Choline deficiency-related multi-omics characteristics are susceptible factors for chemotherapy-induced thrombocytopenia.

The XELOX chemotherapy protocol that includes capecitabine and oxaliplatin is the routine treatment for colorectal cancer (CRC), but it can cause chemotherapy-related adverse events such as thrombocytopenia (TCP). To identify predictive biomarkers and clarify the mechanism of TCP susceptibility, we conducted integrative analysis using normal colorectal tissue (CRT), plasma, and urine samples collected before CRC patients received adjuvant XELOX chemotherapy. RNA-sequencing and DNA methylation arrays were performed on CRT samples, while liquid chromatography-mass spectrometry was performed on CRT, plasma, and urine samples. Differentially expressed features (DEFs) from each uni-omics analysis were then subjected to integrative analysis using Multi-Omics Factor Analysis (MOFA). Choline-deficiency in plasma and CRT was found as the most critical TCP-related feature. Based on bioinformatic analysis and literature research, we further concluded that choline-deficiency was the possible reason for most of the other TCP-related multi-omics DEFs, including metabolites representing reduced sphingolipid de novo synthesis and elevated solute carrier-mediated transmembrane transportation in CRT and plasma, DNA hypermethylation and elevated expression of genes involved in neuronal system genes. In terms of thrombocytopoiesis, these TCP-related DEFs may cause atypical maintenance and differentiation of megakaryocyte, resulting a suppressed ability of thrombocytopoiesis, making patients more susceptible to chemotherapy-induced TCP. At last, prediction models were developed and validated with reasonably good discrimination. The area under curves (AUCs) of training sets were all > 0.9, while validation sets had AUCs between 0.778 and 0.926. In conclusion, our results produced reliable marker systems for predicting TCP and promising target for developing precision treatment to prevent TCP.

TGF-ß/YB-1/Atg7 axis promotes the proliferation of hepatic progenitor cells and liver fibrogenesis.

Hepatic fibrosis is characterized by excessive extracellular matrix deposition and ductular reactions, manifested as the expansion of hepatic progenitor cells (HPCs). We previously reported that the Y-box binding protein 1 (YB-1) in HPCs is involved in chronic liver injury. In this study, we constructed YB-1f/f Foxl1-Cre mice and investigated the role of YB-1 in HPC expansion in murine choline-deficient, ethionine-supplemented (CDE), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) models. Liver injury and fibrosis were measured using hematoxylin and eosin (HE), Masson, and Sirius Red staining. HPC proliferation was detected using EdU and immunofluorescence (IF). Autophagic flow was measured by mCherry-GFP-LC3B staining and transmission electron microscopy (TEM). YB-1 expression was measured by immunofluorescence and western blotting. CUT & Tag analysis, chromatin immunoprecipitation, and RT-PCR were performed to explore the regulation of autophagy-related protein 7 (Atg7) transcription by YB-1. Our results indicated that liver injury was accompanied by high expression of YB-1, proliferative HPCs, and activated autophagy in the CDE and DDC models. YB-1f/f Cre+/- mice displayed less liver injury and fibrosis than YB-1f/f Cre-/- mice in the CDE and DDC models. YB-1 promoted proliferation and autophagy of HPCs in vitro and in vivo. Transforming growth factor-ß (TGF-ß) induced YB-1 nuclear translocation and facilitated the proliferation and autophagy of HPCs. YB-1 nuclear translocation promoted the transcription of Atg7, which is essential for TGF-ß/YB-1 mediated HPCs expansion in vitro and in vivo. In summary, YB-1 nuclear translocation induced by TGF-ß in HPCs promotes the proliferation and autophagy of HPCs and Atg7 participates in YB-1-mediated HPC-expansion and liver fibrosis.

Prenatal choline deficiency does not enhance hippocampal vulnerability after kainic acid-induced seizures in adulthood.

Choline is a vital nutrient needed during early development for both humans and rodents. Severe dietary choline deficiency during pregnancy leads to birth defects, while more limited deficiency during mid- to late pregnancy causes deficits in hippocampal plasticity in adult rodent offspring that are accompanied by cognitive deficits only when task demands are high. Because prenatal choline supplementation confers neuroprotection of the adult hippocampus against a variety of neural insults and aids memory, we hypothesized that prenatal choline deficiency may enhance vulnerability to neural injury. To examine this, adult offspring of rat dams either fed a control diet (CON) or one deficient in choline (DEF) during embryonic days 12-17 were given multiple injections (i.p.) of saline (control) or kainic acid to induce seizures and were euthanized 16 days later. Perhaps somewhat surprisingly, DEF rats were not more susceptible to seizure induction and showed similar levels of seizure-induced hippocampal histopathology, GAD expression loss, upregulated hippocampal GFAP and growth factor expression, and increased dentate cell and neuronal proliferation as that seen in CON rats. Although prenatal choline deficiency compromises adult hippocampal plasticity in the intact brain, it does not appear to exacerbate the neuropathological response to seizures in the adult hippocampus at least shortly after excitotoxic injury.

