Tissue selection

The rat liver and kidney tissues that formed the starting material for this investigation were obtained from animals that formed part of a chronic (2 year) toxicity study looking at the effects of Roundup pesticide [17]. Roundup was administered via drinking water to Sprague–Dawley rats at a regulatory admissible dose (50 ng/L glyphosate equivalent dilution) and which is representative of what may be found in contaminated tap water. At this dose, the glyphosate equivalent average daily intake of Roundup was approximately 4 ng/kg bw/day of glyphosate.

Male animals suffered from liver and kidney damage more acutely than females, resulting in an increased rate of premature death [17]. Most male rats were discovered after death had occurred. This resulted in organ necrosis making them unsuitable for further analysis and thus they were excluded from transcriptome profiling. We therefore focused our investigation on female animals where freshly dissected tissues from cohorts of 9–10 euthanised treated and untreated rats were available.

Female control and Roundup-treated animals were respectively euthanized at 701 +/− 62 and 635 +/− 131 days (Additional file 1). Anatomopathological analysis of organs from these animals revealed that the liver and kidneys were the most affected organs [17]. Roundup-treated female rats showed 3 times more anatomical signs of pathology (15 in 8 rats) than the control group (6 in 4 rats). In addition, serum and urine biochemical analysis showed increased levels of serum triglycerides. While no severe anatomical kidney damage was detected, biochemical analysis revealed a decrease in blood Na, Cl, P and K levels and a corresponding increase in urine suggesting ion leakage and decreased urinary creatinine. Taken together these alterations in blood and urinary biochemical parameters suggest an impairment of kidney function. Overall, twice the number of biochemical parameters was disturbed in kidney than what can be expected by chance. Furthermore, a testosterone/estrogen imbalance was evident with testosterone serum levels significantly increased by 97 % by comparison to controls, while estradiol serum levels were decreased by 26 %. These observations together with pituitary gland disturbances suggest endocrine disrupting effects.

Electron microscopic analysis of liver sections from these animals showed nucleolar disruption in hepatocytes (Fig. 1). There was a statistically significant increase of cell and cytoplasmic area. No major cell structural damage was observed. However, the nuclear morphometry analysis showed that hepatocytes of Roundup-treated female rats had a statistically significant higher heterochromatin content, a reduced dense fibrillar component and a concomitant increase in granular component in comparison to controls indicating a disruption of nucleolar function and overall decreased level of transcription (Fig. 1a). In addition, a cytoplasmic dispersion of glycogen was also observed in the Roundup treated group (Fig. 1b).

Fig. 1 Alterations in hepatocyte nuclear architecture in female Roundup-treated rats suggests transcriptional disturbances. Liver from control (C) and Roundup (R) treated female rats were subjected to an ultrastructural electron microscopic analysis to investigate subcellular architecture. a Quantification of morphometric analysis of hepatocytes revealing alterations in subnuclear (heterochromatin, dense fibrillar, granular) compartments indicative of a reduced transcriptional status. Morphometric parameters are represented by their mean and their standard deviation. A two-tailed unpaired t-test was used as a standard test for statistical comparisons (***, p <0.001). b Representative electron micrographs comparing hepatocytes from control (upper panel) and Roundup-treated (lower panel) rats showing a disruption of glycogen dispersion (G). N nucleus, R rough endoplasmic reticulum Full size image

Transcriptome patterns segregate liver and kidney samples based on Roundup treatment

