Download Mechanisms of cell toxicity and in vitro toxicology

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
”es no ps e“R
Unconscious
Deep Sleep
Asleep
Giddy
Happy
No effect
Dose
(at target tissue)
How do cells die ?
Molecular pathways involved in apoptosis
Necrosis
* Doesn’t require energy
* Unregulated
* Damaging to surrounding
cells
Adaptation
Apoptosis
Concentration
Necrosis
e.g. osmotic
Likely effect
of detergents
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
bromobenzene
proposed RI
formula
Br
type of toxicity
O
Br
liver necrosis
O
CH2
vinyl chloride
aniline
CH2
CHCl
HO
H2N
dimethylnitrosamine
carbon tetrachloride
(CH3)2N
CCl4
N
O
CHCl
NH
liver cancer
methaemoglobinaemia
H3C+
carcinogenesis
C*Cl3
liver necrosis
(free radical)
chloroform
CHCl3
C*Cl3
renal necrosis
Bromobenzene - Reactive metabolite and glutathione depletion
• Solvent for heavy liquids
• Intermediate for organic synthesis - agrochemicals and pharmaceuticals
• Motor oil additive
• Volatile ---- inhalation
Br
• Hepatotoxin
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
factors
Damaging
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
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
Twitching rat muscle fibres in vitro
Testing Carcinogenicity of Tungsten Alloys
Human HSkMC
Tungsten 97%
Nickel 2%
Cobalt 1%
Tungsten 91%
Nickel 6%
Cobalt 3%
Rat L6 C11
Transcriptomic approach
WNiCo
Control
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
er 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
understand 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
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 ?
Effect species specific ?
Role of metabolism ?
Trans-generational effects ?
What is the mechanism of toxicity ?
Make chemicals as safe
to use as possible
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