How are pesticides affecting our immune system?

Methods

Summary

Remember that our hypothesis posits that the combined exposure to Glyphosate and Chlorpyrifos adversely affects immune cells, impairing their normal anti-tumor functions.

To contrast this hypothesis, we are planning both in vitro and in vivo approaches.

In vitro Methodology: For the in vitro component, we plan to isolate peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, and T cells from the blood of healthy donors, utilizing various isolation techniques. 

I will show you how we commonly isolate NK cellsn using the RossettSep technique. This technique employs antibodies specifically designed to target all "contaminant" cells, such as red blood cells, neutrophils, monocytes, and lymphocytes.

Glyphosate and Chlorpyrifos Exposure: Isolated NK cells will undergo exposure to glyphosate-based herbicides (e.g., RoundUP) and Chlorpyrifos-based insecticides. This exposure will occur individually or in combination overnight at 37°C in a CO2 incubator. Following pesticide exposure, cells will be subjected to centrifugation to remove residual pesticides. Subsequently, the NK cells will be co-cultured with human tumor cell lines. This step is crucial to assess whether NK cells retain their ability to effectively target and eliminate susceptible tumor cells even after exposure to the pesticides. The evaluation will extend to the functional capabilities of the exposed NK cells. Specifically, we will assess their capacity to produce essential substances such as interferons and other cytokines. This analysis aims to provide insights into the impact of pesticide exposure on the immune response against tumor cells.

These are the initial experiments we have in mind. Additionally, we will conduct experiments on T cells to assess whether their proliferation capacity and phenotype are affected by pesticide exposure. 

In vivo methodology: Please remind we are working following strict standards of care and animal welfare.

Since a) glyphosate is the most widely used pesticide, b) there is evidence on its carcinogenic potential  and c)  that we need to use the smallest number of animals to follow bioethical standards, and d) our hypothesis is that commercial formulations based on glyophosate (like RoundUP) negatively impact on immunesurveillance against cancer favoring its growth, we will start with this experimental design.

We expect that animals exposed to RoundUP (in any dose used) will show an increased tumor growth compared to control animals (which drink water), probably showing reduced numbers osf activated immune cells in the tumor (infiltrating immune cells).

Challenges

The experiments we are planning closely resemble those conducted in the lab for over two decades. However, working with pesticides poses unique challenges. Previous experiments have revealed that pure glyphosate is less toxic than commercial formulations containing glyphosate (such as RoundUP and others). Additionally, pesticide manufacturers are not obligated to disclose the excipients they use, leaving us unaware of our exposure. This lack of information raises concerns about the potential risks.

If we can demonstrate that a commercial formulation is harmful to the immune system, companies might alter the excipients and claim that the issue is resolved. This underscores the need for us to investigate various commercial formulations containing glyphosate and other pesticides. Simultaneously, we will assess the impact of pure compounds on human cells. This comprehensive approach allows us to explore potential risks associated with both commercial formulations and isolated compounds