mechanisms of cell toxicity and in vitro toxicology...detoxification repair toxicity adaptive...
TRANSCRIPT
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Mechanisms of cell toxicity and in vitro toxicology
Nik Hodges
School of Biosciences
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Cell Toxicity
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The dose “makes” the poison
"dosis sola facit venenum" - dosage alone makes the poison.
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Dose-response curves
Dose(at target tissue)
“Respon
se” No effect
Giddy
Asleep
Deep Sleep
Unconscious
Depressed breathing
Dead
Happy
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How do cells die ?
Concentration
Adaptation Apoptosis Necrosis
Molecular pathways involved in apoptosis
e.g. osmotic
Likely effect of detergents
Necrosis
* Doesn’t require energy* Unregulated* Damaging to surrounding cells
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Measuring cell death
N
MTT assayMitochondrial function
Adenylate kinase (AK) assayMembrane integrity
Cyt c
Caspase 3/7
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How do chemicals cause toxicity ?
1) 3D Shape:enzyme inhibitionreceptor mediated effects - activation of transcription factors resulting ininappropriate changes in gene expressionother specific interactions
2) Reactivity:covalent binding
DNA - mutations - cancerProtein - altered protein function – immune responses
reaction with other biomolecules -depletion of protective factorsglutathione depletionlipid peroxidation
*Interfere with/compromise normal cellular functioning*
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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
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Chemical
Metabolism
ToxicMetabolite
Cellular targets
Cellular Damage
Detoxification
RepairToxicity
Adaptive response
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Formation of reactive intermediates from xenobiotics
compound formula type of toxicity
bromobenzene Br BrO
proposed RI
liver necrosis
vinyl chloride CH2 CHCl CH2CHCl
O
liver cancer
aniline H2N HO NH methaemoglobinaemia
dimethylnitrosamine (CH3)2N N O H3C+ carcinogenesis
carbon tetrachloride CCl4 C*Cl3liver necrosis
(free radical)
chloroform CHCl3C*Cl3 renal necrosis
Reactive metabolites are often critical
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Bromobenzene - Reactive metabolite and glutathione depletion
Br
Depletion of glutathione
• Solvent for heavy liquids• Intermediate for organic synthesis - agrochemicals and pharmaceuticals• Motor oil additive• Volatile ---- inhalation• Hepatotoxin
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Relationship between cellular glutathione and bromobenzene liver toxicity
Bromobenzene
%
Cellular glutathione Protein binding
Liver damage
Threshold (~30%)
• Good correlation between protein binding and hepatotoxicity• Clear existence of a threshold of effect• Assessment of protein adducts potentially useful for biomonitoring of exposure
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Protectivefactors
Damagingspecies
Homeostasis
Toxicity
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Its all about balance…..
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rodentin vivo
rodentin vitro
humanin vitro
Humanin vivo
Strategies for toxicity testing
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Can in vitro systems replace animals ?
Non physiologicalloss of barriers such as blood/brain and placentalloss of complex 3D organ and tissue structuresloss of communication between cells/tissues/organs
Lack of toxicokinetics and (often) metabolism
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The 3 RsRefinement
Reduction
Replacement
FRAME – Fund for replacement of animals in medical experiments
www.frame.org.uk
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Models ?
• Hepatocyte monolayers• Couplets• Co-cultures • Sandwich cultures• Liver spheroids
Liver a complex organ
Aim – to maintain biochemical and structural features
e.g. bile formation, albumin secretion P450 and phase II expression / induction
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Hepatocyte culture models:
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membrane asymmetry bile formation
Couplets
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Liver spheroids
Lee et al, Small 5, 1213-21, 2009
LDH MTT
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Another example in vitro model
24 HoursAfter Seeding
72 HoursDifferentiation
• 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
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Twitching rat muscle fibres in vitro
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Testing Carcinogenicity of Tungsten Alloys
Tungsten 97%Nickel 2%Cobalt 1%
Tungsten 91%Nickel 6%Cobalt 3%
Human HSkMC Rat L6 C11
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Transcriptomic approach
Control
WNiCo
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Toxic alloys: Genes involved in apoptosis signalling pathways
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Epiocular model:* In vitro model of human corneal epithelium using differentiated keratinocytes
Can also model skin, gut, lung epithelia in similar ways
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From FRAME
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
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High throughput screening
What do we want to be able to do ?
Detect all compounds that are toxic andunderstand mechanism
Pie in the sky
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
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Reporter assays
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Can be highly discriminatory
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Micro-array technologies
Can we build profiles of changes in gene expression representativeof exposure to classes of toxins ?
Problems
what cells / tissues ?what dose ? how long ?data analysisdata interpretationvalidation
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1- Metabolism
2 - Depletion of cellular protective factors
3- Cellular/molecular targets
UNDERSTANDMECHANISM
DOSE RESPONSE
MORE RATIONAL RISKASSESSMENT
Modulation by genetic and epigenetic factors
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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 ?
Effect species specific ?
Role of metabolism ?
Trans-generational effects ?
What is the mechanism of toxicity ?
Make chemicals as safeto use as possible
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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