C. Elegans is a worm that is genetically very similar to humans. The worm is often used as a model organism for research into disease mechanisms, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.

Exposure to a commercial formulation of glyphosate led to the degeneration of C. Elegans’ GABA neurons (Negga, 2012). GABA – an abbreviation for gamma-aminobutyric acid – is a so-called neurotransmitter. A neurotransmitter is a signalling substance that transmits nerve impulses between nerve cells. In addition, the researchers show that acute exposure to glyphosate does not lead to the death of dopamine neurons, but chronic exposure does (24 hours is chronic for worms, it appears : )). The researchers conclude that neurotoxicity studies should not only look at dopaminergic neurons but also at other types of neurons. Furthermore, the authors think that C. Elegans is a suitable worm to study the neurotoxicity of (future) pesticides.

The reason for the observed neurodegeneration in C. Elegans is attributed to a reduced activity of the mitochondria and increased oxidative stress (Bailey, 2018).

Not only C. Elegans, but also the zebrafish is widely used as a laboratory animal. The zebrafish is 70 percent genetically similar to humans and is easy to breed.

Glyphosate appears to influence the behaviour of zebrafish. It affects their memory and they become more passive. Zebrafish larvae reduce their ‘exploratory behaviour’ at concentrations of glyphosate currently permitted in surface water. The larvae do not go out as much, which may make them vulnerable as prey.

The researchers not only tested glyphosate as a single substance but also tested the herbicide ‘Roundup’. In addition to glyphosate, this herbicide contains a number of unknown excipients that can lead to a so-called compound toxicity. Both glyphosate and ‘Roundup’ had similar effects.

Oral exposure to glyphosate in rats leads to changes in their central nervous system (CNS) in a dose-dependent manner. A higher exposure leads to lower levels of the neurotransmitters dopamine, norepinephrine and serotonin. The authors argue that this proves that glyphosate can enter the brain through the blood-brain barrier, accumulate there and then exert its neurotoxic effect.

Exposure of female rats to glyphosate led to a toxic accumulation of calcium ions in the hippocampus of her offspring which led to depression and cell death (Cattani, 2017). 

According to the researchers, the toxicity in the offspring is caused by an overactivation of the glutamate metabolism. Glutamate is one of the neurotransmitters in our brain and has a stimulating effect on neurons. Exposure to glyphosate leads to an excess of glutamate between the glutamate neurons and – as a result – also to an excessive activation of the NMDA-glutamate receptor (N-methyl Dextro Aspartic Acid Receptor). Furthermore, the researchers show that glyphosate itself fits the NMDA receptor, which further reinforces the overactivation. When the NDMA receptor is overactivated, an excessive amount of Ca2+ flows into the cells. This in turn leads to oxidative stress. The researchers also showed that the amount of glutathione (GSH) – an antioxidant – in the brains of rats decreased. Increased oxidative stress and a reduced ability to do something about it eventually leads to cell death.

Research on glutamergic neurotransmission in our basal nuclei reveals a complex and interconnected network with back-and-forth interactions between glutamergic and dopaminergic systems. A shortage of dopaminergic neurons can lead to over-stimulation of the glutamergic system, which in turn can lead to all kinds of parkinsonian symptoms (Jenner, 2019).

One of the common prodromal symptoms of Parkinson’s disease are problems in the gastrointestinal tract. προδρομή (prodrome) means ‘running ahead’ in Greek. The prodromal symptoms run in front of the troops and show a possible future.

It is even suggested that a subtype of Parkinson’s disease begins in the intestines (Borghammer, 2019) and various studies show that people with Parkinson’s disease have a different composition of intestinal bacteria than their healthy counterparts (e.g. Boertien, 2019). This altered composition of the intestinal microbiome leads to a decrease in the biosynthesis of aromatic amino acids such as phenylalanine, tyrosine and tryptophan in people with Parkinson’s disease (Pietrucci, 2019). 

For this reason, I found it interesting to consider studies that link glyphosate to changes in the intestinal microbiome. Some hits:

• Glyphosate changes the intestinal flora of bees and increases the susceptibility to infections and bee mortality (Motta, 2018). The researchers showed that the gene encoding EPSPS, the enzyme that is the target of glyphosate, was present in two variants in most of the bees studied. Bees with ‘variant I’ appeared to be much more sensitive to pathogens after exposure.
• Exposure to glyphosate and ‘Roundup’ of female rats – in concentrations that are now considered ‘safe’ – leads to a changing intestinal microbiome in their offspring. (Mao, 2018). These effects were not found in the mother rat herself. The researchers point out that this shows that we have to be extra careful with exposure of pregnant women, children and adolescents.  The effects of glyphosate and ‘Roundup’ were different. Both led to an altered intestinal microbiome, but with a different composition. The authors conclude that it is not only important to understand the individual effects of glyphosate, but also to look at the impact of mixed formulations.
• A study on the effects of glyphosate on various potential pathogens and beneficial bacteria present in the poultry stomach shows that pathogenic bacteria such as Salmonella and Clostridium are resistant to glyphosate. (Shehata, 2012). Useful bacteria such as enterococci, lactobacillus and bifidobacteria appear to be sensitive to herbicide infestation. According to the researchers, a reduction of useful bacteria in the gastrointestinal tract by intake of glyphosate can make poultry vulnerable to infections.
• In a study on glyphosate exposure in mice, the researchers observed an increase in anxiety and depression. At the same time, they saw a change in the composition of intestinal bacteria occur and the authors describe how the observed behaviour may have been triggered by a decrease in the quantity of bacteroids, firmicutes and lactobacilli (Aitbali, 2018).

