Update and Extension of Release Criteria for Canine 117m Sn Treatments.

ABSTRACT: Tin-117 m ( 117m Sn) is used to treat dogs with osteoarthritic joints by radiosynoviorthesis. The internal conversion and Auger electrons emitted by the 117m Sn provide the therapeutic effect. Sn-117 m also emits x rays and gamma rays, of which the most significant is 158.6 keV. Accurate information regarding the interactions of a person with a treated dog is needed to determine the person's total dose and thus regulatory compliance; i.e., a time and motion study. Prior studies have characterized the radiation field emitted by a treated dog, determined the effective dose rates to a person based on those radiation fields, and evaluated dog-human interactions. These studies have been tied together to calculate the prospective dose to the owner of a treated dog. The behavior modifications needed to comply with public dose limits were identified, and a template for written instructions limiting dose was developed. Further calculations based on the written instructions were made to determine the necessary duration of the instructions. The result is guidance that may be used by veterinary practitioners to release treated dogs in accordance with the public dose limits.

Age-dependent Radiation Dose Rates from Canine Sn-117m Treatments.

ABSTRACT: Tin-117 m (Sn-117m) is used to treat dogs with osteoarthritic joints by radiosynoviorthesis. The decay process for Sn-117m is internal conversion wherein IC electrons and auger electrons provide the therapeutic effect. Additionally, the most prominent gamma emission is 158.6 keV. The effective dose rate received by a person interacting at close distances with a treated dog is needed to determine the person's total dose and thus regulatory compliance. Simple measurement of the dose rate at a given distance does not provide an accurate measurement of the effective dose to a person due to the non-uniform nature of the radiation field at close distances. MNCP models of the interactions of five ages of humans at three distances were created to determine the effective dose rates using the methodology from NRC Regulatory Guide 8.40. Ratios of the effective dose rate to the person to the measured dose rate at 1 m from the same source were calculated.

Anisotropy of the Radiation Field Following Canine Sn-117m Treatment.

ABSTRACT: Tin-117m (117mSn) is used to treated dogs with osteoarthritic joints by radiosynoviorthesis. The internal conversion and Auger electrons emitted by the 117mSn provide the therapeutic effect. Sn-117m also emits gamma rays, of which the most significant is 158.6 keV. The external radiation field around a treated dog is of interest to limit the dose to the owners/caretakers of the dog. The dog's torso attenuates the radiation being emitted toward the opposite side of the dog's body. This leads to a radiation field that is significantly non-isotropic. This study characterizes the anisotropy of this field to permit maximum dose rate measurements to be used to calculate the dose to individuals in the vicinity of the dog. Measurements were made in nine directions and at two distances, 0.3 and 1.0 m, to characterize common distances and spatial orientations for human-dog interactions. From these measurements, the percent reduction in the average dose rate compared to the maximum dose rate was determined. From a radiation safety perspective, the important factor is the minimum amount of shielding effectiveness or percent reduction that can be expected. A reasonable measure for this value is the fifth percentile of the shielding effectiveness distribution. The fifth percentile shielding effectiveness measures are 27% and 21% at 0.3 and 1.0 m, respectively.

Right Ventricle Has Normal Myofilament Function But Shows Perturbations in the Expression of Extracellular Matrix Genes in Patients With Tetralogy of Fallot Undergoing Pulmonary Valve Replacement.

Background Patients with repair of tetralogy of Fallot (rToF) who are approaching adulthood often exhibit pulmonary valve regurgitation, leading to right ventricle (RV) dilatation and dysfunction. The regurgitation can be corrected by pulmonary valve replacement (PVR), but the optimal surgical timing remains under debate, mainly because of the poorly understood nature of RV remodeling in patients with rToF. The goal of this study was to probe for pathologic molecular, cellular, and tissue changes in the myocardium of patients with rToF at the time of PVR. Methods and Results We measured contractile function of permeabilized myocytes, collagen content of tissue samples, and the expression of mRNA and selected proteins in RV tissue samples from patients with rToF undergoing PVR for severe pulmonary valve regurgitation. The data were compared with nondiseased RV tissue from unused donor hearts. Contractile performance and passive stiffness of the myofilaments in permeabilized myocytes were similar in rToF-PVR and RV donor samples, as was collagen content and cross-linking. The patients with rToF undergoing PVR had enhanced mRNA expression of genes associated with connective tissue diseases and tissue remodeling, including the small leucine-rich proteoglycans ASPN (asporin), LUM (lumican), and OGN (osteoglycin), although their protein levels were not significantly increased. Conclusions RV myofilaments from patients with rToF undergoing PVR showed no functional impairment, but the changes in extracellular matrix gene expression may indicate the early stages of remodeling. Our study found no evidence of major damage at the cellular and tissue levels in the RV of patients with rToF who underwent PVR according to current clinical criteria.

