Neurotoxins: how we test chemical hazards

Sep 21, 2017 | Trends

“I’m on a juice only diet to flush out the toxins in my body.”

Living in California has many perks, from the sun, to the food, to the diversity of people you meet. One phenomenon I could probably do without, however, are the fad diets! The term “toxin” is tossed about as often and as easily as a frisbee! Here we’ll talk about what toxins are, focusing on neurotoxins, and describe how we test for them. 

What is a “toxin”?

Let’s review the dictionary definition of a toxin:

“[A]n antigenic poison or venom of plant or animal origin, especially one produced by or derived from microorganisms and causing disease when present at low concentration in the body.”

From this, we can conclude that for something to be considered a toxin it must:

  1. Illicit an immune response;
  2. Be capable of entering the body by ingestion, injection, absorption or similar;
  3. Be of plant or animal origin and perhaps be made by or as a resulting from microbes;
  4. Causes disease in the body even at low concentrations.

Some examples are bee stings and botulinum toxin.

What is a neurotoxin?

The damaging effects of neurotoxin’s toxic effects are primarily seen in the nervous system. Examples of neurotoxins are lead, mercury, cocaine, hexane, ethanol, tetanus toxin and tetrodotoxin (from puffer fish). Neuron or axon injury, demyelination (loss of axon insulation) and/or interference with neurotransmission lead to the symptoms of disorders associated with neurotoxin exposure. These disorders include impaired intelligence and the regulation of emotional responses, behavioural problems including attention deficit and hyperactivity disorders, depression, anxiety, memory formation, impaired physical coordination and increased risk of neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease.

If you’re like me, at this point you’re feeling a little concerned about how likely you are to be exposed to a neurotoxin! Let’s go over how the US government and chemical manufacturers test for chemical hazards like neurotoxins and hopefully put your (and my!) mind at ease.

How do we test for chemical hazards?

“In the most effective approach to primary prevention of neurotoxic disease of environmental origin, a potential hazard is identified through premarket testing of new chemicals before they are released into commerce and the environment.”

“Disease is prevented by restricting or banning the use of chemicals found to be neurotoxic or by instituting engineering controls and imposing the use of protective devices at points of environmental release.”

(Environonmental Neurotoxicology, 1992)

The EPA has over 65,000 chemicals listed as toxic. Identifying and controlling neurotoxins and toxic substances generally is a two-part process. Firstly, existing substances that adversely affect the nervous system are identified and steps are taken to minimize human exposure to them. Secondly, new neurotoxic substances in use are identified and either their manufacture is prevented or human exposure and the release into the environment is restricted.

There are many methods for neurotoxic testing but they generally fall into the categories of human subjects, animal models and tissue and cell culture testing. Clearly testing on human subjects is the most representative for how a substance affects humans. However human testing is more expensive and is ethically questionable so the other testing methods are preferred. Mice and rats are the most commonly used animal models being biologically comparable to humans. Since human biologic responses are complex, a variety of testing methodologies is ideal and thus in vitro methods are used to complement animal testing. In vitro models also facilitate rapid testing far more cheaply, quickly and ethically than animal models.

Animal models

When using animal models, a variety of factors must be considered including:

  • the relationship between dose and response;
  • the effects at animal from the molecular level up to the whole organism;
  • Study result reproducibility;
  • The effects of structurally similar substances on humans or animals;
  • Any known metabolic differences between humans and the test species that could affect response;
  • Statistical uncertainties and difficulties in extrapolating to a low dose;
  • Sex of the animal;
  • Route of administration.

The goal is to identify whether or not the substance has primarily altered the nervous system and to identify the dose at which this effect is no longer observed. After an animal has been exposed to the potential toxin, behavioral, biochemical and histopathological assessments are used to determine the effects.

Animal testing is slow, costly and labor intensive and there are far too many chemicals to test in this manner alone. In addition, there are concerns about animal welfare in large-scale testing scenarios. Thus there has been an increasing push towards the utilization and improvement of in vitro models.

In vitro models

In vitro models include:

  • Primary cells such as neurons and glia (microglia, oligodendrocytes and astrocytes) from different brain regions.
  • Cell lines including neuroblastoma, astrocytoma, glioma and pheocromocytoma.
  • Brain slices like the hippocampus.
  • Reaggregating neuronal and glial cell cultures.
  • Organotypic (3D) cultures, usually co-cultures (meaning cultures of more than one type of cell).
  • Neural stem (progenitor) cells: primary cell cultures and cell lines.

Let’s look at cell lines as an example.

Quantitative high-throughput screening of cell culture lines

Here, large populations of genetically-defined cell lines are exposed to various concentrations of chemical hazards to evaluate cytotoxicity and apoptosis. This high-throughput method creates robust, reproducible results in a relatively short space of time. It is useful in determining which substances to prioritize for testing in animal models and in building predictive models for toxicity. The presence and concentration of apoptosis and cytotoxicity molecules, such as caspase 3/7 and intracellular ATP,  indicate toxicity. Intensiometric biosensors are used to measure such toxicity. These biosensors can be genetically encoded into the cell line you’re studying. Such biosensor assays are used to detect cell cytotoxicity through a variety of pathways, for example TempoMito™ measures calcium fluctuations in the mitochondria that indicate mitochondrial dysfunction and TempoATP™ shows cellular fluctuations in ATP both of which are indicative of cytotoxicity.

Further reading:

Rethink how chemical hazards are tested

Neurotoxicity


Article by Olwen Reina. Contact Olwen at olwen@tempobioscience.com.

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