Ancestral exposures can promote the onset of disease in future generations. A study in rats has shown that glyphosate exhibits such a transgenerational effect. The grandchildren and great-grandchildren (who had never been exposed themselves) showed striking effects such as obesity, various types of cancer and premature births.

A number of case studies in which exposure to glyphosate is linked to parkinson’s disease (isme) are reported in the scientific literature:

• Parkinsonism
  Barbosa (2001) reports the case of a 54-year-old man who accidentally sprayed himself with glyphosate and who developed parkinsonism a month later. A year later, this patient showed a slowly resting tremor in his left hand and arm, accompanied by a limitation of his short term memory.
• Parkinsonism
  Wang (2011) reports a case of parkinsonism following chronic exposure to glyphosate in a previously healthy 44-year-old woman who worked for 3 years in a chemical plant in the department where glyphosate was produced. She showed rigidity, slowness and resting tremor in the limbs, without affecting short-term memory.
• Reversible parkinsonism
  Zheng (2018) describes a case study of reversible Parkinsonism after acute exposure to glyphosate. A previously healthy 58-year-old woman comes to the hospital with a headache and in the coming days develops inertia, tremors and difficulty walking. Fifteen days later she has a mask face, bradykinesia, the gear phenomenon, a clearly curved posture, reduced arm swing and a russtremor in all limbs. She had been spraying glyphosate for more than 3 hours a day for a week without using any protective equipment. After being treated with ATP, pralidoxime iodide and scopolamine hydrobromide, her headache and parkinsonism disappeared.
• Parkinsonisme
  A man who wanted to commit suicide by drinking 200 ml of glyphosate, at the age of 34, develops parkinsonism four years later (Eriguchi, 2019). He swallowed the glyphosate and after 10 minutes he vomited. His stomach was pumped at the hospital and he was given activated carbon. Four years later, he developed right-sided parkinsonian symptoms that responded well to the administration of levodopa.

C. Elegans is a worm that is genetically very similar to humans. The worm is often used as a model organism for research into disease mechanisms, including neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.

C.Elegans’ GABA neurons appear to be extremely sensitive to exposure to MZ. GABA – an abbreviation for gamma-aminobutyric acid – is a so-called neurotransmitter. A neurotransmitter is a signalling substance that transmits nerve impulses between nerve cells. The authors conclude that in toxicity studies not only dopaminergic neurons should be considered, but also other neurons.

A pre-treatment of C. Elegans with a dopamine transporter antagonist protects dopaminergic neurons against chronic MZ-treatment (24 hours). A dopamine transporter is a transmembrane protein, responsible for the reuptake of dopamine from the synaptic cleft tween two neurons. A DAT antagonist blocks the reuptake of dopamine and all other substances that may use this channel, such as presumably MZ. The authors conclude that DAT transporters may play a role in the neurotoxicity of MZ for dopamine neurons.

At higher levels of MZ, the behavior of C. Elegans changes. The worm stops laying eggs.

The concentration used by the researchers was within the current LC50 value for MZ (LC50 is the concentration in the air that causes death in inhalation in 50 % of the test animals. LC stands for Lethal Concentration).

The authors conclude that behavioural disorders can be a biomarker of the toxicity of MZ.

Researchers find increased levels of manganese in the brain of common carps exposed to MZ. At non-lethal concentrations, they see perceptible behavioural changes.

In addition, the researchers see a significant increase in ROS (Reactive Oxygen Species) levels in the brain of carp after exposure to MZ.

Futhermore, they see a significant increase in the amount of Nrf2 protein in the brain (Nuclear Factor Erythroid 2-Related Factor 2). Nrf2 is a so-called transcription factor (or sequence-specific DNA binder). A transcription factor is a protein that determines the rate at which genetic information from DNA is read and converted into messenger RNA. Nrf2 activates a large number of so-called cell-protective genes. For example, Nrf2 triggers the transcription of the body’s own antioxidant and detoxification enzymes. The increase in Nrf2 may expose a mechanism by which organisms try to limit damage from MZ.

In addition to neurons, other cells exist in the brain. These cells are called glia cells (glia is Greek for “glue”). One of the types of glia cells are astrocytes. Astrocytes are also part of the blood-brain barrier.