Gene expression profiling in the developing secondary palate in the absence of Tbx1 function.

BACKGROUND: Microdeletion of chromosome 22q11 is associated with significant developmental anomalies, including disruption of the cardiac outflow tract, thymic/parathyroid aplasia and cleft palate. Amongst the genes within this region, TBX1 is a major candidate for many of these developmental defects. Targeted deletion of Tbx1 in the mouse has provided significant insight into the function of this transcription factor during early development of the cardiac and pharyngeal systems. However, less is known about its role during palatogenesis. To assess the influence of Tbx1 function on gene expression profile within the developing palate we performed a microarray screen using total RNA isolated from the secondary palate of E13.5 mouse embryos wild type, heterozygous and mutant for Tbx1. RESULTS: Expression-level filtering and statistical analysis revealed a total of 577 genes differentially expressed across genotypes. Data were clustered into 3 groups based on comparison between genotypes. Group A was composed of differentially expressed genes in mutant compared to wild type (n = 89); Group B included differentially expressed genes in heterozygous compared to wild type (n = 400) and Group C included differentially expressed genes in mutant compared to heterozygous (n = 88). High-throughput quantitative real-time PCR (RT-PCR) confirmed a total of 27 genes significantly changed between wild type and mutant; and 27 genes between heterozygote and mutant. Amongst these, the majority were present in both groups A and C (26 genes). Associations existed with hypertrophic cardiomyopathy, cardiac muscle contraction, dilated cardiomyopathy, focal adhesion, tight junction and calcium signalling pathways. No significant differences in gene expression were found between wild type and heterozygous palatal shelves. CONCLUSIONS: Significant differences in gene expression profile within the secondary palate of wild type and mutant embryos is consistent with a primary role for Tbx1 during palatogenesis.

Oxygen gradients can determine epigenetic asymmetry and cellular differentiation via differential regulation of Tet activity in embryonic stem cells.

Graded levels of molecular oxygen (O2) exist within developing mammalian embryos and can differentially regulate cellular specification pathways. During differentiation, cells acquire distinct epigenetic landscapes, which determine their function, however the mechanisms which regulate this are poorly understood. The demethylation of 5-methylcytosine (5mC) is achieved via successive oxidation reactions catalysed by the Ten-Eleven-Translocation (Tet) enzymes, yielding the 5-hydroxymethylcytosine (5hmC) intermediate. These require O2 as a co-factor, and hence may link epigenetic processes directly to O2 gradients during development. We demonstrate that the activities of Tet enzymes display distinct patterns of [O2]-dependency, and that Tet1 activity, specifically, is subject to differential regulation within a range of O2 which is physiologically relevant in embryogenesis. Further, differentiating embryonic stem cells displayed a transient burst of 5hmC, which was both dependent upon Tet1 and inhibited by low (1%) [O2]. A GC-rich promoter region within the Tet3 locus was identified as a significant target of this 5mC-hydroxylation. Further, this region was shown to associate with Tet1, and display the histone epigenetic marks, H3K4me3 and H3K27me3, which are characteristic of a bivalent, developmentally 'poised' promoter. We conclude that Tet1 activity, determined by [O2] may play a critical role in regulating cellular differentiation and fate in embryogenesis.

Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents.

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 µg/L or 59 µM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol-1) compared to estradiol (-25.79 kcal mol-1), which suggests that activation of this receptor by glyphosate is via a ligand-independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERα in vitro, albeit at relatively high concentrations.

Editor's Highlight: Transcriptome Profiling Reveals Bisphenol A Alternatives Activate Estrogen Receptor Alpha in Human Breast Cancer Cells.

