Lecture 5: How pollutants exert their toxicity

Toxicology (background)

• Paracelsus established the role of chemistry in medicine and is responsible for two founding concepts:

  • A toxic agent is a chemical entity

  • The distinction between beneficial and harmful properties of chemical is to the magnitude of exposure/dose

Toxicology

Toxicology is the study of adverse effects of chemicals on biological systems

*biological systems span from individual cells to whole ecosystem

Environmental toxicology

Environmental toxicology is the multidisciplinary study of the impacts of chemicals on human health and the environment.

Important considerations of environmental toxicology

• What is the source of the chemical to that environment?

• How are organisms exposed?

• How can the chemical effect cells – organisms – populations – ecosystems?

• What are the risks, and how can they be mitigated?

One Health

An emerging concept which encompasses Environmental Toxicology – considers human and environmental health together for good management practice (everything is interconnected, so it should be treated as such)

Types of toxic effects

Some effects are harmful, whereas others are not (e.g. medicine delivered at a therapeutic dose produces a desired effect). Types of toxic effects:

• Allergic reactions

• Idiosyncratic reactions

• Immediate v. delayed toxicity

• Reversible v. irreversible toxic effects

• Local v. systemic toxicity

Allergic reactions

An adverse reaction of the immune system to a chemical in response to a previous exposure to that chemical or to a structurally similar one

Idiosyncratic reactions

Abnormal reactivity of an individual to a chemical based on its genetics or other individual sensitivity factors (e.g. lactose intolerance)

Immediate v. delayed toxicity

Toxic effects can develop rapidly after a single exposure (e.g., pesticides cause immediate death of insects) or may be delayed (e.g., carcinogens can cause tumours decades after exposure)

Reversible v. irreversible toxic effects

Some tissues are able to repair themselves (e.g., liver and gastrointestinal injuries have high ability to regenerate). Other tissues such as CNS are unable to regenerate after damage. Cancers and birth defects cause by chemical exposure are considered irreversible toxic effects

Local v. systemic toxicity

Local effects occur where contact is first made by the chemical and biological system (e.g., a rash occurring on dermal contact). Systemic effects require the absorption and distribution of a chemical from its entry point to a distant site where the toxic effect takes place

Guidelines for toxicity testing

Toxicity testing involves exposing an organism or groups of organisms, to varying doses of chemicals to see what happens

How is toxicity measured?

• Dose–response relationships are the association between the amount of a chemical a biological system is exposed to, and the extent to which an effect is observed

• Dose is expressed as mass or concentration (unit of measurement relative to exposure route)

  • dermal = per surface area of skin expose (e.g. mg/cm²)

  • inhaled = per volume air breathed (e.g. ng/L, mg/L)

  • ingestion = per L water (e.g. ng/L, mg/L) per mass food (g.g. mg/kg, μg/g) per kg of body weight (e.g. mg/kg, μg/g)

Dose-response curve

Characterised by a continuous scale of doses that lead to an increase in the magnitude of a specific response. Common effect includes enzyme inhibition, change in behaviour, feeding inhibition, growth inhibition, death and reproductive inhibition. There are two types of dose-response relationships:

• Graded response (for individuals)

• Quantal response (for populations)

Example of a graded dose-response relationship

The dose-response relationship between different doses of the organophosphate insecticide chlorpyrifos and esterase enzyme inhibition. Testing the effect on chlorpyrifos on the inhibition of:

• Cholinesterase in the brain (blue)

• Carboxylesterase in the liver (red)

Degree of inhibition is dose-related, but the degree of inhibition per unit of concentration is different between enzymes.

At a dose of 3 mg/kg chlorpyrifos:

• brain cholinesterase will be inhibited by ~50%

• there is no effect on liver carboxylesterase

At a dose of 5 mg/kg chlorpyrifos:

• both enzymes inhibited, but to different extents

• primary toxicological response related to cholinesterase inhibition in brain

Example of a quantal dose-response relationship

The distribution of the observed responses of a group or population of
varying doses of a chemical. Identifies if the effect was observed in an individual, within the population:

• shows biological variation within a species/population

• typically exhibits a Normal or Gaussian distribution (e.g. a Bell curve)

• the bars represent the percentage of organisms that responded at each dose

When looking at a population level, we typically want to know the primary toxicological response

Why do species, and individual species within a population have different responses to dose?

• Individual differences

  • Chemical more toxic one individual than another.

• Species differences

  • Chemical more toxic to one species than another.

