Mechanisms of cell toxicity and in vitro toxicology. Nik Hodges School of Biosciences

Transcription

1 Mechanisms of cell toxicity and in vitro toxicology Nik Hodges School of Biosciences

2 Cell Toxicity

3 The dose makes the poison "dosis sola facit venenum" - dosage alone makes the poison.

4 Dose-response curves Unconscious Dead Depressed breathing R es nopse Giddy Happy No effect Deep Sleep Asleep Dose (at target tissue)

5 How do cells die? Necrosis Molecular pathways involved in apoptosis * Doesn t require energy * Unregulated * Damaging to surrounding cells e.g. osmotic Likely effect of detergents Adaptation Apoptosis Necrosis Concentration

6 Measuring cell death MTT assay Mitochondrial function Caspase 3/7 Cyt c N Adenylate kinase (AK) assay Membrane integrity

7 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*

8 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

9 Chemical Metabolism Detoxification Toxic Metabolite Cellular targets Cellular Damage Adaptive response Repair Toxicity

10 Reactive metabolites are often critical Formation of reactive intermediates from xenobiotics compound formula proposed RI type of toxicity bromobenzene Br Br O liver necrosis vinyl chloride CH 2 CHCl CH 2 CHCl O liver cancer aniline H 2 N HO NH methaemoglobinaemia dimethylnitrosamine (CH 3 ) 2 N N O H 3 C+ carcinogenesis carbon tetrachloride CCl 4 C*Cl 3 liver necrosis (free radical) chloroform CHCl 3 C*Cl 3 renal necrosis

11 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

12 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

13 Toxicity Protective factors Damaging species Homeostasis

14 Its all about balance..

15 Strategies for toxicity testing rodent in vivo Human in vivo rodent in vitro human in vitro

16 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

17 FRAME Fund for replacement of animals in medical experiments The 3 Rs Refinement Reduction Replacement

18 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

19 Hepatocyte culture models:

20 Couplets membrane asymmetry bile formation

21 Liver spheroids Lee et al, Small 5, , 2009 LDH MTT

22 Another example in vitro model 24 Hours After Seeding 72 Hours 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

23 Twitching rat muscle fibres in vitro

24 Testing Carcinogenicity of Tungsten Alloys Human HSkMC Rat L6 C11 Tungsten 97% Nickel 2% Cobalt 1% Tungsten 91% Nickel 6% Cobalt 3%

25 Transcriptomic approach WNiCo Control

26 lloys: Genes involved in apoptosis signalling pathways

27 Epiocular model: * In vitro model of human corneal epithelium using differentiated keratinocytes Can also model skin, gut, lung epithelia in similar ways

28 r more complex in vitro models: e.g. Skin co-culture models Epiderm TM 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

29 High throughput screening What do we want to be able to do? Detect all compounds that are toxic and understand mechanism Pie in the sky ight be able to identify chemicals common mechanisms of action e.g. genotoxins, enzyme inducers etc. pproaches - 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

30 Reporter assays

31 Can be highly discriminatory

32 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?

33 Modulation by genetic and epigenetic factors 1- Metabolism 2 - Depletion of cellular protective factors 3- Cellular/molecular targets UNDERSTAND MECHANISM DOSE RESPONSE MORE RATIONAL RISK ASSESSMENT

34 Some things good toxicologists think about What is the mechanism of toxicity? Toxicity reversible or irreversible? Relationship with exposure: - e.g. is there a threshold of effect? Are there susceptible sub-populations? Target organs? Effect species specific? Make chemicals as safe to use as possible Role of metabolism? Trans-generational effects?

35 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