Butyrate rescues chlorpyrifos-induced social deficits through inhibition of class I histone deacetylases

Reviewer #1 (Public review):

Summary:

In this study, the authors examine the effect of Chlorpyrifos (CPF) exposure on zebrafish social development. They expose larval zebrafish to CPF (0 - 3 dpf), and report social deficits at juvenile stages. They show that the gut microbial metabolite butyrate can rescue these social deficits, proposing that butyrate acts as a histone deacetylase (HDAC) inhibitor, given that inhibition of some HDACs can also rescue social deficits. They also show that CPF changes neuronal gene expression, and butyrate partially rescues these changes. Finally, they demonstrate changes in gut microbiome and metabolome composition, pointing to potential modulation of nitrogen metabolism pathways. They then hypothesise that NO can modulate HDAC activity and attempt to link the NO pathway to social behavior.

Strengths:

The authors demonstrate an interesting link between early Chlorpyrifos (CPF) exposure and later-life social deficits, such as changes in neuronal gene expression, including some autism-related genes, and provide solid evidence that butyrate and epigenetic modulation (histone deacetylase inhibition) may be involved.

They also comprehensively characterise the microbiome and metabolome of CPF-exposed zebrafish, providing a useful resource for further investigation into its gut-brain mechanisms.

They are cautious in framing some of their conclusions as a hypothesis and provide some suggestions for future analyses.

Weaknesses:

The claim that butyrate's effects on CPF-induced social deficits and neuron activity changes are mediated by histone deacetylase inhibition is lacking some additional controls and, hence, is not completely supported.

Details on the social behavior assay performed and other potential morphological or behavioral changes were not provided.

Claims on the mechanism of action of CPF are inconclusive. The causal role of the gut microbiome is not established, especially since gut microbial dysbiosis may also be a downstream consequence of direct effects of CPF on the host, such as changes in host gut gene expression. Evidence for the role of nitrogen metabolism is also incomplete, and the authors have not discussed or ruled out the potential alternative mechanism of reduced butyrate production due to gut microbiome changes.

Reviewer #2 (Public review):

Summary:

This paper by Diaz et al. uses the zebrafish model to examine how early embryonic exposure to Chlorpyrifos (CPF), a widely used organophosphate pesticide, induces social behavior deficits later in life. This paper combined behavioral testing, pharmaceutical treatment, genetic manipulation, and multi-omics to test the hypothesis that early CPF increases the abundance of denitrifying bacteria, Pseudomonas, which, in turn, enhances nitric oxide production and induces selective inhibition of HDAC8 and abnormal gene expression in the brain.

Strengths:

(1) The observation that early embryonic CPF exposure causes behavior deficits in juvenile zebrafish is very intriguing. It is especially exciting to see that CPF-induced behavior deficits can be reversed by overnight treatment with butyrate or HDAC1 inhibitors in juvenile zebrafish. In humans, CPF exposure during pregnancy causes brain abnormalities and neurological disorders such as Autism. Though it is far away from the zebrafish experimental study to human application, the experimental effects reported in the paper are still quite thought-provoking.

(2) The authors performed RNA sequencing experiments on control zebrafish, CPF-exposed zebrafish, and CPF-exposed zebrafish that were treated with Butyrate. The data not only showed large-scale transcriptomic changes in the juvenile zebrafish brain in response to embryonic CPF exposure but also showed that many CPF-induced genetic alterations can be alleviated by butyrate exposure later in life.

(3) The authors also performed untargeted metabolomics on zebrafish gut and metagenomic analysis in zebrafish feces samples. The results are interesting and support the conclusion that increased Intestinal Nitric oxide metabolism and the abundance of denitrifying bacteria, such as Pseudomonas, are associated with CPF exposure.

(4) The large datasets presented in the paper will be useful to other researchers interested in understanding how CPF or butyrate alters brain and gut function. It might be useful to generate new hypotheses to power other research lines.

(5) The social preferences, behavior testing, and experimental paradigm used by the paper may also be used by other researchers to investigate the interaction among gene, environmental factors, and brain function.

Weaknesses:

(1) The presented link between gut microbiome and CPF-induced behavior and genetic alteration is an association, but not causation. Although the research data align with the hypothesis, the hypothesis is not fully supported or tested by the data presented in the paper in the current state.

(2) The authors performed several large omic studies. However, some of the presented analyses are relatively simple and incomplete. For example, the authors performed shotgun metagenomic analysis on zebrafish feces. However, the paper only displayed the bacterial taxa differences. Are there any differences in bacterial genetic pathways, especially the pathways associated with microbial nitrogen metabolism? What is the alpha and beta diversity looking like when comparing different experimental groups?