• Target organisms

  • Some substances are very toxic to one organism or group of organisms while being relatively harmless to others

Threshold approach to evaluating dose–response curves

• No Observed Adverse Effect Level (NOAEL)

  • the highest dose an organism was exposed to, but no significant adverse effect was observed

• Lowest Observed Adverse Effect Level (LOAEL)

  • the lowest dose an organism was exposed to that a significant adverse effect was observed

  • this is for the primary toxicological effect

Statistical/probability distribution models approach to evaluating dose–response curves

• Benchmark Dose (BMD)

  • the dose that produces an effect at a specified
    response level

  • typically, up to 10% (e.g. BMD5 is the dose that produces an effect in 5% of the population)

  • specific to the primary toxicological effect

• Effective dose (ED)

  • the dose that produces an effect at a specified response level (e.g. ED20 is the dose that produces an effect in 20% of the population)

  • not specific to the primary toxicological effect

  • can be for therapeutic or toxic effects

• Lethal Dose (LD)

  • the dose that causes death in a specified percentage
    of the population (e.g. the effect is death)

Advantages of statistical models over threshold
approaches

• Advantages

  • they consider information from the entire dose-response curve

    • rather than focusing on a single test dose such as is done with the NOAEL/LOAEL approach (e.g. uses all available relevant information)

  • uses a consistent benchmark-response level that crosses a range of studies and endpoint

  • is less influenced by the arbitrary selection of doses

    • allows potency comparison between endpoints

• Disadvantages

  • it may not be possible to define the shape of the dose-response curve

    • because of limited dose groups or the number of animals per group

  • requires greater statistical expertise than threshold approach

Therapeutic Index (TI)

The ratio of the dose required to produce a toxic effect (TD) and the dose required to produce a therapeutic effect (ED)

𝑻𝒉𝒆𝒓𝒂𝒑𝒆𝒖𝒕𝒊𝒄 𝒊𝒏𝒅𝒆𝒙 = 𝑻𝑫_𝟓𝟎/𝑬𝑫_𝟓𝟎

Used in clinical toxicology, TI is an index of comparative toxicity of two
different chemicals

• It’s an estimation of the relative safety of a drug

• The larger the ratio, the greater the relative safety

Disadvantage of Therapeutic Index

Therapeutic Index doesn’t consider the slope of the dose–response curves

Margin of safety

Margin of safety can overcome the pitfalls of TI by using:

• ED99 for the desired effect

• LD1 for the undesired effect

𝑻𝒉𝒆𝒓𝒂𝒑𝒆𝒖𝒕𝒊𝒄 𝒊𝒏𝒅𝒆𝒙 = L𝑫_𝟓𝟎/𝑬𝑫₉₉

Shapes of dose-response curves

• It is often assumed that chemicals will elicit a monotonic response

  • where increased dose causes a steady increasing or decreasing response

• U-shape or J-shape can be observed when some chemicals are required for normal physiological function and/or survival

• inverted U-shape or multiple inflection points with variable slope indicate detoxification mechanism or fast rate of repair in species

Dose-response curve for homeostasis

Nutrient and vitamin concentrations (Cu, Mg, Zn) need to be maintained within a specific range that allows for homeostasis. At very low doses there is an adverse effect (deficiency) and at very high doses responses appear from toxicity e.g. vitamin A can cause liver toxicity and birth defects at high doses, and immune and skin issues at deficiency

Hormesis dose-response

Hormesis is when a toxicant imparts a beneficial or stimulatory effect at low doses, but produces adverse effects at higher doses e.g. induction of enzymatic pathways that protect against oxidative stress

When do toxic effects occur?

A toxic effect can only occur when a chemical:

• Reaches an appropriate site for a reaction/interaction to occur

• The chemical is present at that site in a high enough dose

• The reaction occurs for a long enough time

Factors contributing to a chemical’s toxicity

• Physico-chemical properties

  • chemical structure, molecular weight, water/fat solubility (log P), viscosity, pH, ionisation potential (pKa), environmental persistence/degradation

• Exposure

  • effect do not necessarily occur immediately

  • it may take time for sufficient effect to build up

• Metabolism

  • the organism may metabolise or detoxify before effect takes place

• Overall suceptibility of the individual/population

Mechanism of toxicity

After the chemical is exposed to an organism:

• Delivery of the chemical to its target site

  • e.g. chemical is ingested and absorbed into the
    blood stream where it is delivered to its target site

• The chemical-biological interaction takes
  place

  • e.g. the chemical binds to its target receptor

• Toxic effects take place

  • e.g. enzyme inhibition, impaired growth, impaired
    reproduction

• Inability to repair, or the organism adapts

  • e.g. irreparable organ damage and cellular death
    or detoxification/sequestration mechanisms occur and damage is repaired

Two forms of cell death

• Necrosis: unprogrammed cell death (dangerous)

  • Passive cell death, induced by accidental damage
    of tissue (and baseline toxicity)

  • Does not involve activation of any specific cellular pathway

    • early loss of plasma membrane integrity and
      swelling of the cell body followed by bursting of
      cell

    • burst cell expels substances that can be
      damaging to surrounding cells

• Apoptosis: programmed cell death (not as
  dangerous)

  • Active form of cell death enabling individual cells to
    shut down.

