerne.gif Effects of cytostatics on ALGAL GROWTH (Zounková et al. 2010 Chemosphere 81:253-260) 1212569_21823227.jpg logo_mu_cerne.gif Toxicity of PAHs & their N-derivatives to plants (Pašková et al. 2006 Environmental Chemistry and Ecotoxicology 25:3238–3245) 1212569_21823227.jpg logo_mu_cerne.gif Toxicity of PAHs & their N-derivatives to plants (Pašková et al. 2006 Environmental Chemistry and Ecotoxicology 25:3238–3245) 1212569_21823227.jpg logo_mu_cerne.gif Toxicity of PAHs & their N-derivatives to plants (Pašková et al. 2006 Environmental Chemistry and Ecotoxicology 25:3238–3245) 1212569_21823227.jpg logo_mu_cerne.gif EFFECTS on DECOMPOSERS bacteria, microorganisms Key component for global GEO-BIO-CHEMICAL CYCLES http://askabiologist.asu.edu/sites/default/files/image/ecosystems/ecosystem_movement.jpg 1212569_21823227.jpg logo_mu_cerne.gif 1) Unicellular (or small in general) large specific surface – easy uptake of chemicals - 2) Relativelly good protection (cell wall) 3) Fast division and proliferation - generally good ADAPTATION of populations (antimicrobial resistencies) Specific notes on ecotoxicity to microorganisms http://upload.wikimedia.org/wikipedia/commons/thumb/3/32/EscherichiaColi_NIAID.jpg/210px-Escherichi aColi_NIAID.jpg 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif Therapeutic antibiotics … and resistance 1212569_21823227.jpg logo_mu_cerne.gif Spread of ARG (antibiotic resistence genes) … also at waste water treatment plants 1212569_21823227.jpg logo_mu_cerne.gif 1212569_21823227.jpg logo_mu_cerne.gif WHO Report: The Review of Antimicrobial Resistance, Chaired by Jim O’Neil, UK, 2014 1212569_21823227.jpg logo_mu_cerne.gif Total 10 million deaths per year