Abstract Background A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult male zebrafish following an acute 24 hr high dose exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride or sodium dichromate concentrations corresponding to their respective 96 hr LC[20], LC[40] and LC[60]. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal. Results Comparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor (TF) enrichment algorithms revealed a number of key biological processes perturbed by metal poisonings and the master transcriptional regulators mediating gene expression changes. Metal poisoning differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation, and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel poisoning did not induce any morphological alterations in male zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx, and intestinal mucosa. A number of enriched transcription factors mediated the observed gene response to metal poisoning, including known targets such as p53, HIF1α, and the myc oncogene, and novel regulatory factors such as XBP1, GATA6 and HNF3β. Conclusions This work uses an experimentally innovative approach to capture global responses to metal poisoning and provides mechanistic insights into metal toxicity. Keywords: Metals, Toxicity mechanisms, Zebrafish, Whole organism, Nickel, Chromium, Cobalt, Toxicogenomics Background Toxicogenomics is a powerful tool for evaluating toxicity profiles of known and potentially hazardous compounds. The zebrafish, a classic model for developmental toxicity, has recently proven to be an effective model organism for chemical screening [[35]1,[36]2] and environmental sentinel applications, including sewage testing and chemical hazard detection [[37]3-[38]6]. The low husbandry costs, small size, ease of genetic manipulation, and wealth of genome database resources distinguish the zebrafish as a highly promising model organism for toxicological studies. Responses to toxic insults usually affect multiple organs and tissues, supporting a role for gene profiling in the whole animal to evaluate toxic responses. Although whole organism toxicogenomics has routinely been conducted in invertebrate models such as the worm Caenorhabditis elegans and the fly Drosophila melanogaster[[39]7-[40]9] and in ecoindicator species such as Daphnia magna and Pimpephales promelas[[41]10,[42]11], only one study to date has evaluated whole organism gene profiling in adult zebrafish [[43]12]. In this study, robust expression signatures differentiated between potent aryl hydrocarbon and estrogen receptor agonists, and accurately identified target tissues. We hypothesized that gene profiling in whole adult zebrafish could also be used to infer toxic responses to hazardous chemicals. Nickel, cobalt and chromium are environmentally ubiquitous metals with recognized human health hazards [[44]13]. Recently, applications in mining, smelting, industry, medicine, and agriculture have increased the environmental distribution of these metals, elevating elevated exposure risk and incidences of occupational exposure. Nickel, cobalt, or chromium exposure can cause incapacitating acute toxicity and/or long-term damage (e.g., carcinogenesis) [[45]14-[46]16]. Primary mechanisms of metal toxicity include the production of free radicals which can trigger oxidative stress, induce mutagenesis by DNA-metal interactions, and impair protein function by covalently modifying proteins or competing with metal binding sites. Metal-derived reactive oxygen species (ROS) may perturb a number of tightly regulated cellular processes (e.g., cell growth and proliferation), activate transcription factors and genes, and trigger cellular adaptive programs including metal stress response, DNA repair mechanisms, and inflammation [[47]17]. Numerous studies have examined gene responses to acute poisoning by nickel, cobalt, or chromium [[48]18,[49]19], but besides a few studies in invertebrates, most of these studies measure gene responses in isolated tissues or tissue-derived cell lines [[50]8,[51]19-[52]23]. Although analyzing isolated tissues is the ideal approach to unambiguously identify gene changes in an organ of interest, it is experimentally impractical to microdissect and to analyze all potentially affected organs from zebrafish individually. Alternatively, a whole-organism approach with post hoc gene ontology enrichment analysis has the advantage of predicting biomolecular pathways linked to observed histopathologic endpoints for informing later organ-specific experiments. In this study, we used a whole-organism approach exposing adult male zebrafish to increasing concentrations of nickel chloride, cobalt chloride, and sodium dichromate, and evaluating whole genome transcriptional responses using DNA microarrays. We identified differentially regulated biological processes using gene ontology enrichment analysis in order to infer toxicity mechanisms [[53]21]. We also identified transcription factors upstream of the differentially enriched genes which are predicted to directly activate or repress gene expression in order to characterize regulatory processes involved in metal toxicity. Histopathological changes in the whole organism were