New study challenges regulatory assumptions and practices. Report by Claire Robinson
Mixtures of pesticide residues commonly found in foods in the EU can have adverse effects on health even when each individual pesticide is present at a level considered safe by regulators, a new study shows.
The study also found that the use of molecular analytical techniques known as “omics” can reveal adverse effects on health that are missed by the standard toxicological measures used to support regulatory authorisations of pesticides.
The study is the first to directly compare in-depth profiling of an organism’s molecular components using “omics” analytical techniques with the standard toxicological measures that regulators rely upon to assess the health risks of pesticides.
The researchers set out to see if omics would reveal signs of ill health in rats fed a mixture of pesticides over the relatively short-term period of 90 days. The mix was made up of six pesticide active ingredients, which are among the most frequently detected at the highest levels in foods in the EU – azoxystrobin, boscalid, chlorpyrifos, glyphosate, imidacloprid and thiabendazole.
A mixture was tested because this reflects real-world conditions, where we are exposed to cocktails of pesticides. Each individual pesticide was present at the EU acceptable daily intake (ADI) level, meaning the level that is deemed by regulators to be safe to consume on a daily basis.
The study found that the standard toxicological measures – analysis of water and feed consumption, body weight, histology (microscopic examination of tissues), and blood biochemistry – showed little or no evidence of harm. But in contrast, the omics analyses showed biochemical changes in the gut and blood and gene function changes in the liver that indicated the possible onset of harm.
For a detailed technical account of the omics findings, see the section at the foot of this article, “Omics findings in detail”.
Metabolomics analysis of the blood of the pesticide-exposed rats showed that many metabolites had their levels altered by exposure to the pesticide mixture. In particular, a decrease in blood pyridoxal (a form of vitamin B6) was seen. This may indicate that exposure to the pesticide mixture could in the long term result in vitamin B6 deficiency, which has been linked with autism spectrum disorder.
Omics analysis of the biochemical signalling between the gastrointestinal tract and the animal’s body as a whole suggested a cell danger response. This response included adaptation to oxidative stress, which manifested as alterations in tryptophan-nicotinamide biochemical pathways. Oxidative stress results from excessive production of reactive oxygen, an imbalance in the body that can damage vital molecules and cell structures, which in turn can lead to serious disease such as cancer.
The results showed a link between the gut biochemical disturbance and overall health status – what was seen in the gut correlated with the blood biochemistry and liver profiling.
Transcriptomics analysis, which investigates total patterns of gene function, of the liver showed that 257 genes had their expression changed. Gene functions affected included those involved in the regulation of response to hormones.
Analysis of the DNA methylation (a process that can change the activity of DNA without changing the gene sequence) of the liver showed a metabolic adaptation that could exceed the cells’ capacity for restoring balance, ultimately leading to disease such as liver damage or cancer.
Commenting on the findings, lead researcher Dr Michael Antoniou of King’s College London said, “Our starting hypothesis, that omics analysis can be used to identify predictors of ill health following a relatively short period of exposure to pesticides, was confirmed. This suggests that it is in the public interest that regulators adopt in-depth omics profiling as part of pesticides risk assessment policy, since the measures they currently rely upon are evidently lacking in sensitivity.”
The new study has not yet been peer-reviewed but is published on the pre-print site bioRxiv. It was conducted by an international team of scientists based in the UK, Italy, France, and the Netherlands.
Regulatory “safe” levels challenged
The new research challenges current regulatory “safe” limits set for pesticides. Dr Antoniou explained, “In our pesticide mixture, each pesticide was present at the ADI, which is set at least 100 times below the level at which the standard industry tests found no effect. So according to regulators we should have seen nothing. But the mixture did cause changes in blood and gut biochemistry that indicate the potential onset of health problems. These changes were missed by the standard measurements performed on pesticides to justify regulatory authorisation.”
The effects found in this study were seen over a period of 90 days, compared with the two years or more taken by standard industry long-term safety tests on pesticides. Dr Antoniou said, “By using omics techniques one can do relatively short-term studies with fewer animals, which saves money and has animal welfare benefits. That should please both industry and regulators.”
EFSA drags its feet
In the EU, the risks posed by pesticides to health are assessed by the European Food Safety Authority (EFSA). The authority has given a cautious welcome to omics techniques, calling them “a valuable addition in some aspects of risk assessment of food and feed products and the environment”.
However, EFSA is dragging its feet on incorporating omics into risk assessments for pesticides and genetically modified (GM) foods. It held a scientific colloquium on the topic in 2018, which concluded that interpretation of omics data was “challenging”, due to a lack of standardised protocols of collection, analysis, and interpretation, though it points out that several international groups are working on such standardization, including the OECD, which is responsible for issuing global Guidelines for the Testing of Chemicals. EFSA added, “The barriers reside in the complexity of the data and its analysis combined with the fact that omics focus on sublethal endpoints rather than lethality.”
Sublethal effects are effects that don’t kill but can make you very sick. Current pesticide risk assessment focuses on gross harms such as deaths or obvious tumours. It can miss less obvious harms, such as the liver dysfunction highlighted by omics in the new study, even though those harms can cause serious health impacts. EFSA’s concern that sublethal effects may be identified via omics hinges on the fact that “this information has the potential to identify associations that may not be causative, may reflect adaptive mechanisms and/or may diverge under certain conditions”.
In other words, EFSA is worried that the pesticide under test may be wrongly accused by omics data, in that it may not be causing the effect seen; or the effect seen may be an adaptive response to a stress, rather than outright disease; or the omics results may indicate the potential onset of a disease but not actually result in the development of the disease.
