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Published in final edited form as: Sci Total Environ. 2023 Dec 17;912:169271. doi: 10.1016/j.scitotenv.2023.169271

Transcriptomic and behavioral analyses reveal unique target tissues and molecular pathways associated with embryonic exposure to low level glyphosate and metal mixtures in zebrafish

Remy Babich a, Ilaria Merutka b, Emily Craig a,b, Akila Harichandara a, P Mangala C S De Silva c, T D K Sameera C Gunasekara c, Nishad Jayasundara a,b
PMCID: PMC10964846  NIHMSID: NIHMS1953635  PMID: 38114029

Abstract

Investigation of developmental molecular events following exposure to environmentally relevant agrochemical mixtures is critical to predicting their potential long-term ecological and human health risks. Here, we sought to uncover transcriptomic changes during zebrafish (Danio rerio) embryonic development following exposure to glyphosate and co-exposure to metals. Glyphosate is widely used globally with an allowable drinking water limit of 700 ppb. We examined effects of glyphosate (10 ppb) alone and when co-exposed to a metal mixture containing low levels of arsenic (4 ppb), lead (5 ppb), cadmium (2 ppb), and vanadium (15 ppb). This mixture was derived based on behavioral and morphological toxicity findings and environmentally relevant concentrations found in agricultural regions where glyphosate and metals are ubiquitously present. Gene expression patterns coupled to a single-cell transcriptomic dataset revealed that developmental exposure (28 – 72 hours post fertilization) to glyphosate dysregulates expression of developmental genes specific to the central nervous system. Subsequent studies indicated significant suppression of larval zebrafish movement with 10 ppb glyphosate exposure. Studies with glyphosate + metals mixture and metals mixture alone showed unique developmental transcriptomic patterns and behavioral changes compared to glyphosate exposure alone. However, some outcomes (e.g., changes in expression of genes involved in epigenetic regulation and extracellular matrix patterning) were common across all three exposures compared to the control. Notably, glyphosate + metals co-exposure distinctly suppresses lysosomal transcripts and targets renal developmental genes. While further studies are required to uncover the precise nature of the interactions between glyphosate and metals, our study shows that glyphosate at very low levels is a behavioral and neurotoxicant that changes when metals are present. Given this herbicide affects distinctive physiological processes, including renal development and lysosomal dysregulation when co-exposed with metals, we conclude that environmental cation levels should be considered in glyphosate toxicity and risk assessment.

Keywords: Pesticide, Developmental Exposure, RNASeq, Renal Development, Lysosomal Dysregulation

Graphical Abstract

graphic file with name nihms-1953635-f0006.jpg

1. Introduction

Glyphosate is the most commonly used herbicide among both farming and suburban communities around the world (Benbrook, 2016) and evidence of glyphosate toxicity and persistence continue to emerge (CDC, n.d.; Costas-Ferreira et al., 2022; Gunarathna et al., 2018). Glyphosate is considered low-risk as it was designed to inhibit the plant-specific shikimate pathway, which is absent in animal species (Steinrücken and Amrhein, 1980).

Currently, there are multiple active lines of investigation into off-target effects from glyphosate exposure. These have identified concerns for many organ systems, especially neural, renal, and immune systems (CDC, n.d.; Costas-Ferreira et al., 2022).

Glyphosate, an organophophate (OP) pesticide, may affect neurobehavior sice there is some evidence that exposure can affect AChE expression or activity in vertebrate brains (Bali et al., 2019; Glusczak et al., 2006; Martins-Gomes et al., 2022). Additionally, glyphosate is considered to increase oxidative stress and affect mitochondrial function via multiple mechanisms (Upamalika et al., 2022; Uren Webster and Santos, 2015). Evidence suggests that early life exposure to glyphosate impacts neurological, renal, and hematological development in vertebrate models and can lead to lasting consequences even in the absence of acute toxicity (Coullery et al., 2020a; Forner-Piquer et al., 2021a).

The U.S. Environmental Protection Agency (EPA) maximum contaminant level for glyphosate in drinking water is 700 ppb (4.14 uM), and it is often present in environmental samples at concentrations much lower (Gillezeau et al., 2019). Most existing mechanistic studies into glyphosate toxicity use concentrations at or above 700 ppb and may be underestimating low level impacts, especially in environmental mixture contexts.