Perinatal choline deficiency produces abnormal sensory inhibition in Sprague-Dawley rats.

Adequate choline levels in rodents during gestation have been shown to be critical to several functions, including certain learning and memory functions, when tested at adulthood. Choline is a selective agonist for the alpha7 nicotinic receptor which appears in development before acetylcholine is present. Normal sensory inhibition is dependent, in part, upon sufficient numbers of this receptor in the hippocampus. The present study assessed sensory inhibition in Sprague-Dawley rats gestated on normal (1.1 g/kg), deficient (0 g/kg) or supplemented (5 g/kg) choline in the maternal diet during the critical period for cholinergic cell development (E12-18). Rats gestated on deficient choline showed abnormal sensory inhibition when tested at adulthood, while rats gestated on normal or supplemented choline showed normal sensory inhibition. Assessment of hippocampal alpha-bungarotoxin to visualize nicotinic alpha7 receptors revealed no difference between the gestational choline levels. These data suggest that attention to maternal choline levels for human pregnancy may be important to the normal functioning of the offspring.

Prenatal choline deficiency increases choline transporter expression in the septum and hippocampus during postnatal development and in adulthood in rats.

Supplementation of maternal diet with the essential nutrient, choline, during the second half of pregnancy in rats causes long-lasting improvements in spatial memory in the offspring and protects them from the memory decline characteristic of old age. In contrast, prenatal choline deficiency is associated with poor performance in certain cognitive tasks. The mechanism by which choline influences learning and memory remains unclear; however, it may involve changes to the hippocampal cholinergic system. Previously, we showed that the hippocampi of prenatally [embryonic days (E) 11-17] choline-deficient animals have increased synthesis of acetylcholine (ACh) from choline transported by the high-affinity choline transporter (CHT) and reduced ACh content relative to the control and to the E11-17 choline-supplemented rats. In the current study, we found that, during postnatal period [postnatal days (P) 18-480], prenatal choline deficiency increased the expression of CHT mRNA in the septum and CHT mRNA and protein levels in the hippocampus and altered the pattern of CHT immunoreactivity in the dentate gyrus. CHT immunoreactivity was more prominent in the inner molecular layer in prenatally choline-deficient rats compared to controls and prenatally choline-supplemented animals. In addition, in all groups, we observed a population of hilar interneurons that were CHT-immunoreactive. These neurons are the likely source of the hippocampal CHT mRNA as their number correlated with the levels of this mRNA. The abundance of hippocampal CHT mRNA rose between P1 and P24 and then declined reaching 60% of the P1 value by P90. These data show that prenatal availability of choline alters its own metabolism (i.e., CHT expression). While the upregulated CHT expression during the period of prenatal choline deficiency may be considered as a compensatory mechanism that could enhance ACh synthesis when choline supply is low, the persistent upregulation of CHT expression subsequent to the brief period of prenatal deprivation of choline in utero might be beneficial during choline deficiency in adulthood.

Choline cannot be replaced by propanolamine in mice.

Choline is an important nutrient for humans and animals. Animals obtain choline from the diet and from the catabolism of phosphatidylcholine made by phosphatidylethanolamine N-methyltransferase (PEMT). The unique model of complete choline deprivation is Pemt(-/-) mice that are fed a choline-deficient diet. This model, therefore, can be used for the examination of choline substitutes in mammalian systems. Recently, propanolamine was found to be a replacement for choline in yeast. Thus, we tested to see whether or not choline can be replaced by propanolamine in mice. Mice were fed a choline-deficient diet and supplemented with either methionine, 2-amino-propanol, 2-amino-isopropanol and 3-amino-propanol. We were unable to detect the formation of any of the possible phosphatidylpropanolamines. Moreover, none of them prevented liver damage, reduction of hepatic phosphatidylcholine levels or fatty liver induced in choline-deficient-Pemt(-/-) mice. These results suggest that choline in mice cannot be replaced by any of the three propanolamine derivatives.

Review of the carcinogenic activity of diethanolamine and evidence of choline deficiency as a plausible mode of action.

