Mechanisms of cell toxicity and in vitro toxicology Nik Hodges School of Biosciences [email protected] Cell Toxicity The dose “makes” the poison "dosis sola facit venenum" - dosage alone makes the poison. Dose-response curves Dead Depressed breathing Unconscious Deep Sleep Asleep R “ e s Giddy p o n Happy s e ” No effect Dose (at target tissue) How do cells die ? Necrosis Molecular pathways involved in apoptosis e.g. osmotic * Doesn’t require energy * Unregulated * Damaging to surrounding cells Likely effect of detergents Adaptation Apoptosis Necrosis Concentration Measuring cell death MTT assay Mitochondrial function Cyt c Caspase 3/7 N Adenylate kinase (AK) assay Membrane integrity How do chemicals cause toxicity ? 1) 3D Shape: enzyme inhibition receptor mediated effects - activation of transcription factors resulting in inappropriate changes in gene expression other specific interactions 2) Reactivity: covalent binding DNA - mutations - cancer Protein - altered protein function – immune responses reaction with other biomolecules -depletion of protective factors glutathione depletion lipid peroxidation *Interfere with/compromise normal cellular functioning* Why is it important to understand the mechanism of toxicity ? Understanding of mechanism facilitates: • Extrapolation of animal data and in vitro data to the humans • Biological monitoring and screening • Understanding and predicting toxicity of new substances • Risk assessment • Make chemicals safer Chemical Metabolism Detoxification Toxic Metabolite Cellular targets Adaptive response Cellular Damage Repair Toxicity Reactive metabolites are often critical Formation of reactive intermediates from xenobiotics compound formula proposed RI type of toxicity O bromobenzene Br Br liver necrosis O vinyl chloride CH2 CHCl CH2 CHCl liver cancer aniline H2N HO NH methaemoglobinaemia dimethylnitrosamine (CH ) N N O 3 2 H3C+ carcinogenesis C*Cl liver necrosis carbon tetrachloride CCl4 3 (free radical) renal necrosis C*Cl3 chloroform CHCl3 Bromobenzene - Reactive metabolite and glutathione depletion • Solvent for heavy liquids • Intermediate for organic synthesis - agrochemicals and pharmaceuticals • Motor oil additive • Volatile ---- inhalation • Hepatotoxin Br Depletion of glutathione Relationship between cellular glutathione and bromobenzene liver toxicity Cellular glutathione Protein binding Liver damage % Threshold (~30%) Bromobenzene • Good correlation between protein binding and hepatotoxicity • Clear existence of a threshold of effect • Assessment of protein adducts potentially useful for biomonitoring of exposure Toxicity Protective Damaging factors species Homeostasis Its all about balance….. Strategies for toxicity testing rodent in vivo Human in vivo rodent in vitro human in vitro Can in vitro systems replace animals ? Non physiological loss of barriers such as blood/brain and placental loss of complex 3D organ and tissue structures loss of communication between cells/tissues/organs Lack of toxicokinetics and (often) metabolism FRAME – Fund for replacement of animals in medical experiments www.frame.org.uk The 3 Rs Refinement Reduction Replacement Liver a complex organ Aim – to maintain biochemical and structural features e.g. bile formation, albumin secretion P450 and phase II expression / induction Models ? • Hepatocyte monolayers • Couplets • Co-cultures • Sandwich cultures • Liver spheroids Hepatocyte culture models: Couplets membrane asymmetry bile formation Liver spheroids Lee et al, Small 5, 1213-21, 2009 LDH MTT Another example in vitro model 24 Hours 72 Hours After Seeding Differentiation • Cells can be differentiated in culture • Have “normal” muscle phenotype – both structurally and biochemically e.g. they form muscle fibres that twitch, they store glycogen Twitching rat muscle fibres in vitro Testing Carcinogenicity of Tungsten Alloys Human HSkMC Rat L6 C11 Tungsten 97% Nickel 2% Cobalt 1% Tungsten 91% Nickel 6% Cobalt 3% Transcriptomic approach WNiCo Control Toxic alloys: Genes involved in apoptosis signalling pathways Epiocular model: * In vitro model of human corneal epithelium using differentiated keratinocytes Can also model skin, gut, lung epithelia in similar ways Other more complex in vitro models: e.g. Skin co-culture models EpidermTM • In vivo like growth and morphological characeteristics • Highly reproducible • Replicates many of the structures found in vivo • Validated • Rapid easy, quick clear testing protocols From FRAME High throughput screening What do we want to be able to do ? Detect all compounds that are toxic and Pie in the sky understand mechanism Might be able to identify chemicals common mechanisms of action – e.g. genotoxins, enzyme inducers etc…. Approaches - fluorescent probes (GSH, Ca++) - nuclear translocation of tagged stress proteins e.g. nrf2 - reporter assays e.g. activation of p53, stress response genes - transcriptomics, proteomics, metabonomics Reporter assays Can be highly discriminatory Micro-array technologies Problems what cells / tissues ? what dose ? how long ? data analysis data interpretation validation Can we build profiles of changes in gene expression representative of exposure to classes of toxins ? Modulation by genetic and epigenetic factors 1- Metabolism 2 - Depletion of cellular protective factors UNDERSTAND MECHANISM 3- Cellular/molecular targets DOSE RESPONSE MORE RATIONAL RISK ASSESSMENT Some things good toxicologists think about Toxicity reversible or irreversible ? Relationship with exposure: - e.g. is there a threshold of effect ? Are there susceptible sub-populations ? Target organs ? Make chemicals as safe to use as possible Effect species specific ? Role of metabolism ? Trans-generational effects ? What is the mechanism of toxicity ? Examples of current toxicological challenges Extrapolating from in vitro to in vivo and animal to human.. Nanoparticles Mixtures Better in vitro and in silico models Low dose effects e.g. hormesis, practical thresholds of effect Endocrine disruptors.