Mechanisms of cell toxicity and in vitro toxicology

Nik Hodges

School of Biosciences

n.hodges@bham.ac.uk

Cell Toxicity

The dose “makes” the poison

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

Dose-response curves

Dose(at target tissue)

“Respon

se” No effect

Giddy

Asleep

Deep Sleep

Unconscious

Depressed breathing

Dead

Happy

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

Measuring cell death

N

MTT assayMitochondrial function

Adenylate kinase (AK) assayMembrane integrity

Cyt c

Caspase 3/7

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*

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

ToxicMetabolite

Cellular targets

Cellular Damage

Detoxification

RepairToxicity

Adaptive response

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

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

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

Protectivefactors

Damagingspecies

Homeostasis

Toxicity

Its all about balance…..

rodentin vivo

rodentin vitro

humanin vitro

Humanin vivo

Strategies for toxicity testing

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

The 3 RsRefinement

Reduction

Replacement

FRAME – Fund for replacement of animals in medical experiments

www.frame.org.uk

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

Hepatocyte culture models:

membrane asymmetry bile formation

Couplets

Liver spheroids

Lee et al, Small 5, 1213-21, 2009

LDH MTT

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

Twitching rat muscle fibres in vitro

Testing Carcinogenicity of Tungsten Alloys

Tungsten 97%Nickel 2%Cobalt 1%

Tungsten 91%Nickel 6%Cobalt 3%

Human HSkMC Rat L6 C11

Transcriptomic approach

Control

WNiCo

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

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

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

Reporter assays

Can be highly discriminatory

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

1- Metabolism

2 - Depletion of cellular protective factors

3- Cellular/molecular targets

UNDERSTANDMECHANISM

DOSE RESPONSE

MORE RATIONAL RISKASSESSMENT

Modulation by genetic and epigenetic factors

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

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