Mancozeb appears to be cytotoxic to astrocytes, isolated from the hippocampus of rats, in a dose-dependent manner. If cells were exposed to MZ for 1 hour, they would not recover. With atomic absorption, the researchers showed a significant accumulation of manganese in the astrocytes.

If the researchers administered chelators – compounds that form complexes with metal ions – and antioxidants together with MZ, then neurotoxicity did not occur. This seems to indicate that neurotoxicity is at least partly caused by an accumulation of manganese.

Male mice were exposed to low and high doses of MZ (30 and 90 mg/kg body weight respectively) from late pregnancy to adolescence. The hypothalamus of the mice was then examined for neurotoxicity. The researchers saw cell death, neuroinflammation and demyelination (loss of myelin, the substance that can be present as an insulating layer around nerves and that accelerates the conduction of impulses). In addition, they saw an imbalance in the proportions of the neurotransmitters glutamate and GABA (gamma-aminobutyric acid) (Moralles-Ovales, 2018).

A neurotransmitter is a signalling substance that transmits nerve impulses between neurons in the synapses. Some neurotransmitters stimulate signal transmission (such as glutamate) and others inhibit it (such as GABA). In this study, with an increasing dose of MZ, the ratio of stimulating to inhibitory neurotransmitters increased, leading, according to the authors, to increased toxicity and neuronal vulnerability.

Two additional articles with the same type of conclusions:

• On exposure to MZ, an increase in oxidative stress markers (ROS, reactive oxygen species) and mitochondrial inhibition has been observed in rats (Domico, 2012). 
• In cell cultures of the cerebral cortex of the mouse, mitochondria are damaged and there are signs of neuroinflammation. According to the authors, this can lead to neuronal damage (Pena-Contreras, 2016)

Maneb and Mancozeb are both equally toxic to cell cultures grown from human intestinal epithelium. Exposure leads to metal accumulation of manganese, copper and zinc in and between large intestinal cells called HT-29 and Caco2. The authors conclude that the rapid increase in metal concentrations affects the ability of individual cells to maintain a healthy level of metals in the cell. Metal overload occurs, which in turn leads to oxidative stress and cell death. According to the authors, the toxicity of MZ is caused both by the metal part and by the organic part of the polymer complex.

A lymphocyte is a type of white blood cell and lymphocytes play an important role in our immune system. In cell cultures of human lymphocytes, exposure to MZ leads to DNA damage and cell death. The lymphocytes tested were isolated from blood from healthy, non-smoking blood donors. The cytotoxic effects of MZ were studied at various concentrations (0.2-10μg/ml) at 6, 12, and 24-hour exposure.

As exposure increased, the researchers found that cytochrome c, ROS (Reactive Oxygen Species) and caspace-9 and caspace-3 increased, among other things. Cytochrome c and caspaces each play their own role in ‘programmed cell death’. Different signals can incite the cells to ‘suicide’ via a process called apoptosis. When mitochondria are damaged, cytochrome C can signal that it is time for ‘suicide’. Caspaces are proteins that help break down enzymes. In this case, the researchers saw an increase in the breakdown of the DNA repair enzyme PARP (poly (ADP-ribose) polymerase). The breakdown of PARP meant that DNA damage could not be repaired. The research also shows the involvement of oxidative stress in MZ-induced genotoxicity and cell death.

A small 2005 study with 13 winegrowers working with Mancozeb and 13 control subjects showed that exposure to a low concentration of MZ changed the functioning of the immune system. First, the researchers showed that the farmers were actually exposed by measuring the level of ethylenethiourea (ETU) in urine. ETU is a degradation product of MZ. In addition, they took blood samples and looked at the samples for factors that say something about the functioning of the immune system. For example, in exposed farmers, they saw an increase in the amount of white blood cells and also in CD19+ cells, a sign that the amount of B cells was increasing. A decrease in CD25+ cells indicated that the number of T-helper cells actually decreased. They also saw a decrease in TNF-alpha (Tumor Necrosis Factor), a substance that normally increases with an infection.

Researchers have used data from the cohort study AGRICAN to investigate relationships between the occurrence of Parkinson’s disease and exposure to certain pesticide/pesticide mixtures. Research among 1732 farmers with Parkinson’s disease showed, among other things, that the risk of Parkinson’s disease increases with exposure to MZ.

This study provides much more information. The researchers saw that Parkinson’s disease is more common among farmers who use pesticides anyway, regardless of the crop or cattle with which they farm. The study provides further support for already established relationships between certain pesticides and Parkinson’s disease, such as rotenone, diquat and paraquat, bipyridil herbicides and the dithiocarbamate fungicides maneb, mancozeb and ziram. The researchers also found – for the first time – positive associations between Parkinson’s disease and exposure to other dithiocarbamate fungicides such as cupreb, ferbam, mancopper, metiram, propineb, thiram and zineb.