In the aquatic environment, glyphosate may exist with metal cations. Metals and glyphosate chemically interact and form glyphosate-metal chelation complexes that increase glyphosate’s half-life from 90 days to 7 years in water and 47 days to 22 years in soil and may change glyphosate’s biological toxicity (Costas-Ferreira et al., 2022; Mertens et al., 2018). Furthermore, as mitochondrial toxicants and oxidative stressors, glyphosate and many metals share overlapping mechanisms of toxicity (Upamalika et al., 2022) and may contriute to adverse human and health wildlife health outcomes. For example, in agricultural communities in Sri Lanka glyphosate and metal mixtures in the drinking water is emerging as a link to kidney disease incidence (Babich et al., 2020; Ulrich et al., 2023) Synergistic effects and environmental persistence increase the risk of human exposure to these agrochemicals and warrant further examination of health implications of their mixture.

In this study, we use RNAseq analysis to evaluate changes in molecular pathways after developmental exposure to glyphosate both alone and in mixture with metals. We leverage the zebrafish as an in vivo model to understand both molecular and neurodevelopmental consequences of early exposures to chemicals and chemical mixtures (Shankar et al., 2019; Zheng et al., 2018). High fecundity and ease of rearing allow for high-throughput exposure studies and larval behavioral assays can indicate neurological impacts of early chemical exposures (He et al., 2014; Nishimura et al., 2015; Ogungbemi et al., 2021). Both molecular and functional changes in the zebrafish model have been related across species (Nishimura et al., 2015).

Our study aims to identify molecular pathways and tissues targeted in response to environmentally relevant concentrations of glyphosate and metals. Importantly, these levels are at or below concentrations considered safe in drinking water in the United States. We specifically examine how the biological response to glyphosate-metal complexes differs from that of glyphosate alone and of the metals mixture alone, in order to understand the contributions of individual components to effects of glyphosate + metals co-exposure. Notably, we demonstrated treatment group specific highly complex and unique molecular initiating events associated with altered neurobehavioral outcomes.

2. Materials and Methods

2.1. Zebrafish care and treatment paradigm

One cell AB strain zebrafish were incubated at 28.5 °C in egg water (1 embryo/1 mL) until 6 hpf (behavior) or 28 hpf (gene expression) at which time the embryos were screened for normal development and randomly distributed into treatment solutions. Complex mixtures were designed to understand how glyphosate alone (GLYPH) and in the presence of metals (GLYPH_ METALS) may be altering gene expression profiles and neurodevelopment. Metal concentrations used were chosen as representative of concentrations found in drinking water samples in agricultural regions in Sri Lanka (Babich et al., 2020). Zebrafish embryos were reared in four treatment conditions: clean egg water, GLYPH (10 ppb), METALS (NaAsO4 (4 ppb), CdCl2 (2 ppb), V (15 ppb), and PbCl2 (5 ppb), and GLYPH_METALS (glyphosate & metals). Metals (Cd, Sigma Aldrich Cat# 202908, As, Sigma Aldrich Cat# S7400, V, Sigma Aldrich Cat# 262935, and Pb, Sigma Aldrich Cat# 268690) and glyphosate (Sigma Aldrich Cat# 45521, fresh stock made bi-weekly) were dissolved in egg water to create the treatment solutions. Embryos remained undisturbed in treatment solutions until 72 hpf or 5 dpf for gene expression and behavioral assays respectively. After the behavior test was complete, fish were euthanized in MS-222. The zebrafish research was approved by UMaine IACUC committee proposal number A2017–05-04.

2.2. RNA Preparation

RNA was extracted from whole larvae at 72 hpf, euthanized in MS-222, using Qiashredder and RNAeasy minikit (Qiagen, Valencia, CA) following manufacturers protocol. Each RNA extraction contained a total of 10 larvae. A total of 5–8 RNA extractions were completed per treatment (50–80 individuals). High quality RNA (as determined by nanodrop), was sent to the Duke sequencing facility. Five samples of control, GLYPH, METALS, and GLYPH_METALS treatments underwent quality control and were sequenced on the Illumina Novaseq 6000 with S-prime 50 bp full flow cell. Library preparation was performed following the methodology by Peters et al., 2020.