Adaptive responses are danger signal
Dr Antoniou thinks EFSA’s concerns are misplaced. He said, “An adaptive response is a sign that the body is under toxicological stress – a danger signal that the chemical is causing harm, especially in more vulnerable individuals – for example, the very young and old or those with other chronic ailments. It’s something we want to avoid. As we say in our paper, over time such an ‘adaptive response’ could lead to cumulative cellular or organ damage and serious disease. Such responses need to be taken seriously as a warning sign that all is not well and that the chemical or mixture under test is not safe, at least at the dose administered.”
It is dispiriting that EFSA appears to see any increased emphasis on sublethal effects as a problem rather than a benefit. In reality, such an emphasis would only be a problem for industry, which would rightly bear the burden of proving that any given effect seen by omics analysis is not in fact real. For the public, such an early warning system in pesticides testing would be a benefit and would help fulfil the aim of the precautionary principle, which is enshrined in the EU pesticides regulation.
Omics a “threat” to pesticide industry
In this light, it is significant that EFSA also said that use of omics in risk assessment may be perceived as “a threat”, given that the “lowest observed effect” and “no observed effect” concentrations derived from omics analyses may well be “substantially lower” than those obtained from the standard tests. This would in turn mean that the ADI for any pesticide would need to be far lower than is currently the case, leading to possible restrictions on how much pesticide farmers could spray and when.
But as with the sublethal effects issue, any “threat” in this case would be to industry. Public health could only benefit from the strengthened precautionary approach enabled by the more sensitive omics analysis.
EFSA does not make clear the potential benefits to public health protection that incorporation of omics into risk assessment could offer but instead appears to be primarily concerned about negative impacts on the pesticide industry.
EFSA concluded its analysis by stating that the use of omics technologies “alongside current toxicological or environmental risk assessment approaches is needed to reinforce confidence and expertise before implementation of these datasets as a standalone tool in risk assessment”.
Standard toxicological measurements inadequate
The new study contributes to filling this knowledge gap, as it is the first to directly compare omics analyses with the standard toxicological measurements performed for regulatory purposes. Unfortunately for the existing pesticide risk assessment model, the standard measurements were found to be inadequate, as the omics analyses were able to detect health problems that were not revealed by the standard measurements.
The only way to enable omics outcomes to be confirmed by standard measurements is to extend omics studies to long-term experimental periods, as the authors of the new paper suggest. This would give time for the early warning signs seen in the omics data to potentially develop into gross disease that can be measured by the standard tests. Then the results of the omics and the standard measurements could be compared to see where they agree or diverge. If they agree, as is expected, the predictive value of omics in risk assessment would be confirmed.
Confidence in omics’ predictive ability
Dr Antoniou says that there is already enough evidence for a high level of confidence in omics’ predictive ability: “Omics researchers do not interpret their results blindly in an ad hoc manner. They scrutinize them against a huge and ever-increasing database stemming from thousands of studies using this technology, which provide insights into omics profiles representative of health and different disease states. This allows accurate correlations between the results obtained and the health or disease status of the organism under study.”
In this light, Dr Antoniou says the international bodies that are working on standardization of omics should set a short timeframe (less than 5 years) to perform whatever validation studies they believe are needed: “Regulatory agencies need to come into the 21st century and embrace omics as part of chemical risk assessment without further delay.”
It appears that anything less risks their being guilty of dereliction of their duty to protect public health.
While we wait for pesticide risk assessment to catch up with scientific developments, the public is well advised to minimize exposure to pesticides by eating organic food and avoiding the use of these chemicals in their homes and gardens.
Omics findings in detail
The metabolomics analysis of the blood of the pesticide-exposed rats showed that many metabolites had their levels altered by exposure to the pesticide mixture. The most striking changes corresponded to alterations in tryptophan-nicotinamide biochemical pathways, which are important in maintaining vitamin B3 nutrition, among other functions. In addition, a decrease in blood pyridoxal (a form of vitamin B6) was seen.
Metabolomics analysis of the gut microbiome also showed an alteration in tryptophan-nicotinamide biochemistry, as highlighted by increased levels of serotonin, which has both neurotransmitter and hormone functions and is synthesized from tryptophan, as well as increased levels of pyridoxal (note this increased in the gut parallel to decreasing in the blood) and nicotinamide riboside. These observations suggest that the effects of the pesticide mixture in the gut microbiome can be linked to the observed disruptions in serum metabolite levels.
Overall, metabolomics analysis of the gut and blood showed that the biochemical signaling between the gastrointestinal tract and the animal’s body as a whole suggested a cell danger response. This response included adaptation to oxidative stress (highlighted by alterations in nicotinamide and tryptophan metabolism), an imbalance in the body that over time can damage vital cell molecules including DNA. This, in turn, can lead to serious diseases such as cancer. In addition, the decrease in blood pyridoxal is also noteworthy as it may indicate that exposure to the pesticide mixture could in the long term result in vitamin B6 deficiency, which has been linked with autism spectrum disorder.
Transcriptomics analysis, which investigates total patterns of gene function, revealed that in the liver 257 genes had their expression changed. Gene functions affected included those involved in the regulation of response to hormones and alterations in nicotinamide biochemistry.
Analysis of DNA methylation patterns (a process that can change the activity of genes without changing their base unit sequence) of the liver showed widespread changes (totaling 4,255) in the distribution of this epigenetic marker, which could explain at least some of the changes in gene function revealed by the transcriptomics.
In summary, the metabolomics results showed a link between the gut and blood biochemical disturbances and health status – what was seen in the gut correlated with the blood biochemistry. In addition, changes in liver gene expression patterns were also consistent with these gut and blood metabolomics results.
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