In an attempt to confirm the anatomopathological effects in a more quantitative manner and to gain insight into the gene expression profiles that are associated with the signs of pathology observed in the liver and kidneys, we conducted a full transcriptome microarray analysis of these organs. We began by undertaking an unsupervised Principal Component Analysis (PCA) of the dataset, which reduces a high-dimensional expression profile to single variables (components) retaining most of the variation (Fig. 2a). As control and Roundup-treated animals were sacrificed at various ages, we initially conducted a PCA analysis where individual animals were sorted by age (oldest vs youngest), to ascertain if differences in transcriptome profiles correlate with this parameter (Additional file 2). This showed that the statistical control values were weak, with no segregation of the oldest and youngest animals in either control or Roundup-treated groups, indicating that age was not a major source of difference. By contrast, a clear separation was observed between control and Roundup-treated rats based on treatment (Fig. 2a; Additional files 3 and 4). Even if the principal components only account for ~30 % of the observed variation, which seems quite low, this high level view of the data showed a homogeneous segregation and a low intragroup variability, as well as the absence of outliers. Figure 2b shows the statistical significance (by Student’s t-tests) of differential transcript cluster expression by volcano plots along with respective fold changes (FC) (Fig. 2b), where a transcript cluster constitutes a group of exon clusters (each exon cluster composed of different probes) corresponding to a known or putative gene. Overall, gene expression changes varied from −3.5 to 3.7 fold in liver and from −4.3 to 5.3 in kidneys. The expression of 57 and 226 transcript clusters were respectively disturbed in liver and kidney over an FC of 2. Akr1b1 (FC of −4.3, p = 2.2E-5) and Ten1 (FC of −3.5, p = 7.3E-4) were the most down-regulated genes respectively in liver and kidneys The most up-regulated transcripts were small nucleolar RNAs (snoRNAs), ENSRNOT00000053015 in liver (FC = 3.7, p = 3.0E-6) and ENSRNOT00000068958 in kidneys (FC = 5.3, p = 8.6E-7). Large statistical significance (p-values up to 2.3E-10 in liver) was observed. A large number of transcript clusters were significantly disturbed below stringent q-value thresholds (Table 1). In addition, the statistical analysis of simulated random samples confirms that the degree of statistical difference between control and Roundup treatment groups are far greater than what can be expected by chance (Table 1).

Fig. 2 Wide-scale transcriptome profile alteration in liver and kidneys of Roundup-treated female rats. Liver and kidneys from control rats and animals receiving 0.1 ppb Roundup (50 ng/L glyphosate equivalent dilution) in drinking water were subjected to a full transcriptome microarray analysis. a PCA analysis of transcript cluster expression profiles shows a distinct separation into groups of treated (red) and control (green) rats in both kidney and liver samples. Numbers by data points denote age at time of death in days. b Volcano plots of the liver and kidney transcriptome profiles showing the log 2 fold changes and the –log10 p-values in transcript cluster expression induced by Roundup exposure compared to controls. Data were selected at the cut off values p <0.01 and fold change >1.1. Red dots represent genes commonly disturbed between liver and kidney samples Full size image

Table 1 Number of transcript clusters whose expression is disturbed at different cut-off threshold p-values Full size table

Data used in functional analysis were selected at the cut off values of p < 0.01 and q < 0.08 with FC >1.1 for use with large gene lists as previously recommended [19]. A Venn diagram comparing liver and kidney transcript cluster expression profiles at an FC >1.1 (Fig. 3a) indicates that even if most of the disturbances were tissue specific, 1319 transcript clusters were commonly disturbed between the two organs. A comparison at a frequently selected cut off threshold of FC > 2 again results with most changes in gene expression being specific to either liver or kidney but with a total of 20 genes being commonly disturbed in both organs (Fig. 3b). The FC, p-value and q-value for all genes whose expression is commonly disturbed in both liver and kidney are shown in Additional file 5.

Fig. 3 Large spectrum of transcript cluster expression is commonly disturbed in liver and kidney by Roundup. Venn diagrams showing numbers of genes commonly disturbed in liver and kidneys as revealed by transcriptome analysis at cut off threshold values of p <0.01 and fold change >1.1 (a) and >2 (b) Full size image

Out of these, 18 genes were randomly selected for validation of the microarray results by RT-qPCR (Additional file 6). The overall pattern of the RT-qPCR results confirmed the microarray data with 86 % of the genes found to be similarly up- or down-regulated by both methods.