2.3. RNASeq Bioinformatics and Analyses

Quality of raw reads was checked using FastQC. It was determined that per base sequence quality scores were > 28 with an average sequence quality score of 36 across all sequences. Reads were processed, which included Truseq adaptor trimming and mapping to the zebrafish reference genome, by following the protocol by Pertea et al. 2016. Annotated gene counts were imported into R studio (Vs 4.1.2) where edgeR was used to normalize zebrafish gene counts. Gene counts were normalized by TMM method. Following normalization, common dispersion was estimated, and tagwise dispersion was applied. Final normalized log2 counts per million (CPM) were used to determine fold change relative to control using topTags for pairwise comparisons. Genes were considered differentially expressed with an FDR adjusted p-value < 0.01 and absolute log2FC > 1.

To determine biological function of differentially expressed genes, genes were annotated using online software gprofiler. A principal component analysis was used to determine clustering of samples. Normalized log2 CPM gene expression data were inserted into the PCA for individual samples. Principal components 1 and 2 were extracted per sample, plotted, and overlayed with treatment condition. Inputs for the PCA included expression data for all genes sequenced as well as genes that were determined to be differentially expressed.

2.4. Tissue specificity analysis based on Embryonic Single Cell Transcriptomic data

In order to identify potential tissue specificity between each treatment and examine identified gene changes in the context of early zebrafish development, we leveraged a catalog of gene sets from a published single-cell transcriptome atlas that is curated to represent tissue-specific changes in expression during development (Farnsworth et al. 2020). Normalized RNAseq expression data from our dataset were compared against 31 tissue-specific gene sets and used in the competitive gene set test CAMERA (R studio v 4.1.2) to identify tissue gene-sets particularly dysregulated by each treatment. A developmental gene set was considered differentially expressed between treatment and controls with an FDR adjusted p-value < 0.01. The p-value was further used to determine biological ranking of gene sets (i.e., tissue-specific genes sets that had the greatest differential expression between treatment and control were correlated to the lowest p-value). An excel file containing tissue-specific differentially expressed gene sets is included in Supplemental Excel File Table S1.

2.5. Larval Behavior Analysis

To assess the neurodevelopmental toxicity of GLYPH, METALS, and GLYPH_METALS, a locomotor response to the light dark stimulus (LMR-L/D) assay was conducted using DanioVision TM (Noldus, Wageningen, The Netherlands). After a 4-day exposure to treatment solutions, 5 dpf larvae were screened for mortality and developmental abnormalities such as pericardia edema, yolk sack edema, curved spine, and stunted growth (Table A.1). As the LMR-L/D Daniovision system tracks larval movement, non-deformed larvae were randomly chosen and used for subsequent behavioral studies to avoid confounding behavioral differences with gross developmental deformity. 12 randomly selected zebrafish larvae from a given treatment solution were transferred into a 96-well petri dish with one larvae and 200 uL egg water per well. This was repeated two times for each treatment and control (n=24). Stratification of treatment groups by sex was not possible, as zebrafish larvae at this stage (5 dpf) do not possess sexual differentiation.

The light-dark test consisted of the following parameters: 10-minute dark period of habituation, 10-minute light period, 10-minute dark period, 10-minute light period, and 10-minute dark period for a total of 50 minutes per trial. EthoVision XT13 (Noldus, Wageningen The Netherlands), a software that is coupled with DanioVision, measures the location of the larvae in each well every 0.016 seconds, and records mean velocity and total distance traveled (Bailey et al., 2016). Raw data was exported as total distance traveled (TD) (mm/minute) over the course of 50 minutes per well. The TD was compared between treatment groups by ANOVA with Tukey’s post-hoc.

3. Results

3.1. Overview of Differentially Expressed Genes

Treatment with GLYPH resulted in 105 differentially expressed genes (DEGs), the fewest of any treatment. The mixture of the metals As, Cd, V, and Pb (total metal burden 26 ppb) resulted in the greatest abundance of 339 differentially expressed genes relative to controls. Strikingly, the addition of glyphosate to the metal mixture reduced the number of DEGs from 339 (metals mixture) to 196 (glyphosate + metals mixture). While some DEGs overlapped, 46 genes were specific to GLYPH, 103 for GLYPH_METALS, and 228 for METALS treatments (Figure 1A). Volcano plots were used to visualize scattering of differentially expressed transcripts among treatments relative to control and show that all treatments produced comparable magnitudes of fold change in DEGs, though total number of DEGs vary between treatments (Figure 1B–D). An overview of the direction of DEG changes between all groups is included in Appendix Figure A.1.