compared with gene changes. Overall, our study provides (i) insight into transcriptomic changes corresponding to toxic indicators of metal poisoning and (ii) an experimental evaluation of whole organism toxicogenomics in the zebrafish model. Methods Research was conducted in compliance with the United States Animal Welfare Act, and other Federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals (NRC 2011) in facilities that are fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International. Approvals were granted for this study by USACEHR’s Institutional Animal Care and Use Committee. Water quality All husbandry and aquatic exposures are performed using USACEHR’s well water which is processed to ensure proper conditioning of the water supply. The water is supplied from a mix of onsite ground water wells and from municipal tap (domestic) water. Domestic water is used to produce “RO permeate” which is later mixed with raw well water to produce water of appropriate hardness and alkalinity (150 – 210 mg/L as CaCO[3]; 110 – 180 mg/L as CaCO[3] respectively). Domestic water is carbon filtered to remove chlorine levels (maintained below 0.1 mg/L), treated by a water softener, processed through reverse osmosis (RO) membranes then stored for distribution. Prior to use, the RO processed domestic water is blended with well water, filtered through a 10 μm particle filter, carbon filtered, then heated and aerated to near 100% saturation at 25 ± 1°C. This processed water is then passed through another 10 μm particle filter and UV sterilized prior to distribution throughout the facility. This water is continuously monitored to maintain the following ranges: pH = 6.5 – 8.5; alkalinity = 110 – 180 mg/L CaCO[3]; hardness = 150 – 210 mg/L as CaCO[3]; conductivity = 400 – 1000 mS/cm; total ammonia < 0.1 mg/L as NH[3]; dissolved oxygen (DO) = 80 – 100% saturation (6.8 – 8.5 mg/L at 25°C). Contaminant analysis is performed quarterly by our in-house analytical chemistry department as well as annually by an external, certified testing facility. Fish exposures Exposures were conducted using USACEHR well water in 5-gallon glass aquaria adapted for flow-through use (60 mL/min; 5.4 turnovers/day) and maintained at 25°C with a 12 hr:12 hr (light:dark) photoperiod. During both acclimation and exposure periods, water quality for each tank is monitored daily (temperature, pH, alkalinity, hardness, DO, and conductivity (data not shown). We estimated the concentration of each metal necessary for 20% (LC[20]; low), 40% (LC[40]; mid) and 60% lethality (LC[60]; high) in 96 hr range-finding experiments. Exposures were conducted for 24 hr using control (no toxicant) plus the high, mid and low concentrations of each metal (Table [54]1). Our intent with this exposure paradigm was to evaluate levels of toxicant sufficient to induce a measurable intoxication response without producing lethality at 24 hours. Metal concentrations in the test tanks were verified by our analytical chemistry department before and after exposures. Table 1. Nominal versus measured concentrations of metals Treatment Nominal LC[ 20 ](low) Measured LC[ 20 ](low) Nominal LC[ 40 ](mid) Measured LC[ 40 ](mid) Nominal LC[ 60 ](high) Measured LC[ 60 ](high) NiCl[2] __________________________________________________________________ 45 __________________________________________________________________ 42.4 __________________________________________________________________ 54 __________________________________________________________________ 51.0 __________________________________________________________________ 62 __________________________________________________________________ 64.0 __________________________________________________________________ CoCl[2] __________________________________________________________________ 39 __________________________________________________________________ 39.7 __________________________________________________________________ 50 __________________________________________________________________ 46.3 __________________________________________________________________ 65 __________________________________________________________________ 59.5 __________________________________________________________________ Na[2]Cr[2]O[7] 53 56.5 65 69.9 76 80.6 [55]Open in a new tab Nominal and actual concentrations (mg/L) of metals used in the definitive 24-hour zebrafish exposures as estimated from 96-hour range finding studies. Only male zebrafish were included in the analysis because of concern that RNAs encoding vitellogenin and other liver-abundant egg proteins found in breeding females [[56]24,[57]25] might confound global gene expression studies. Therefore, we initially selected 25 adult (6–9 months) presumptive male zebrafish per condition to ensure that 20 male fish were available for subsequent microarray analysis and histopathology. During exposure, animals received a pre-measured quantity of food twice per day (1X flake food, 1X brine shrimp). After the exposure period, fish were euthanized by immersion in a lethal concentration (0.5 g/L, pH 7.2) of MS-222. Five fish per condition were immediately preserved in a modified Davidson’s solution for histological examination. For transcriptional