Gene function alterations involved in mitochondrial respiration, spliceosome function and chromatin structure modification is associated with Roundup treatment

We next conducted an ontology analysis of the 1319 transcript clusters commonly deregulated in liver and kidney using the DAVID gene functional classification tool to reveal the most affected gene categories. As a result 868 genes were recognised. The 8 clusters of functional disturbances having enrichment scores over 2 are presented in Table 2. All have significant p-values and Benjamini corrected p value (q-values). A total of 3 major affected gene networks were identified.

Table 2 Functional clustering of genes derived using the DAVID gene functional classification tool Full size table

First, two clusters were related to spliceosome function. This included genes encoding cleavage and polyadenylation specific factors (Cpsf2, Cpsf3 and Cpsf7), heterogeneous nuclear ribonucleoproteins (Hnrnpl, Hnrnpf) and splicing factors (Sf3b5, Sf3a1). Expression of all of these genes was upregulated with the exception of Cpsf3. Other genes involved in RNA splicing, such as Luc7l3, Pnn, Prpf4b, Pnisr, Prpf39, Srek1, Ddx39b and Ddx39a, were significantly upregulated. Additionally, expression of at least 160 non-coding snoRNAs were found to be altered with almost all being upregulated with a large FC of up to 5.32 for ENSRNOT00000068958in kidneys. Second, two clusters consisted of members of the chromatin modification family of enzymes, in particular histone-lysine N-methyltransferases. Expression of the 7 genes (Men1, Setdb1, Suv420h2, Dot1l, Ehmt1, Ehmt2, Nsd1) belonging to this cluster was upregulated. Other genes with a related ontology (Mll2, Mll4, Tet3, Baz2a, Dnmt3a, Brd1, Brd4, Ino80d or Arid4b), which are not taken into account in the cluster of enriched biological functions, were also upregulated. Most also belong to the family of histone-lysine N-methyltransferase complexes that specifically methylate lysine residues of histone H3 (Lys-4, 9, 20 or 79) or H4 (Lys-20) among others, tagging them for chromatin condensation. In addition, most of these are also included in a larger disturbed cluster of 40 genes involved in negative regulation of macromolecule biosynthetic processes.

Third, functional disturbance of genes involved in mitochondrial metabolism was represented by two clusters, especially related to respiratory chain complex I and the TCA cycle. Expression of most of these genes was repressed. A total of 7 genes encoding NADH dehydrogenase (ubiquinone) complex I of the mitochondrial respiratory chain were found disturbed, with 6 of them being downregulated. Additionally, the genes encoding isocitrate dehydrogenases (Idh3B and Idh3g), succinate dehydrogenase (Sdhc), succinate-CoA ligases (Sucla2 and Suclg2) and mitochondrial F1 complex ATP synthases (Atp5b and Atp5d) were downregulated. These data suggest that activities of mitochondrial complexes, in particular respiratory activity, are depressed.

These 3 major enriched biological functions are presented by organ-specific heat maps using hierarchical clustering of samples (Fig. 4). Our results show genes related to mitochondrial respiration and the TCA cycle are mostly repressed while those involved in mRNA splicing and histone modification are upregulated.

Fig. 4 Heatmaps of the three major ontologically enriched biological functions from transcriptome analysis of liver and kidneys. The ontologically enriched biological functions (Table 2) derived from the alteration in gene expression patterns commonly disturbed in liver and kidneys from Roundup treated female rats (Figs. 2 and 3) with respect relative to mRNA splicing via spliceosome (GO:0000398, in blue), histone modification (GO:0016570, in yellow) and cellular respiration and TCA cycle (GO:0045333 and GO:0006099, in pink), were grouped on organ-specific heatmaps using hierarchical clustering of samples (C, control; R, Roundup) and variables (gene symbols). A distinct separation based on direction (up- or down-regulation) of gene expression, biological function and organ between Roundup-treated and control animals is discernible Full size image