Plasticizers with estrogenic activity, such as bisphenol A (BPA), have potential adverse health effects in humans. Due to mounting evidence of these health effects, BPA is being phased out and replaced by other bisphenol variants in "BPA-free" products. We have compared estrogenic activity of BPA with 6 bisphenol analogues [bisphenol S (BPS); bisphenol F (BPF); bisphenol AP (BPAP); bisphenol AF (BPAF); bisphenol Z (BPZ); bisphenol B (BPB)] in 3 human breast cancer cell lines. Estrogenicity was assessed (10-11-10-4 M) by cell growth in an estrogen receptor (ER)-mediated cell proliferation assay, and by the induction of estrogen response element-mediated transcription in a luciferase assay. BPAF was the most potent bisphenol, followed by BPB > BPZ ∼ BPA > BPF ∼ BPAP > BPS. The addition of ICI 182,780 antagonized the activation of ERs. Data mining of ToxCast high-throughput screening assays confirm our results but also show divergence in the sensitivities of the assays. Gene expression profiles were determined in MCF-7 cells by microarray analysis. The comparison of transcriptome profile alterations resulting from BPA alternatives with an ERα gene expression biomarker further indicates that all BPA alternatives act as ERα agonists in MCF-7 cells. These results were confirmed by Illumina-based RNA sequencing. In conclusion, BPA alternatives are not necessarily less estrogenic than BPA in human breast cancer cells. BPAF, BPB, and BPZ were more estrogenic than BPA. These findings point to the importance of better understanding the risk of adverse effects from exposure to BPA alternatives, including hormone-dependent breast cancer.

Differences in the coronal proteome acquired by particles depositing in the lungs of asthmatic versus healthy humans.

Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.

Transcriptome and metabolome analysis of liver and kidneys of rats chronically fed NK603 Roundup-tolerant genetically modified maize.

BACKGROUND: A previous 2-year rat feeding trial assessing potential toxicity of NK603 Roundup-tolerant genetically modified maize revealed blood and urine biochemical changes indicative of liver and kidney pathology. In an effort to obtain deeper insight into these findings, molecular profiling of the liver and kidneys from the same animals was undertaken. RESULTS: Transcriptomics showed no segregation of NK603 maize and control feed groups with false discovery rates ranging from 43 to 83% at a cut-off p value of 1%. Changes in gene expression were not reflective of liver and kidney toxic effects. Metabolomics identified 692 and 673 metabolites in kidney and liver, respectively. None of the statistically significant disturbances detected (12-56 for different test groups) survived a false discovery rate analysis. Differences in these metabolites between individual animals within a group were greater than the effect of test diets, which prevents a definitive conclusion on either pathology or safety. CONCLUSIONS: Even if the biological relevance of the statistical differences presented in this study is unclear, our results are made available for scrutiny by the scientific community and for comparison in future studies investigating potential toxicological properties of the NK603 corn.

Evaluation of the Myocilin Mutation Gln368Stop Demonstrates Reduced Penetrance for Glaucoma in European Populations.

PURPOSE: Sequence variations in the myocilin (MYOC) gene account for approximately 2% to 4% of glaucoma cases. One particular MYOC mutation, Gln368Stop (dbSNP accession number: rs74315329), is the most common genetic mutation causing glaucoma by increasing intraocular pressure (IOP). The objective of this study was to evaluate the effect of this MYOC mutation on IOP using data from large-scale European population panels (directly sequenced and imputation based). DESIGN: Cross-sectional, cohort study. PARTICIPANTS: For this study, the penetrance of the variant rs74315329 was estimated in 2 population-based cohorts, the TwinsUK (N = 6092) and the Rotterdam Study (RS) (N =11 189). METHODS: Carriers of the risk allele for rs74315329 were identified using whole-genome sequencing and imputation data (based on 1000 Genomes Project and Haplotype Reference Consortium panels). The penetrance of this variant was evaluated using IOP measurements and data on visual field testing/a diagnosis of glaucoma (if available). MAIN OUTCOME MEASURES: The penetrance of the variant rs74315329 was estimated from the percentage of the carriers of the risk allele of the variant who had high IOP (ocular hypertension) or glaucoma. RESULTS: In our study, the observed penetrance of the variant rs74315329 in relation to increased IOP was 12.5% and 19.4% in the TwinsUK and the RS, respectively. Thus, our study suggests a much lower penetrance for rs74315329 for ocular hypertension (and thus glaucoma), in comparison with that reported previously. CONCLUSIONS: The significance of this finding is that higher numbers of healthy individuals in the population are expected to be carriers of this mutation, which in turn reduces the utility of identifying carriers of this mutation as a screening tool for glaucoma.

Induced Pluripotent Stem Cell Differentiation and Three-Dimensional Tissue Formation Attenuate Clonal Epigenetic Differences in Trichohyalin.

