Multiomics reveal non-alcoholic fatty liver disease in rats following chronic exposure to an ultra-low dose of Roundup herbicide

The impairment of liver function by low environmentally relevant doses of glyphosate-based herbicides (GBH) is still a debatable and unresolved matter. Previously we have shown that rats administered for 2 years with 0.1 ppb (50 ng/L glyphosate equivalent dilution; 4 ng/kg body weight/day daily intake) of a Roundup GBH formulation showed signs of enhanced liver injury as indicated by anatomorphological, blood/urine biochemical changes and transcriptome profiling. Here we present a multiomic study combining metabolome and proteome liver analyses to obtain further insight into the Roundup-induced pathology. Proteins significantly disturbed (214 out of 1906 detected, q < 0.05) were involved in organonitrogen metabolism and fatty acid β-oxidation. Proteome disturbances reflected peroxisomal proliferation, steatosis and necrosis. The metabolome analysis (55 metabolites altered out of 673 detected, p < 0.05) confirmed lipotoxic conditions and oxidative stress by showing an activation of glutathione and ascorbate free radical scavenger systems. Additionally, we found metabolite alterations associated with hallmarks of hepatotoxicity such as γ-glutamyl dipeptides, acylcarnitines, and proline derivatives. Overall, metabolome and proteome disturbances showed a substantial overlap with biomarkers of non-alcoholic fatty liver disease and its progression to steatohepatosis and thus confirm liver functional dysfunction resulting from chronic ultra-low dose GBH exposure

Stat2 loss disrupts damage signalling and is protective in acute pancreatitis

The severity of sterile inflammation, as seen in acute pancreatitis, is determined by damage-sensing receptors, signalling cascades and cytokine production. Stat2 is a type I interferon signalling mediator that also has interferon-independent roles in murine lipopolysaccharide-induced NF-κB-mediated sepsis. However, its role in sterile inflammation is unknown. We hypothesised that Stat2 determines the severity of non-infective inflammation in the pancreas. Wild type (WT) and Stat2-/- mice were injected intraperitoneally with caerulein or L-arginine. Specific cytokine-blocking antibodies were used in some experiments. Pancreata and blood were harvested 1 h and 24 h after the final dose of caerulein and up to 96 h post L-arginine. Whole-tissue phosphoproteomic changes were assessed using label-free mass spectrometry. Tissue-specific Stat2 effects were studied in WT/Stat2-/- bone-marrow chimera and using Cre-lox recombination to delete Stat2 in pancreatic and duodenal homeobox 1(Pdx1)-expressing cells. Stat2-/- mice were protected from caerulein- and L-arginine-induced pancreatitis. Protection was independent of type I interferon signalling. Stat2-/- mice had lower cytokine levels including TNFα and IL-10 and reduced NF-kB nuclear localisation in pancreatic tissue compared to WT. Inhibition of TNFα improved (inhibition of IL-10 worsened) caerulein-induced pancreatitis in WT but not Stat2-/- mice. Phosphoproteomics showed down-regulation of mitogen-activated protein kinase (MAPK) mediators but accumulation of Ser412-phosphorylated Tak1. Stat2 deletion in Pdx1-expressing acinar cells (Stat2flox/Pdx1-cre ) reduced pancreatic TNFα expression, but not histological injury or serum amylase. WT/Stat2-/- bone-marrow chimera mice were protected from pancreatitis irrespective of host or recipient genotype. Stat2 loss results in disrupted signalling in pancreatitis, upstream of NF-κB in non-acinar and/or bone marrow derived cells. This article is protected by copyright. All rights reserved

The effects of hydroxycarbamide on the plasma proteome of children with sickle cell anaemia

We investigated changes in the plasma proteome of children with sickle cell anaemia (SCA) associated with hydroxycarbamide (HC) use, to further characterize the actions of HC. Fifty‐one children with SCA consented to take part in this study. Eighteen were taking HC at a median dose of 22 mg/kg, and 33 were not on HC. Plasma was analysed using an unbiased proteomic approach and a panel of 92 neurological biomarkers. HC was associated with increased haemoglobin (Hb) (89·8 vs. 81·4 g/l, P = 0·007) and HbF (6·7 vs. 15·3%, P 1·3 increased concentration. HC use was associated with reduced haemolysis (lower α, β, δ globin chains, haptoglobin‐related protein, complement C9; higher haemopexin), reduced inflammation (lower α‐1‐acid glycoprotein, CD5 antigen‐like protein, ceruloplasmin, factor XII, immunoglobulins, cysteine‐rich secretory protein 3, vitamin D‐binding protein) and decreased activation of coagulation (lower factor XII, carboxypeptidase B2, platelet basic protein). There was a significant correlation between the increase in HbF% on HC and haemopexin levels (r = 0·603, P = 0·023). This study demonstrated three ways in which HC may be beneficial in SCA, and identified novel proteins that may be useful to monitor therapeutic response

An integrated multi-omics analysis of the NK603 Roundup-tolerant GM maize reveals metabolism disturbances caused by the transformation process