Figure 1: Differentially expressed genes from RNA seq analysis.

Figure 1:

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Venn diagram showing the number DEGs that are unique to metals (red), glyphosate (yellow), and glyphosate & metals (green) as well as those that are shared among treatments. Volcano plot displaying DEGs (red) compared to all genes between glyphosate and controls (B), glyphosate & metals and controls (C), and metals and controls (D). Genes are plotted by log2 Fold change (x-axis) and -log10 p-value (y-axis).

PCA plots were used to explore clustering of treatments based upon gene expression. To understand drivers of the total expression landscape, normalized expression counts for all 23,437 transcripts sequenced were used as inputs for the PCA. Two principal components (PC) were extracted, PC1 explained 36.215% of variance and PC2 explained 9.503% of variance, explaining a cumulative 45.719% of variance. Extracted regression factors PC1 and PC2 were used to build a scatter plot, which showed clear clusters of the metals mixture and glyphosate + metals treatments (Figure 2A). In a PCA analysis using all 509 DEGs identified as inputs, PC1 explained 32.139% of variance and PC2 explained 18.136% of variance, explaining a cumulative 50.275% of variance. The clear clustering and spatial relationships of control, GLYPH, METALS, and GLYPH_METALS treatments demonstrates transcriptional profiles are related to chemical components (Figure 2B). It is important to note that compared to other treatment groups, glyphosate treatment resulted in the most varied PCA distribution across both all transcripts measured (Figure 2A) and all DEGs (Figure 2B).

Figure 2:

Figure 2:

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Principal component plots representing normalized transcript counts of (A) all transcripts sequenced and (B) DEGs identified across all treatments. Each point indicates a sequenced sample (10 larvae combined). Clusters are labeled by treatment condition for control (blue), glyphosate (yellow), glyphosate & metals mixture (green), and metals mixture (red).

3.2. Gene Ontology Analysis

Gene ontology analysis identified clear overlap between biological processes affected by glyphosate and metals as toxicants, but revealed evident shifts in GO profile when glyphosate and metals are combined in solution. Significant GO terms fell broadly into the categories of DNA remodeling (67 DEGs), organization of the extracellular matrix (91 DEGs), lysosomal processing (14 DEGs), and all other GO terms gathered in an uncategorized group (162 DEGs).

Collectively, all three treatments shared the GO terms DNA geometric change, DNA duplex unwinding, and DNA conformation change, and the category of DNA remodeling had the most significant GO terms across all treatments. Within this category, there is a clear divergence between the glyphosate treatment and metals mixture (Figure 3A). The METALS mixture uniquely downregulated genes attributed to functional terms related to chromatin and epigenetic regulation of transcription. Conversely, GLYPH and the GLYPH_METALS mixture predominantly upregulated genes related to helicase activity and recombination.

Figure 3.

Figure 3.

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Significant GO terms identified from treatment to glyphosate, the metals mixture, and glyphosate + metals co-treatment by category A) DNA remodeling, B) Collagen and ECM, C) Lysosomal activity, and D) remaining uncategorized GO terms. Bars indicate the number of DEGs identified by that GO term that were down (blue) or up (red) regulated by treatment to each solution.

The second category of GO terms identified is organization of the extracellular matrix (ECM) and collagen architecture (Figure 3B). Terms related to peptidase activity were included in this category due to 96.8% overlap between DEGs in those GO terms and the GO terms explicitly related to collagen and the ECM. Here, the GLYPH_METALS treatment paralleled the GLYPH treatment with the GO terms hydrolase activity and anatomical structure homeostasis, and paralleled the METALS mixture with GO terms related to activity in the extracellular space. However, the GLYPH_METALS treatment produced a unique expression profile of 15/35 DEGs in this category that was uncorrelated to either METALS or GLYPH treatment (Appendix Figure A.2).

Finally, the glyphosate + metals co-treatment uniquely perturbs genes related to lysosomal processing of cellular cargo and downregulates all DEGs identified in this category (Figure 3C). While GLYPH downregulates 2 DEGs in this category, the METALS mixture did not significantly change any DEGs in GO terms related to lysosomal processing. In the remaining uncategorized GO terms, each treatment produces a distinct response (Figure 3D). GLYPH alone stimulates GO terms related to organic cyclic compound response, non-membrane bound organelles, and metabolic process. The METALS mixture induces genes in calcium signaling, neuropeptide receptor, and vitamin B transport. The GLYPH_METALS treatment downregulated genes related to RUNX1 and necroptosis.