analysis, the remaining 15 zebrafish were immersed whole in liquid nitrogen and stored at -80°C until RNA processing. Histopathology Slides were prepared by Experimental Pathology Laboratories, Inc. (EPL, Inc., Sterling, VA). Briefly, the fish were initially preserved in modified Davidson’s solution, washed in 70% ethanol, and then transferred to 10% neutral buffered formalin for transport. Fish required additional decalcification prior to sectioning and were placed in Formical 2000® decalcification fluid for seven hours. Tails were removed from each fish, followed by processing and embedding in paraffin. Vertical longitudinal sections were obtained at five different levels: 1) left lateral, 2) left paramedian, 3) midline sagittal, 4) right paramedian, and 5) right lateral. Two serial sections were obtained at each level for a total of 10 sections per fish that were H&E stained. The following tissues were evaluated (if present) for each zebrafish: bone (vertebra), brain, corpuscle of Stannius, esophagus, eye, gallbladder, gills, heart, gonad (ovary), gonad (testis), hematopoietic tissue, interrenal tissue, intestine, kidney, liver, mesonephric duct, nares, pancreas, peripheral nerve, pineal organ, pituitary, pseudobranch, skeletal muscle, skin, spinal cord, spleen, stato-acoustic organ, swim bladder, thymus, thyroid, and ultimobranchial body. The following tissues occasionally were not present in the sections that were evaluated: corpuscle of Stannius, esophagus, gallbladder, interrenal tissue, mesonephric duct, pineal organ, pituitary, spleen, thymus, thyroid, and ultimobranchial body. Occasional absence of these tissues is a condition inherent in the sectioning method and did not appear to affect the overall evaluation of the histopathology data. Microarray analysis RNA processing Whole frozen fish were pulverized under liquid nitrogen using a SPEX 6750 freezer mill (SPEX Sample Prep, Metuchen, NJ). Total RNA was isolated from the pulverized material using Trizol® (Invitrogen, Carlsbad, CA) with an extra clarification centrifugation step to remove bone, scales, lipid, and other insoluble debris followed by column purification with RNeasy® Midi kits (Qiagen, GmbH, Germany) to remove residual salt and organic solvents. Total RNA quality and quantity were evaluated using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA) and verified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE). A portion of each total RNA preparation was reverse transcribed into cDNA using the Advantage® RT-for-PCR Kit (Clontech, Mountain View, CA) and screened against a primer panel designed to verify that RNA was isolated from male fish. Specifically, we measured levels of transcripts coding for vitellogenin 1 (vit1, expressed only in female liver, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which was used as an internal control for normalizing the sample RNA and cDNA concentrations. Our initial PCR screen was critical as multiple RNA samples were pooled for microarray analysis (see below) and the presence of female RNA within the pool would complicate analysis. Microarray hybridization To maximize statistical power and minimize cost, we pooled equal amounts of total RNA from four or five fish within each exposure condition to create a biological replicate pool and hybridized each replicate pool to a separate microarray; generating four biological replicate pools per experimental condition for a total of 16 microarrays per toxicant screened (i.e. four control replicates, four low dose replicates, four mid dose replicates and four high dose replicates). Statistical modeling demonstrates that performing microarray analysis on four biological replicates comprised of RNA pooled from five samples approaches the statistical power attained by analyzing 20 individual samples [[58]26]. Numerous theoretical discussions of the pooling procedure can be found in the literature [[59]27-[60]29]. Although pooling eliminates the ability to assess fish-to-fish variability in gene expression, it does provide a statistically powerful approach to identify clear toxicant responses, which is the main focus of the current work. The microarrays used in this study were custom designed in-house using the eArray microarray design tool ([61]https://earray.chem.agilent.com/earray/; Agilent Technologies, Inc.) and manufactured by Agilent. Each array contains 44,000 60-mer oligonucleotides representing 21,904 zebrafish gene targets derived from Ensembl build 46 (Zv7 genome build) and Vega build 26. Two probes were designed per transcript wherever possible; only 94 target transcripts have only one probe. Probes were designed using genes that are annotated, i.e., matched to named genes in the published databases, and represent good coverage of the whole zebrafish genome. Microarrays were processed following Agilent’s One-Color Microarray-Based Gene Expression Analysis Protocol (Version 5.5, February, 2007) for processing 4 x 44 K microarray slides using an initial 1 μg pooled RNA input and an 18 hr overnight hybridization at 65°C. A final step for preventing ozone related degradation of signal using the Stabilization and Drying solution (Agilent Technologies, Inc.) was included after the required