Glyphosate tolerant genetically modified (GM) maize NK603 was assessed as ‘substantially equivalent’ to its isogenic counterpart by a nutrient composition analysis in order to be granted market approval. We have applied contemporary in depth molecular profiling methods of NK603 maize kernels (sprayed or unsprayed with Roundup) and the isogenic corn to reassess its substantial equivalence status. Proteome profiles of the maize kernels revealed alterations in the levels of enzymes of glycolysis and TCA cycle pathways, which were reflective of an imbalance in energy metabolism. Changes in proteins and metabolites of glutathione metabolism were indicative of increased oxidative stress. The most pronounced metabolome differences between NK603 and its isogenic counterpart consisted of an increase in polyamines including N-acetyl-cadaverine (2.9-fold), N-acetylputrescine (1.8-fold), putrescine (2.7-fold) and cadaverine (28-fold), which depending on context can be either protective or a cause of toxicity. Our molecular profiling results show that NK603 and its isogenic control are not substantially equivalent

The effects of hydroxycarbamide on the plasma proteome of children with sickle cell anaemia

We have investigated changes in the plasma proteome of children with sickle cell anaemia (SCA) associated with hydroxycarbamide (HC) use, to further characterize the actions of HC. 51 children with SCA consented to take part in this study. 18 were taking HC at a median dose of 22mg/kg, and 33 were not on HC. Plasma was analyzed using an unbiased proteomic approach and a panel of 92 neurological biomarkers. HC was associated with increased haemoglobin (Hb) (89.8g/l vs 81.4g/l, P=0.007) and HbF (15.3 vs 6.7%, P&lt;0.001). 17 proteins were decreased on HC compared to controls by a factor of &lt;0.77, and six proteins showed &gt;1.3 fold increased concentration. HC use was associated with reduced haemolysis (lower globin chains, haptoglobin related protein, complement C9; higher hemopexin), reduced inflammation (lower -1-acid glycoprotein, CD5 antigen-like protein, ceruloplasmin, factor XII, immunoglobulins, cysteine-rich secretory protein 3, vitamin D-binding protein) and decreased activation of coagulation (lower factor XII, carboxypeptidase B2, platelet basic protein). There was a significant correlation between the increase in HbF% on HC and hemopexin levels (R=0.603, P=0.023). This study demonstrated three ways in which HC may be beneficial in SCA, and identified novel proteins, which may be useful to monitor therapeutic response

Proteomic analysis of plasma from children with sickle cell anemia and silent cerebral infarction

Silent cerebral infarction is the most common neurological abnormality in children with sickle cell anemia, affecting 30-40% of 14 year olds. There are no known biomarkers to identify children with silent cerebral infarcts, and the pathological basis is also unknown. We used an unbiased proteomic discovery approach to identify plasma proteins differing in concentration between children with and without silent cerebral infarcts. Clinical parameters and plasma samples were analysed from 51 children (mean age 11.8 years, range 6-18) with sickle cell anemia (HbSS). A total of 19 children had silent cerebral infarcts and 32 normal MRI; the children with silent infarcts had lower HbF levels (8.6 vs. 16.1%, P=0.049) and higher systolic blood pressures (115 vs. 108.6, P=0.027). Plasma proteomic analysis showed 13 proteins increased more than 1.3 fold in the SCI patients, including proteins involved in hypercoagulability (α2-antiplasmin, fibrinogen−γ chain, thrombospondin-4), inflammation (α2-macroglobulin, complement C1s and C3), and atherosclerosis (apolipoprotein B-100). Higher levels of gelsolin and retinol-binding protein 4 were also found in the population with silent infarcts, both of which have been linked to stroke. We investigated the genetic basis of these differences by studying 359 adults with sickle cell disease (199 with silent cerebral infarcts, 160 normal MRIs), who had previously undergone a genome-wide genotyping array. None of the genes coding for the differentially expressed proteins were significantly associated with silent infarction. Our study suggests that silent cerebral infarcts in sickle cell anemia may be associated with higher systolic blood pressure, lower HbF levels, hypercoagulability, inflammation and atherosclerotic lipoproteins

Proteomic analysis of plasma from children with sickle cell anemia and silent cerebral infarction

Silent cerebral infarction is the commonest neurological abnormality in children with sickle cell anemia, affecting 30-40% 14 year olds. There are no known biomarkers to identify children with silent cerebral infarcts and the pathological basis is also unknown. We used an unbiased proteomic discovery approach to identify plasma proteins differing in concentration between children with and without silent cerebral infarcts. Clinical parameters and plasma samples were analysed from 51 children (mean age 11.8 years, range 6-18) with sickle cell anemia (HbSS). 19 children had silent cerebral infarcts and 32 normal MRI; the children with silent infarcts had lower HbF levels (8.6 vs. 16.1%, P=0.049) and higher systolic blood pressures (115 vs 108.6, P=0.027). Plasma proteomic analysis showed 13 proteins increased more than 1.3 fold in the SCI patients, including proteins involved in hypercoagulability (α2-antiplasmin, fibrinogen-γ chain, thrombospondin-4), inflammation (α2-macroglobulin, complement C1s and C3) and atherosclerosis (apolipoprotein B-100). Higher levels of gelsolin and retinol-binding protein 4 were also found in the population with silent infarcts, both of which have been linked to stroke. We investigated the genetic basis of these differences by studying 359 adults with sickle cell disease (199 with silent cerebral infarcts, 160 normal MRIs), who had previously undergone a genome-wide genotyping array. None of the genes coding for the differentially expressed proteins were significantly associated with silent infarction. Our study suggests that silent cerebral infarcts in sickle cell anemia may be associated with higher systolic blood pressure, lower HbF levels, hypercoagulability, inflammation and atherosclerotic lipoproteins.</p