In this blogpost, Fola Ogungbemi talks about the effects of mixtures of neuroactive substances. Risk assessment of chemicals is usually conducted for individual chemicals whereas mixtures of chemicals occur in the environment. Considering that neuroactive chemicals are a group of contaminants that dominate the environment, it is then imperative to understand the effects of mixtures of neuroactive substances.

The dose makes the poison

Have you ever imagined why you feel sleepy after having a heavy lunch?

Do you want to know why taking coffee during or after lunch helps to keep you alert after lunch?

Do you want to know why drinking 10 bottles of beer knocks you out quickly?

Whether it is beer, coffee or potatoes, these food substances can affect your nervous system directly or indirectly and the overall outcome of the way you feel is mostly determined by the mixture consumed (I.e., beer and coffee mixture), amount consumed, and duration of consumption.

You might ask how coffee and beer relates to mixture toxicity in the environment. Oh well! Did you know that neuroactive chemicals (such as insecticides and pharmaceuticals) are one of the largest groups of chemicals occurring in European surface? Just like a person’s alertness after having lunch and a cup of coffee is determined by the combination of the 2 food substances, the neurotoxic effect of these largely occurring neuroactive substances is driven by all the substances occurring simultaneously – the so-called something from nothing principle (Figure 1). However, risk assessment of chemicals is usually conducted for a single chemical. This could lead to a large discrepancy between the observed effect in the environment and the expected effect based on single chemical risk assessment.

One might also ask why neurotoxicity is an important problem. In recent times, there has been an increase in the number of people suffering from neurological diseases (i.e. Parkinson and autism) and incidents of nervous system-related diseases are increasingly associated with exposure to pesticides and pharmaceuticals. Neuroactive chemicals have also been identified as the main driver of biodiversity loss in aquatic invertebrate communities. This indicates the need to emphasize on screening neuroactive chemicals from environmental samples as well as the assessment of neurotoxic risk of new chemicals to protect human and environmental health.

Tackling this neurotoxicity problem

The goal of this research was to investigate the predictability of neuroactive mixture effects based on similarity/dissimilarity of mechanisms of action. For example, if one eats a big lunch, one might experience the so called post-lunch slump (let’s call it hypoactivity). In order to avoid this slump, one might take one or two cups of coffee to stay alert after lunch (let’s call it hyperactivity). In reality, this is a mixture of the food and coffee with opposing effects (both hyper and hypoactivity) in the body. Another scenario is when one goes for a 3-course dinner at a restaurant and drink several bottles of beer at the same time. The food and the beer will act in a similar way to make the person feel tired (hypoactivity) but these actions will be via different mechanisms. The main question of this research (assuming the food and drinks above were neuroactive chemicals acting similarly or dissimilarly) was how different neuroactive substances will interact to induce a combined effect irrespective of their mechanism of action and whether such effects can be predicted  based on the knowledge of the mixture contents (Figure 2).

How we did it?

Animal model species, such as rodents and fishes, are usually used for neurotoxicity testing. However, exposure of animals to chemicals may inflict pain and distress. Hence, there is an increased pressure to develop alternatives to animal tests because of ethical reasons, as well as to reduce time and cost of these tests. Consequently, the use of animals in toxicity testing is gradually being discouraged in favor of promoting the 3R principle: reduction, refinement and replacement (Russell and Burch (1959)). Zebrafish embryos prove to be a valid alternative due to their small size, transparency and fast development.

The spontaneous tail coiling (STC) of the zebrafish embryo is the earliest observable motor activity generated by the developing neural network. It consists of single or multiple rhythmic contractions and can be observed as early as 17-19 hours post fertilization (Video 1). Therefore, spontaneous coiling of the embryo was measured as a useful endpoint to detect effects on the muscle innervation and generally, the nervous system. In addition to measuring the effect induced by exposing binary and ternary mixtures of neuroactive chemicals to zebrafish embryo, the effects were also modelled using classical mixture models such as concentration addition and independent action models.

Video 1 – Attached is a 60 seconds video of the spontaneous coiling taken from Ogungbemi et al. (2021)

The results

Results showed that mixture toxicity of neurotoxic chemicals such as propafenone and abamectin, as well as chlorpyrifos and hexaconazole were predictable using concentration addition and independent action models. These chemicals are known to show different mechanisms of action but similar effects (i.e., beer and big lunch). Antagonistic effects for mixtures containing substances with opposing effects were also found (i.e., coffee and big lunch).

What the results mean?

The results show that chemicals which have different mechanism of action will still combine to induce a mixture effect. Most important, this result gives information on how such mixtures can be predicted without the need to expose animals. Finally, it is discussed in our paper how the spontaneous coiling of zebrafish embryo may be used to amend neurotoxic risk assessment.

The paper was authored by Afolarin Ogungbemi, Riccardo Massei, Rolf Altenburger, Stefan Scholz and Eberhard Küster, and published open access in Toxics journal.