Diethanolamine (DEA) is a chemical used widely in a number of industries and is present in many consumer products. Studies by the National Toxicology Program (NTP) have indicated that lifetime dermal exposure to DEA increased the incidence and multiplicity of liver tumors in mice, but not in rats. In addition, DEA was not carcinogenic when tested in the Tg.Ac transgenic mouse model. Short-term genotoxicity tests have yielded negative results. In view of these apparent inconsistencies, we have critically evaluated the NTP studies and other data relevant to assessing the carcinogenic potential of DEA. The available data indicate that DEA induces mouse liver tumors by a non-genotoxic mode of action that involves its ability to cause choline deficiency. The following experimental evidence supports this hypothesis. DEA decreased the hepatic choline metabolites and S-adenosylmethionine levels in mice, similar to those observed in choline-deficient mice. In contrast, DEA had no effect in the rat, a species in which it was not carcinogenic at a maximum tolerated dose level. In addition, a consistent dose-effect relationship had been established between choline deficiency and carcinogenic activity since all DEA dosages that induced tumors in the NTP studies were also shown to cause choline deficiency. DEA decreased phosphatidylcholine synthesis by blocking the cellular uptake of choline in vitro, but these events did not occur in the presence of excess choline. Finally, DEA induced transformation in the Syrian hamster embryo cells, increased S-phase DNA synthesis in mouse hepatocytes, and decreased gap junctional intracellular communication in primary cultured mouse and rat hepatocytes, but all these events were prevented with choline supplementation. Since choline is an essential nutrient in mammals, this mode of action is qualitatively applicable to humans. However, there are marked species differences in susceptibility to choline deficiency, with rats and mice being far more susceptible than other mammalian species including humans. These differences are attributed to quantitative differences in the enzyme kinetics controlling choline metabolism. The fact that DEA was carcinogenic in mice but not in rats also has important implications for human risk assessment. DEA has been shown to be less readily absorbed across rat and human skin than mouse skin. Since a no observed effect level for DEA-induced choline deficiency in mice has been established to be 10 mg/kg/d, this indicates that there is a critical level of DEA that must be attained in order to affect choline homeostasis. The lack of a carcinogenic response in rats suggests that exposure to DEA did not reach this critical level. Since rodents are far more sensitive to choline deficiency than humans, it can be concluded that the hepatocarcinogenic effect of DEA in mice is not predictive of similar susceptibility in humans.

Potential mechanisms of tumorigenic action of diethanolamine in mice.

Diethanolamine (DEA), a secondary amine found in a number of consumer products, reportedly induces liver tumors in mice. In an attempt to define the tumorigenic mechanism of DEA, N-nitrosodiethanolamine (NDELA) formation in vivo and development of choline deficiency were examined in mice. DEA was administered with or without supplemental sodium nitrite to B6C3F1 mice via dermal application (with or without access to the application site) or via oral gavage for 2 weeks. Blood levels of DEA reflected the dosing method used; oral greater than dermal with access greater than dermal without access. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Choline, phosphocholine and glycerophosphocholine were decreased

Inhibitory effects of N,N'-diphenyl-p-phenylenediamine on the early stage of the enhanced hepatocarcinogenesis caused by coadministration of ethionine and a choline-deficient L-amino acid-defined diet in rats.

Effects of N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, on liver carcinogenesis caused by a choline-deficient L-amino acid-defined (CDAA) diet containing ethionine were studied in Fischer 344 rats. Male animals, 6 weeks old, were fed a CDAA diet, a choline-supplemented L-amino acid-defined (CSAA) diet or a CDAA diet containing 0.05% ethionine with or without 0.2% DPPD. Histological changes and lesions positive for gamma-glutamyltransferase (GGT) were analyzed 12 weeks after the beginning of the experiment. The levels of 8-hydroxyguanine (8-OHGua) in DNA and 2-thiobarbituric acid-reacting substances (TBARS) were measured as the parameters for cellular oxidative damage after 4 and 11 days of treatment. Expression of c-myc and c-Ha-ras was also investigated in relation to cell proliferation after 2, 4, 8 and 11 days. Histologically, development of diffuse fatty liver observed in rats fed a CDAA diet was inhibited, while massive oval cell proliferation and cholangiofibrosis resulted from the addition of ethionine with/without DPPD. The sizes but not numbers of GGT-positive lesions seen in the liver of rats fed a CDAA diet were increased and the levels of 8-OHGua formation and TBARS generation were also increased by the ethionine supplement. Both numbers and sizes of GGT-positive lesions were decreased and the level of TBARS, but not 8-OHGua, was decreased by adding DPPD. The increased expression of c-myc and c-Ha-ras detected in the liver of rats fed a CDAA diet was further increased by addition of ethionine and again reduced by DPPD. These results indicate that an antioxidant DPPD can inhibit the early stage of enhanced hepatocarcinogenesis caused by coadministration of ethionine and a CDAA diet, by blocking cellular oxidative damage as well as c-myc and c-Ha-ras expression.