The epigenetic background of pluripotent stem cells can influence transcriptional and functional behavior. Most of these data have been obtained in standard monolayer cell culture systems. In this study, we used exome sequencing, array comparative genomic hybridization (CGH), miRNA array, DNA methylation array, three-dimensional (3D) tissue engineering, and immunostaining to conduct a comparative analysis of two induced pluripotent stem cell (iPSC) lines used in engineering of 3D human epidermal equivalent (HEE), which more closely approximates epidermis. Exome sequencing and array CGH suggested that their genome was stable following 3 months of feeder-free culture. While the miRNAome was also not affected, ≈7% of CpG sites were differently methylated between the two lines. Analysis of the epidermal differentiation complex, a region on chromosome 1 that contains multiple genes involved in skin barrier maturation (including trichohyalin, TCHH), found that in one of the iPSC clones (iKCL004), TCHH retained a DNA methylation signature characteristic of the original somatic cells, whereas in other iPSC line (iKCL011), the TCHH methylation signature matched that of the human embryonic stem cell line KCL034. The difference between the two iPSC clones in TCHH methylation did not have an obvious effect on its expression in 3D HEE, suggesting that differentiation and tissue formation may mitigate variations in the iPSC methylome.

T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

The NLRP3 inflammasome controls interleukin-1ß maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1ß secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses.

Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner.

BACKGROUND: The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. METHODS: Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. RESULTS: Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. CONCLUSIONS: We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

Quantifying the magnitude of the oxygen artefact inherent in culturing airway cells under atmospheric oxygen versus physiological levels.

To date, in vitro studies assessing the pulmonary toxicity of inhaled particles have provided poor correlation with in vivo results. We explored whether this discrepancy reflected cellular adaptations in pulmonary cells cultured under atmospheric oxygen concentrations (21%) compared with in vivo alveolar concentrations (100 mm Hg, ~ 13%) and whether this blunted cellular responses to nanoparticle challenge. At 21% oxygen, A549 cells had augmented intracellular glutathione concentrations, with evidence of increased tolerance to CuO nanoparticles, with reduced reactive oxygen species production, blunted transcriptional responses and delayed cell death, compared to cells cultured at 13% oxygen. These data support the contention that standard cell culture conditions pre-adapt cells to oxidative insults and emphasize the necessity of ensuring normoxic conditions in model systems to improve their predictive value.

Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels.

The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2-Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.

Application of the pMHC Array to Characterise Tumour Antigen Specific T Cell Populations in Leukaemia Patients at Disease Diagnosis.

Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms' Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8-1.4 x 10(6)). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1(126-134) (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1(950-958) epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.

Transcriptome profile analysis reflects rat liver and kidney damage following chronic ultra-low dose Roundup exposure.

BACKGROUND: Glyphosate-based herbicides (GBH) are the major pesticides used worldwide. Converging evidence suggests that GBH, such as Roundup, pose a particular health risk to liver and kidneys although low environmentally relevant doses have not been examined. To address this issue, a 2-year study in rats administering 0.1 ppb Roundup (50 ng/L glyphosate equivalent) via drinking water (giving a daily intake of 4 ng/kg bw/day of glyphosate) was conducted. A marked increased incidence of anatomorphological and blood/urine biochemical changes was indicative of liver and kidney structure and functional pathology. In order to confirm these findings we have conducted a transcriptome microarray analysis of the liver and kidneys from these same animals. RESULTS: The expression of 4224 and 4447 transcript clusters (a group of probes corresponding to a known or putative gene) were found to be altered respectively in liver and kidney (p < 0.01, q < 0.08). Changes in gene expression varied from -3.5 to 3.7 fold in liver and from -4.3 to 5.3 in kidneys. Among the 1319 transcript clusters whose expression was altered in both tissues, ontological enrichment in 3 functional categories among 868 genes were found. First, genes involved in mRNA splicing and small nucleolar RNA were mostly upregulated, suggesting disruption of normal spliceosome activity. Electron microscopic analysis of hepatocytes confirmed nucleolar structural disruption. Second, genes controlling chromatin structure (especially histone-lysine N-methyltransferases) were mostly upregulated. Third, genes related to respiratory chain complex I and the tricarboxylic acid cycle were mostly downregulated. Pathway analysis suggests a modulation of the mTOR and phosphatidylinositol signalling pathways. Gene disturbances associated with the chronic administration of ultra-low dose Roundup reflect a liver and kidney lipotoxic condition and increased cellular growth that may be linked with regeneration in response to toxic effects causing damage to tissues. Observed alterations in gene expression were consistent with fibrosis, necrosis, phospholipidosis, mitochondrial membrane dysfunction and ischemia, which correlate with and thus confirm observations of pathology made at an anatomical, histological and biochemical level. CONCLUSION: Our results suggest that chronic exposure to a GBH in an established laboratory animal toxicity model system at an ultra-low, environmental dose can result in liver and kidney damage with potential significant health implications for animal and human populations.