3.3. Tissue-specific Gene Set Analysis

In order to identify whether the treatments may target different tissues during embryonic development, we compared expression data for each treatment to curated tissue-specific developmental gene lists from a published single-cell RNAseq database (Farnsworth et al., 2020). The relative enrichment of each tissue gene set was then biologically ranked for each treatment. A total of 31 tissue-specific gene sets were analyzed (Figure 4). 11 out of 31 gene sets were statistically differentially expressed in GLYPH treatment relative to control. Both GLYPH_METALS and the METALS mixture significantly differentially expressed 27 of 31 tissue gene sets relative to control. For GLYPH treatment the top five differentially expressed gene sets, ranked in order by those most represented to least are pharyngeal arch, neuron, tailbud, pharyngeal endoderm, and central nervous system (FDR adjusted p-value < 4.28e-4). Interestingly, the gene sets for central nervous system, neuron, pineal gland, and pancreas were only significantly affected by GLYPH treatment and not the METALS or GLYPH_METALS treatment. Rather, GLYPH_METALS treatment was significantly associated with gene sets representing thymus, spleen, intestine, tailbud, and integument (FDR adjusted p-value < 1.64e-410). METALS treatment was significantly associated with genes representing tailbud, thymus, pectoral fin bud, paraxial mesoderm, and lens placode (FDR adjusted p-value < 1.63e-12).

Figure 4:

Figure 4:

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Developmental tissue-specific gene set enrichment. Multi-variable dot plot representing the biological rank (x-axis) of a tissue type (y-axis) identified for transcripts in each treatment condition by the CAMERA test. The size of the dot represents the number of transcripts sequenced present in a given tissue gene set. The color of the dot reflects treatment condition of glyphosate (yellow), glyphosate & metals (green), or metals (red). If a tissue gene set was not determined to be significantly different from controls for a given treatment, a dot was not included for that tissue type.

3.4. Behavioral Assay

Given the concern for glyphosate as a neurodevelopmental toxicant, we evaluated the impact of our treatment solutions on larval swimming behavior at 5 dpf. Treatment with GLYPH markedly reduced the total distance traveled, while the METALS and GLYPH_METALS treatments did not significantly impact larval swimming (Figure 5A). Additionally, GLYPH drastically modified the response to light stimulus. Control larvae and larvae reared in both mixture treatments reduce their activity in light (10–20 mins, 30–40 mins) but regain some activity throughout the period, while larvae reared in GLYPH show a severe startle response and do not resume movement until well into the next dark period (Figure 5B). To resolve the role of each of the mixture components in the behavioral patterns we detected, we compared all compounds individually and in mixture across a range of fixed-ratio doses and found that GLYPH_METALS treatment forms a unique dose-response relationship from both the METALS and the GLYPH treatments alone (Appendix Figure A.3)

Figure 5:

Figure 5:

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A) Total distance traveled (mm) in 50 minutes by 5 dpf larvae exposed to treatment solutions (n=18–24). B) Distance traveled (mm) each minute during the larval activity assay in periods of dark (0–10 minutes, 20–30 mins, and 40–50 mins) and light (10–20 mins, 30–40 mins). Line trace and ribbon represent mean and standard error of distance traveled by all larvae in each group within each minute (n=18–24). Statistical difference between TD of each group was evaluated by ANOVA with Tukey’s post-hoc and differing letters between groups indicate p<0.05.

4. Discussion

Considering the ubiquitous use of glyphosate, it is of utmost importance to examine potential adverse outcomes of exposure. While emerging research shows that glyphosate can cause cytotoxicity and disrupt development in vertebrates, most studies use high concentrations of glyphosate or directly apply compounds to cells to resolve mechanisms of toxicity (Costas-Ferreira et al., 2022). There remains a significant gap in understanding the processes disrupted by exposure to glyphosate (with and without metal co-exposure) at low levels, comparable to those found in urine and drinking water, and interpretation using targeted mechanistic studies.