Roundup-associated changes may occur via sex-hormone signaling pathways

The gene ontology analysis (Table 2) indicates a modulation of cell signalling pathways has taken place. The GO biological processes GO:0007264 “small GTPase mediated signal transduction” (21 genes, p = 1.2E-3, q = 1.2E-1) and GO:0007242 “intracellular signalling cascade” (57 genes, p = 2.2E-3, q = 1.7E-1) are enriched among genes commonly disturbed in liver and kidneys. Additionally, the networks highlighted by this analysis that may account for the disturbance in gene expression were centred on the transcription factors Creb1 (280 genes regulated, p ~ 0), c-Myc (159 genes regulated, p ~ 0), Yy1 (113 genes regulated, p <4.8E-234), Oct3/4 (94 genes regulated p <6.7E-194) and Esr1 (83 genes regulated, p <8.E-171) (Additional file 7). These transcription factors are intimately connected in regulation of gene expression and can be involved in hormone signalling pathways.

In this context, it is noteworthy that the gene encoding the androgen receptor is statistically significantly downregulated in liver (FC = −1.4, p = 8.1E-3, q = 7.7E-2) and kidneys (FC = −1.32, p = 2.9E-3, q = 3.8E-2). In addition, the retinoid X receptor beta gene (Rxrb) is significantly upregulated in liver (FC = 1.2, p = 3.5E-5, q = 3.2E-3) and kidneys (FC = 1.36, q = 4.5E-6, q = 1.2E-3). In addition, the KEGG annotation for the mTOR signalling pathway (14 genes, p = 6.4E-3, q = 0.21) and the phosphatidylinositol signalling system (16 genes, p = 1.3E-2, q = 0.29) are enriched in liver. In kidney, the KEGG annotation for the adipocytokine signalling pathway (involving mTOR) (19 genes, p = 2.4E-3, q = 0.06) and the phosphatidylinositol signalling system (21 genes, p = 6E-4, q = 0.05) are enriched. However, expression of Star, which has been suggested by previous studies to mediate endocrine disrupting effects of Roundup in MA-10 Leydig tumor cells [21], and of Esr1, Esr2 and aromatase (Cyp19a1), which were found disturbed in the human HepG2 hepatocyte cell line [22], were not found to be dysregulated in this investigation.

Alterations in transcriptome profile suggest liver and kidney anatomorphopathogy

Scoring maps for pathways and toxicity processes (Fig. 5) indicates that multiple cellular functions could be involved. Out of the 4224 liver and 4447 kidney transcript clusters found to be altered, 2636 and 2933 network objects were respectively recognized by GeneGO Metacore. Mapped pathways related to inflammatory responses, which can be secondary outcomes to organ damage, are enriched (for instance, those involving NF-κB or CD28 signalling). Various pathways associated with the cytoskeleton are also enriched, suggestive of a change in cellular growth in an effort to overcome toxic effects and to regenerate damaged tissues. In this regard the enrichment of the proinsulin C-peptide signalling pathway in liver (14 genes, p = 2.4E-5, q = 1.2E-3) involving mTOR and the phosphatidylinositol signalling systems is of note since it has an established role in cellular proliferation and lipid metabolism. Other maps confirmed the induction of intracellular signalling pathways and an influence on the balance between proliferation and apoptosis. The regulation of translation by EIF4F activity (16 genes, p = 1.2E-6, q = 8.1E-5), another mTOR regulated function, is also disturbed.

Fig. 5 Toxicity ontology analysis of genes disturbed in liver and kidneys of Roundup-treated rats. List of top 10 scoring pathway and toxicity process networks revealed by MetaCore analysis of female liver and kidney transcriptome profiles receiving 0.1 ppb of Roundup in drinking water (p <0.01, fold changes >1.1). The p-values are determined by hyper-geometric calculation Full size image

Furthermore, the GO “metabolic process” and “cellular response to stress” processes have low p-values in liver (respectively p = 3.7E-58 and 3.9E-16) and kidneys (p = 1.5E-49 and 1.0E-14), which strongly suggest a state of metabolic stress. Overall, toxicity process analysis revealed gene expression disturbances associated with apoptosis, necrosis, phospholipidosis, mitochondrial membrane dysfunction and ischemia. Thus the alteration in the transcriptome profile identified in this study correlates with the observed increased signs of anatomical and functional pathology of the liver and kidneys.