specificity washes prior to scanning the arrays. Microarray slides were scanned with a GenePix Autoloader 4200 AL scanner (Molecular Devices, Union City, CA) and raw images processed using GenePix Pro 6.0 (Molecular Devices). All microarray data from this study have been deposited in NCBI’s Gene Expression Omnibus under the accession number [62]GSE50648. Statistical analysis Raw microarray data was analyzed with Partek Genomics Suite software with probe intensities based on the median signal intensity of each feature and signal-to-noise ratio (SNR) data imported from GenePix Pro 6.0. GenePix Pro calculates SNR as the difference between median spot signal and median background divided by the standard deviation of the background signal. Data preprocessing comprised manual inspection of each extracted gene feature and quality control. We selected only unsaturated probes with an SNR greater than or equal to three (SNR ≥ 3) for analysis and performed quantile normalization across arrays to control for inter-array variability. Normalized probe intensities were then log transformed. We also removed probes without Ensembl annotation producing a subset of 15,818 probes which mapped to 7,909 genes (Additional file [63]1: Table S1). We performed three sets of ANOVAs using Partek Genomics Suite to identify probes that were differentially expressed between each treatment group and its respective control. Each set consisted of data from all the replicate pools of fish exposed to a specific metal and the replicate pools of unexposed fish housed in adjacent tanks during the metal exposure. The ANOVA model included terms for treatment (unexposed or exposed), concentration (control, low, mid, or high) and in interaction term for treatment*concentration. Contrasts were performed to determine significance between each concentration and control. We used a step-up Benjamini and Hochberg false discovery rate (FDR) of 0.01 to select differentially expressed probes. An FDR alpha value equal to 0.01 was chosen as the cut-off for the combined datasets of all replicate pools. Probes not meeting this threshold were filtered out and the resultant list was submitted to a second filter specifying a 1.8-fold-difference between treated vs. control samples. Only transcripts for which probes passed these filters were included in the final list (Additional file [64]1: Table S1). Fold changes for each probe pair (single probe transcripts excluded) were then averaged to generate a single value for each transcript. Gene Ontology (GO) enrichment analysis was performed using the web-based tool GOTree Machine (GOTM; [65]http://genereg.ornl.gov/gotm/), which generates a tree-like structure to navigate the GO Directed Acyclic Graph for input gene sets. GOTM supports analysis of the zebrafish genome; however, this analysis had to be performed at the gene rather than the transcript level. GO term and KEGG pathway ([66]http://bioinfo.vanderbilt.edu/webgestalt/) enrichment analyses were then performed on the secondary lists to determine biological processes that are significantly (FDR = 0.1) enhanced or depressed by each metal. The enriched GO terms and KEGG pathways were also manually annotated into top level biological categories to clarify the overarching biological “themes” related to metal-induced gene perturbations. Finally, transcription factor enrichment was performed using MetaCore’s algorithm (GeneGo, Inc.) with settings enabled for identification of node relationships encompassing only direct downstream transcriptional regulation. Zebrafish genes were mapped to their human homologs using the Biomart feature in Ensembl with genes mapping one-to-many discarded from subsequent analysis. Background reference sets comprised the set of all transcripts with SNR ≥ 3 in each platform that could be subsequently mapped in a one-to-one fashion to their human homologs. Thresholds were set at an FDR = 0.1 with at least three DEG target identified for each enriched transcription factor. Metacore does not explicitly provide zebrafish transcription factor regulatory networks; however, we anticipated that there would be high degree of conservation between the zebrafish and human networks [[67]30], and mapped the zebrafish DEGs to their human orthologs before performing enrichment. Each differentially expressed transcript was mapped to its corresponding gene using the Ensembl database and resultant gene lists queried against their appropriate reference gene lists. Background reference sets comprised the set of all transcripts with SNR ≥ 3 in each exposure condition that could be subsequently mapped to Ensembl genes. Significantly enriched GO terms, or those GO terms that are statistically over-represented in each treatment compared to the reference set, were determined using the hypergeometric test with p-values adjusted using the Benjamini & Hochberg FDR correction (α = 0.1) and setting a threshold for the minimum number of genes per category (n = 3). While many statistical tests have been used for GO enrichment evaluation, the hypergeometric distribution provides an appropriate method for modeling data in which genes can be selected only once, i.e. sampling without replacement, as occurs in GO enrichment analysis [[68]31]. Results and discussion Gross changes, behavior, and histopathology Figure [69]1 provides a schematic of the experimental paradigm. During range-finding metal exposure studies, fish were qualitatively assessed for gross changes in behavior and general appearance. At study termination, tissues were stained with hematoxylin and eosin (H&E) to determine the morphological changes associated with each metal poisoning at the concentrations listed in Table [70]1. Figure 1. Figure 1 [71]Open in a new tab Schematic of experimental design. Diagram of the experimental design used for rangefinding and exposures. Nickel-exposed fish appeared fuzzy, an appearance which is generally attributable to the excretion of mucus from goblet cells following irritation (personal communication, Dr. Donald K. Nichols). There were no deaths observed at any of the nickel concentrations nor did any of the nickel concentrations lead to any discernible histopathologic alterations (Table [72]2) although behavioral differences and skin abnormalities were qualitatively different between control and treated fish (i.e. sluggish swimming and fuzzy skin appearance). Previous studies indicate that gills, liver, and kidney are histopathological targets of nickel poisoning [[73]22,[74]23,[75]32-[76]34], but differences in fish species, exposure time and concentration could account for the discrepancy in the histopathological endpoints. Table 2. Summary of histopathology Treatment Morphological alterations NiCl[2] __________________________________________________________________ No significant changes observed in any tissues examined. __________________________________________________________________ CoCl[2] __________________________________________________________________ Acute damage only to the olfactory organs including various inflammatory, degenerative, metaplastic, and necrotic lesions extending from the nasal cavity to the lungs. Intranasal lesions include olfactory epithelium necrosis (5 L, 5 M, 5H), lymphocytic inflammation (3 L, 4 M, 4 H), reactive hyperplasia (0 L, 0 M, 1 H), and olfactory lamellae fusion (0 L, 1 M, 3 H). __________________________________________________________________ Na[2]Cr[2]O[7] Acute damage only to the gills, intestine, and pharynx. Gills exhibited multifocal lesions consisting of lamellar fusion (4 L, 4 M, 5 H), epithelial hyperplasia (3 L, 4 M, 5 H), mononuclear cell infiltration (1 C, 4 L, 4 M, 5 H), epithelial necrosis (0 L, 4 M, 5 H), presence of thrombi in vessels (0 L, 4 M, 3 H) and hemorrhage (0 L, 2 M, 3 H). Intestine exhibits moderate to moderately severe atrophy of the mucosal folds (5 L, 5 M, 5 H), mild mononuclear infiltration of the lamina propria (5 L, 5 M, 5H) and mild necrosis of mucosal epithelium (2 L, 3 M, 3 H). Pharynx exhibits epithelial atrophy characterized by decreased mucosal thickness and loss of mucous secreting cells; also accompanied by mononuclear cell infiltration (0 L, 2 M, 4 H). Necrosis of pharyngeal epithelium occurred in 1 H. [77]Open in a new tab Histopathological perturbations in zebrafish exposed to nickel, cobalt, or chromium for 24 hours. A total of five fish were examined per concentration including the control. In each case the numbers of instances of a finding are indicated in parentheses. C = control, L = low, M = mid and H = high concentration. Chromium poisoning caused visible changes in fish behavior and general appearance, including sluggish swimming, gasping and ulcerations near the tail in some fish. Four zebrafish died at the highest dose. Exposure to all three concentrations of chromium histopathologically affected the gills, intestine, and pharynx (see Table [78]2 and Figure [79]2A). Both gill and pharynx epithelium exhibited mononuclear cell infiltration, which is indicative of acute inflammation. The most prominent change in intestine was moderate to moderately severe atrophy of the mucosal folds and a mild infiltration of the intestinal lamina propria with mononuclear cells. Earlier studies report that the gills, kidney, and liver are histopathological targets of hexavalent chromium exposure in several species of freshwater fish [[80]35,[81]36]. The histopathology associated with this study supports the hypothesis that chromium exposure affects certain physiological processes including respiration, metabolic regulation, and possibly feeding. Figure 2. [82]Figure 2 [83]Open in a new tab Histopathology of affected tissues. Histopathology of affected tissues after (A) chromium – pharynx, intestine, and gill or (B) cobalt – nares and olfactory lamellae – exposure compared to unexposed controls (left panels). Hematoxylin and eosin staining. Arrows indicate regions with metal-induced pathology. During the cobalt range-finding studies, zebrafish exposed to high doses showed less schooling behavior, more surfacing, and less overall movement than the controls. In zebrafish, these behaviors are usually indicative of abnormal respiration and physiological stress. There were six deaths at the high dose and one death at the mid dose. Histopathology confirmed that zebrafish exposed to all three concentrations of cobalt presented with respiratory tract lesions specific to the olfactory epithelium (see