Broadly, developmental exposure to low levels of glyphosate, a mixture of metals (Cd, Pb, As, V), and the combination of glyphosate + metals produced overlapping but distinct transcriptional profiles. There are two primary models to understand transformation of response upon mixture. First, the direct chemical interaction between glyphosate and metals in chelating complexes could facilitate or hinder key interactions of the individual chemicals with cellular machinery. In this way, co-exposures can transform individual chemical toxicity by affecting transport into the cell, changing target tissues, or differentially activating or inhibiting antioxidant enzymes in response to oxidative stress (Wallace and Buha Djordjevic, 2020). For example, the OP pesticide chlorpyrifos can chelate cadmium, facilitate uptake into hepatocytes, and induce novel toxicity compared to separate compounds (He et al., 2015).

The second model is that co-exposure to glyphosate and metals produces a shift in cellular responses; that is, cells integrate information from each chemical and produce a new response profile as opposed to a simple additive effect. Numerous metals are known to interact biologically to produce opposing, synergistic, or novel effects in co-exposure. In an RNAseq dataset comparing low level (<10 ppb) Pb, Hg, As, and Cd exposures, individual metals primarily changed transcripts related to the immune response whereas mixtures targeted antioxidant pathways (Cobbina et al., 2015). Additionally, our previous work with glyphosate and metals mixtures demonstrated differential expression of the kidney development genes pax2a and kim1 between glyphosate alone, metals, and glyphosate + metals mixture, accompanied by divergent effects on mitochondrial respiration (Babich et al., 2020).

4.1. Epigenetic Modification and Regulation of Gene Expression

The most abundant GO categories across all treatments were related to DNA accessibility and epigenetic remodeling. Chemical exposure during early life alters the epigenetic profile. This can alter physiological trajectory of a developing organism and susceptibility to stressors, even without manifesting acute dysfunction (Lapehn and Paquette, 2022; Rager et al., 2020).

While all treatment groups affected DNA regulation, the GLYPH and GLYPH_METALS exposures predominantly upregulated DNA helicase activity. The METALS treatment uniquely downregulated genes associated with chromatin organization, DNA packaging, and epigenetic regulation of gene expression. Metals are versatile DNA regulators; they can bind directly to DNA and alter its structure (Kanellis and dos Remedios, 2018), interact with transcription factors (Blanchard and Manderville, 2016), and interfere with DNA methylation (Park et al., 2020; Ryu et al., 2015). These epigenetic changes can have lasting functional consequences. Although the concentrations of metals used in this study are lower than those typically associated with mutation-driven genotoxicity, low levels of metals may be acting in combination to trigger DNA protective responses or induce epigenetic remodeling to induce changes in gene expression profiles.

4.2. Regulation of extracellular matrix formation

All three treatment groups perturbed genes related to maintenance of the extracellular matrix (ECM) and collagen, which is a major structural protein component of the ECM and acts as a receptor for cell signaling (Karamanos et al., 2021). The ECM plays a role in critical cellular functions including proliferation, migration, differentiation, and cell death, and developmental ECM patterning is tightly controlled to regulate tissue formation (Rozario and DeSimone, 2010). Perturbation of ECM regulation could disrupt proper tissue development, promote pathological tissue fibrosis, or alter cellular capacity to respond to developmental and future stress cues (Ricard-Blum et al., 2018).

Alongside dysregulation of genes involved in extracellular components, the metals mixture induced expression of genes related to degradation of the ECM and peptidase activity. Fibroid cells exposed to cadmium downregulated ECM components (such as collagen) and upregulated ECM degradation (Yan et al., 2021). Metals have been shown to alter ECM dynamics more broadly as well. Arsenic has been positively associated with DNA methylation of ECM remodeling genes (such as matrix metalloproteinases) in children that were exposed in utero (Gonzalez-Cortes et al., 2017). Mice treated with 100 ppb As in drinking water developed a maladaptive mitochondrial phenotype in connective tissue fibroblasts as well as dysfunctional ECM (Anguiano et al., 2020).

GLYPH had a milder impact on ECM regulation than the other treatments, but GLYPH_METALS shifted the profile of metal-induced changes to ECM regulation. The GLYPH_METALS treatment both produced unique DEGs and reverted changes in ECM-related genes that were induced by treatment with the metals mixture (Appendix Figure A.2).There are multiple mechanisms by which glyphosate could impact ECM deposition. Glyphosate may interfere with normal ECM deposition due to structural similarity to glycine, a primary component of collagen (Kay et al., 2021; Martínez et al., 2018). In addition, signaling components of the ECM may respond differently to chelated glyphosate and metals complexes than the individual constituents, which could be evidenced by the loss of DEGs induced by the metals mixture when glyphosate is added.