    • dying cells shrink

    • small membrane-bound apoptotic bodies are
      released, which are phagocytosed by immune
      cells (e.g. macrophages)

• Intracellular constituents are not released where they might have deleterious effects on neighboring cells

Three modes of toxic action

The modes of toxic action are based on the 3 types of primary interactions between environmental pollutants and biomolecules:

• Non-specific

  • (sometimes called baseline toxicity or narcosis)

  • chemicals partition through cell membranes

  • is there is a high conc on the outside v low conc on the inside, diffusion is rapid

• Specific

  • receptors looking for a particular shape to trigger a response

• Chemical reactions

  • forming covalent bonds

Non-specific (baseline toxicity or narcosis)

Hydrophobic pollutants accumulate in biological membranes where they disturb membrane integrity:

• Membrane function acts as a barrier and governs energy transduction (ion gradients that drive endergonic processes) and the formation of matrix for proteins

• When impaired, membrane function decreases, potentials decay (things can cross into membrane too easily) and enzyme activity decreases

Specific toxicity

Compounds that generally interfere (complex, react etc.) with biological ligand (receptors for signalling). Chemical structure is really important:

• Structural similarities to endogenous chemicals (e.g.,
  proteins, hormones, enzymes)

  • Endocrine disruptors

  • Enzyme inhibitors

• Irreversible binding to receptors

• No longer produce signals

• Leave signalling pathways open

• Block signals all together

Endocrine disruption

• Normal function

• Hormones bind to hormone receptors

• Endocrine disruption

• Micropollutants enter as an agonist (binds to receptors and mimics hormones)

  • trigger hormone response

• Micropollutants may also enter as antagonists (block hormones receptors

  • inhibit hormone response

Imidacloprid (example of specific toxicity)

Part of a group of insecticides called Neonicotinoids.

Imidacloprid mode of action in the body is:

• An agonist of the nicotine acetylcholine receptor (nAChR)

  • this receptor is located within post-synaptic membrane of neurons, within the CNS

• Agonist binds irreversibly to the nAChR

• Leaves the Na+ (sodium ion) channel open

  • causes over-excitation of the central nervous system causing the organism to die

Mechanism of toxicity (exposure through dermal contact):

• Delivery

  • transfuses through membranes into CNS due to its water solubility

• Interaction with target

  • imidacloprid binds to nAChR, opening Na+ channel

• Cellular disfunction or injury

  • neuron dies

• Repair or permanent damage

  • damage is irreparable (once damage accumulates to a point, the insect dies)

Chemical reactions (reactive toxicity)

When covalent bonds are made between the chemical pollutant and natural biochemicals in the body:

• Genotoxicity (DNA damage)

• Mutagenicity (permanent genetic change)

• Carcinogenicity (cancer causing)

Binding to biomolecules can cause altered protein functions and trigger altered immune responses (cells have range of defense mechanisms /adaptive stress response pathways):

• Heat shock response

  • produces heat shock proteins to chaperone damaged proteins back to homeostasis

• Hypoxia

  • conserved oxygen by cutting it off elsewhere

• Metal stress

  • induces chelation and sequestration by metallothioneins

• Osmotic stress

  • triggers rapid water efflux and cell shrinkage

• Oxidative stress

  • production of antioxidants, often triggers hormesis effect

DNA damage (example of reactive toxicity)

Associated with:

• Loss of stability

  • bulky adducts with guanine

  • strand breaks due to phosphodiester formation with bulky adducts

• Lethal lesions

  • alkylation of adenenine stops DNA polymerase

• Mutations (most common)

  • alkylation of bases changes hydrogen bond formation and causes base change

DNA repair mechanism include:

• Adaptive response (reverse transfer of adducts)

• Base excision by adduct specific glycosylases

• SOS response (nucleotide excision)

How to use toxicity data?

• Analyse chemical in drinking water

• Review Human Health and Ecological Risk Assessment

• Assess risk of PFAS and other chemicals in food

Australian Drinking Water Guidelines (ADWG)

Drinking water in Queensland is not allowed to exceed the ADWG health-based guidelines. Health-based guidelines are protective of human health, for a lifetime of exposure (chronic):

• Toxic dose from toxicity studies, can be from animal studies

  • very strong preference for this to be a NOAEL

• Body weight = assumed to be 70 kg for adults

• Proportionality factor = proportion of that chemical likely to be consumed

• from drinking water (more often than not is 10%)

• Volume of water consumed = 2 L for an adult

• Safety factor is an adjustment applied to account for things like:

  • variation between animals of the same species (including humans)

  • variation between species

  • use of a LOAEL instead of a NOAEL