Figure [84]2B and Table [85]2). The gill epithelium was unaffected, suggesting that the olfactory epithelial injury is specific to cobalt rather than a nonspecific reaction to waterborne irritants. Consistent with these results, inhalation studies in rats and mice confirm cobalt-specific morphological damage to the olfactory epithelia [[86]37]. NOEL (no-observable-effect-level) could not be determined for any of the structural changes observed in fish treated with cobalt at the concentrations we tested. Transcriptomic responses to metal poisonings To identify differentially expressed genes (DEGs) resulting from nickel, cobalt, or chromium exposure, we compared the expression of genes in whole fish exposed to each metal with unexposed controls. Chromium, cobalt, or nickel exposure significantly altered expression of 696, 461, and 287 genes, respectively (Figure [87]3). There was a steep concentration-response relationship between mortality and metal concentration, with relatively little difference in the gene response to the three poisoning concentrations in the surviving fish as assessed by principal components analysis and analysis of variance (ANOVA). The concentrations were chosen to evoke histopathologic changes in an acute setting. Thus, the observed lack of an appreciative concentration-response curve would result if the doses fell in a nearly vertical region of the typical sigmoidal dose–response curve. Dose-dependent effects may have been lost in experimental variation. Lower doses or a longer time interval may be necessary to elicit a true dose–response curve. Figure 3. [88]Figure 3 [89]Open in a new tab Observed genes altered in expression. Total number of observed genes significantly (FDR = 0.01) altered in expression by at least 1.8 fold in response to chromium (Cr), cobalt (Co), or nickel (Ni) versus untreated controls. Biological processes perturbed by metal exposures DEG lists were further subdivided into secondary lists by direction of response: for example, of the 287 genes induced by nickel, 97 were down-regulated and 190 were up-regulated. GO term enrichment analysis and KEGG pathway enrichment analysis inferred biological processes modified by each metal. The biological processes identified in the gene ontology enrichment analyses (GO biological processes and KEGG pathways) fell into five categories with differential responses for the three metals: protein synthesis and translation; altered reduction-oxidation (redox) levels; inflammation and acute phase stress response; cell cycle regulation and apoptosis; and metabolic depression (Figure [90]4 and Table [91]3). Table [92]3 summarizes the biological processes, chemistries and toxicities and results of pathway enrichment analysis for metals compared to histopathologic findings. Biological processes identified in the transcriptomic analysis were compared at the pathway level to published mechanisms of aquatic toxicology. In general, metals affect the cellular heme content, impairing oxidative function of cells [[93]13,[94]38-[95]40]. Consistent with this observation, all three metals showed down-regulation in biological processes associated with the oxidative stress response, including oxidation-reduction (Figure [96]4A) and metabolic pathways in the oxidative stress response (Figure [97]4B). Further, all three metals induced genes in GO biological processes (most notably ribosome biogenesis) and KEGG pathways regulating protein synthesis and translation (Figure [98]4A and B). Increased demand for newly synthesized proteins may result from enhanced requirements for translation of stress responsive genes particularly those involved in combating oxidative and inflammatory stress. It is also possible that the enhanced protein synthesis is a compensatory mechanism to replenish cells lost through apoptosis in tissues specifically targeted by metal exposures. The literature is conflicting regarding ribosome biogenesis in response to toxicant poisoning [[99]41-[100]46]. The discrepancies in our study between metals may reflect differences in the severity of toxic insult among the metals. Figure 4. [101]Figure 4 [102]Open in a new tab Enrichment analyses. A – Gene ontology term enrichment analysis for biological processes that are significantly (FDR = 0.1) enriched for up-regulated or down-regulated gene sets with at least 3 genes/transcripts present in a category to be considered significant by each metal. NOTE: The yellow highlight indicates that the enriched GO term “metabolic process” contained both up-regulated and down-regulated gene sets. B – KEGG pathway enrichment analysis for biological processes that are significantly (FDR = 0.1) enriched for up-regulated or down-regulated gene sets with at least 3 genes/transcripts present in a category to be considered significant by each metal. Metals listed on each heat map from left to right: nickel (Ni), cobalt (Co) and chromium (Cr) respectively. Color scale is log p-value of enrichment. White is for up-regulated gene sets and blue for down-regulated gene sets. Table 3. Comparison of histopathology, chemistry, toxicity and observed gene response among metals Metal Predicted Histopathology target GO biological process KEGG pathway Genes References