4.3. Glyphosate targets neuronal and CNS development

Glyphosate is considered an OP in chemical structure, but is not a potent acetylcholinesterase inhibitor (Bali et al., 2019; Martins-Gomes et al., 2022). While there are clear neurological impacts from developmental glyphosate exposure in vertebrate models, developmental neurotoxicity from environmentally relevant concentrations as used in this study have not been shown. GLYPH treatment dysregulated genes specific to development of neuronal, central nervous system, neural crest, and lens placode, including fosab, ier2b, and socs3a. fosab has been shown to increase upon early life exposure to the OP DFP as an indication of CNS hyperexcitation (Brenet et al., 2020). In our dataset, fosab was significantly upregulated from GLYPH exposure relative to controls.

We show that these transcriptional changes correspond to hypoactivity uniquely observed in larvae exposed to GLYPH. Previous studies in adult animals have shown that chronic exposure to glyphosate impairs learning and memory in rats and induces neuronal dysfunction via overstimulation of dopaminergic activity at environmentally relevant concentrations (Bali et al., 2019; Faria et al., 2021). Early life exposure to glyphosate is known to cause lasting neurological effects, in the form of decreased motor activity and alterations in microglial synapse pruning (Coullery et al., 2020b; Forner-Piquer et al., 2021b). Here, GLYPH induced transcriptional changes in the developing CNS may have altered axon formation or neuoron outgrowth and/or the function of neuroreceptors resulting in a diminished response to the light/dark cycle and hypoactivity (Ogungbemi et al., 2019).

Strikingly, neither the METALS nor GLYPH_METALS treatments induced DEGs that were enriched in neuron- or CNS-specific gene sets (Figure 4). However, the metals used in this study are established neurotoxicants, but at much higher concentrations. The METALS mixtures could disrupt neurodevelopment and not be captured in this analysis in two ways: these mixtures could change expression of essential regulators that are not specific to neuronal or CNS cell-types; in addition, cell-specific changes could occur at a timepoint after 72 hpf. Indeed, we report significant changes in larval activity at 5 dpf from both the metals mixture and individual metals across a range of low doses (Appendix Figure A.3). This result supports previous evidence that at extremely low concentrations heavy metals can perturb neurodevelopment.

Both metals-containing mixtures target genes specific to development of spleen, kidney, thymus and myeloid lineage (Figure 4). This suggests that development of the innate immune response may be targeted, as these systems are production sites for T and B cells as well as macrophages (Bjørgen and Koppang, 2021). GO analysis highlights the METALS mixture’s impacts on immune response, but GLYPH_METALS treatment does not parallel this and may be targeting other aspects of spleen and kidney development (Figure 3D).

4.4. Glyphosate + metal specific lysosomal disruption

Only the GLYPH_METALS mixture altered genes related to lysosomal processing, reflected by GO terms lysosome, lytic vacuole, and phagosome (Figure 3C). This not only represents novel effects arising from co-treatment, but provides insight into a potential mechanism for underlying cytotoxicity and long term effects from drinking water contaminated with both metals and glyphosate. Cadmium, Arsenic, Lead, Vanadium, and Glyphosate can impair lysosomal function by suppressing lysosomal acidification, autophagosomal fusion, and lysosomal biogenesis (Dodson et al., 2018; Liu et al., 2017; Pi et al., 2017). Mechanistically, this may be mediated by mitochondrial dysfunction. Cd, As, Pb, and glyphosate all reduce efficient ATP production and increase mitochondrial reactive oxygen species (ROS) by multiple mechanisms (reviewed by Upamalika et al., 2022). Mitochondrial and lysosomal function are highly coordinated and their quality control are interlinked.Chronic mitochondrial dysfunction and sustained overproduction of ROS can disrupt key lysosomal maintenance pathways (e.g. Transcription factor EB (TFEB)-mediated signaling), which reduces autophagy and the expression of lysosomal components may lead tothe accumulation of impaired organelles (Demers-Lamarche et al., 2016; Deus et al., 2020; Fernandez-Mosquera et al., 2019; Fernández-Mosquera et al., 2017).

Though these metals and glyphosate are known oxidative toxicants, it is important to note that genes directly related to mitochondrial function and stress response were not enriched in our dataset. Rather than inducing a strong oxidative stress response, we posit that the low level treatments to glyphosate and metals mixtures produce ROS-mediated signaling alongside impacts on mitochondrial function. This model brings the unique impact of glyphosate and metals on lysosomal genes in line with our previous work which shows that exposure to the glyphosate + metals mixture produces different mitochondrial respiration effects compared to glyphosate or metal mixture exposure alone (Babich et al., 2020). In addition, these differences in mitochondrial function are accompanied by dysregulation of renal development genes pax1a and kim1 and vacuolation in developing renal tissue that is most severe from glyphosate + metals co-treatment. Given the links between renal epithelial vacuolation and lysosomal capacity/function, as well as dependence of renal function on lysosomal and mitochondrial function, the combined exposure to glyphosate + metals could undermine renal health in development or over time.

4.5. Study Limitations

The primary limitation of this study is that it is exploratory. Because little is known about molecular implications of low-level, early-life, or mixture interactions of glyphosate with agrochemical metals, fundamental insight into the target processes is needed. A secondary limitation is that we evaluate a complex metal mixture without comparing each constituent metal for its role in driving expression changes. However, the mixtures were designed to be highly relevant to environmental exposures. Additionally, timing of treatment was variable between gene expression analysis (28–72 hpf) and behavior screening (6 hpf-5 dpf). Though consistent timing of treatment would improve correlation between behavioral changes and alterations in gene expression, all endpoints reflectsensitive periods of embryogenesis and treatments may have similarly impacted development of target organ systems (such as the CNS) in both experimental schemes. Future studies should explore individual chemicals and sub mixtures with more targeted analyses to determine what chemicals may be contributing to the clear shifts in biological function pathways that were identified here. In addition, it is crucial to understand the long-term functional consequences of the transcriptional disruption from early life and low-level exposures to these agrochemicals.

4.6. Conclusion

Here, we show that developmental exposure to a very low concentration of glyphosate (i.e., 10 ppb, which is 70 times lower than allowable drinking water limit) dysregulates expression of key developmental genes specific to CNS and neuron tissues and induces hypoactivity. Also, we distinguish transcriptional profiles of GLYPH, METALS, and GLYPH_METALS and demonstrate clear biological interaction between glyphosate and metals even at very low levels. Importantly, data reveal that GLYPH_METALS distinctly suppresses lysosomal transcripts and targets important genes for renal development, and lysosomal dysregulation remains an important unique outcome of glyphosate + metals exposure.

This study provides mechanistic insight into consequences of low-level exposure to these widespread agrochemical pollutants and evinces that chemical interaction between glyphosate and metals may change primary targets of toxicity when exposed together, as in co-contamination of drinking water. Altogether, this study emphasizes the need to consider low-level exposures to glyphosate, metals, and their mixtures in ecological and human health risk assessment.

Supplementary Material

1
2

Highlights:

  • Zebrafish developmental exposure to glyphosate altered expression of genes associated with the central nervous system and suppressed larval movement

  • Exposure to metals mixture and glyphosate + metal co-exposure showed differential gene expression and behavioral outcomes compared to glyphosate exposure

  • Renal developmental genes and lysosomal transcript expression were most impacted by glyphosate + metal co-exposure

Acknowledgments:

We thank the University of Maine Zebrafish facility and Mark Nilan for supplying zebrafish embryos for this project. We thank Dr. Iain Drummond for his knowledge and support in study design. We thank Dr. Gayani Thilakaratne, and Dr. Truls Ostbye at the Duke Global Health Institute for facilitating this research in Sri Lanka, and Prof. Joel Meyer, Duke University, NC, for his feedback. We are also incredibly grateful to Prof. Kamani Wanigasuriya, Manoj Wijesekera, and Yohan Mahagamage from University of Sri Jayawardenapura, and Sakuntha Gunarathna and Isini Ranawake from University of Ruhuna for the immense support in the field. We also thank health officials in endemic region, especially Dr. Chamal Priyantha and Dr. Saman Chandana. This work was supported by the National Science Foundation [grant numbers DGE-1922560, 2019]; National Institute of Environmental Health Sciences [grant numbers T32-ES021432, 2022; grant numbers R01ES033466, 2023]; Duke Global Health Institute and Nicholas School of the Environment Pilot Grant funding.

Footnotes

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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