Identifying Dihydropyrimidine Dehydrogenase as a Novel Regulator of Hepatic Steatosis

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine Dehydrogenase (DPYD) is an enzyme responsible for uracil and thymine catabolism, and DPYD human genetic variability affects clinically observed toxicity following 5-Fluorouracil (5-FU) administration. In an in vitro model of diet-induced steatosis, the pharmacologic inhibition of DPYD resulted in protection from lipid accumulation. Additionally, a gain-of-function mutation of DPYD, created through clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) engineering, led to an increased lipid burden, which was associated with altered mitochondrial functionality in a hepatocarcionma cell line. The studies presented herein describe a novel role for DPYD in hepatocyte metabolic regulation as a modulator of hepatic steatosis.

Identifying Dihydropyrimidine Dehydrogenase as a Novel Regulator of Hepatic Steatosis

Pyrimidine catabolism is implicated in hepatic steatosis. Dihydropyrimidine Dehydrogenase (DPYD) is an enzyme responsible for uracil as well as thymine catabolism, and human genetic variability in this enzyme has been described in relation to clinically observed toxicity following 5-Fluorouracil (5-FU) administration. We have demonstrated that pharmacologic inhibition of DPYD is protective in a human in vitro model of diet-induced steatosis. A gain-of-function mutation in DPYD through CRISPR-Cas9 engineering leads to an increased lipid burden associated with altered mitochondrial functionality in a hepatocarcionma cell line. These studies uncovered a novel role DPYD plays in regulating the metabolic phenotype of hepatocytes and therefore identifies DPYD as a key modulator of hepatic steatosis.

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines

ABSTRACTCyanuric acid was likely present on prebiotic Earth, may have been a component of early genetic materials, and is synthesized industrially today on a scale of more than one hundred million pounds per year in the United States. In light of this, it is not surprising that some bacteria and fungi have a metabolic pathway that sequentially hydrolyzes cyanuric acid and its metabolites to release the nitrogen atoms as ammonia to support growth. The initial reaction that opens thes-triazine ring is catalyzed by the unusual enzyme cyanuric acid hydrolase. This enzyme is in a rare protein family that consists of only cyanuric acid hydrolase (CAH) and barbiturase, with barbiturase participating in pyrimidine catabolism by some actinobacterial species. The X-ray structures of two cyanuric acid hydrolase proteins show that this family has a unique protein fold. Phylogenetic, bioinformatic, enzymological, and genetic studies are consistent with the idea that CAH has an ancient protein fold that was rare in microbial populations but is currently becoming more widespread in microbial populations in the wake of anthropogenic synthesis of cyanuric acid and others-triazine compounds that are metabolized via a cyanuric acid intermediate. The need for the removal of cyanuric acid from swimming pools and spas, where it is used as a disinfectant stabilizer, can potentially be met using an enzyme filtration system. A stable thermophilic cyanuric acid hydrolase fromMoorella thermoaceticais being tested for this purpose.

Short Term Effect of Caffeine on Purine, Pyrimidine and Pyridine Metabolism in Rice (Oryza sativa) Seedlings

As part of our studies on the physiological and ecological function of caffeine, we investigated the effect of exogenously supplied caffeine on purine, pyrimidine and pyridine metabolism in rice seedlings. We examined the effect of 1 mM caffeine on the in situ metabolism of 14C-labelled adenine, guanine, inosine, uridine, uracil, nicotinamide and nicotinic acid. The segments of 4-day-old dark-grown seedlings were incubated with these labelled compounds for 6 h. For purines, the incorporation of radioactivity from [8-14C]adenine and [8-14C]guanine into nucleotides was enhanced by caffeine; in contrast, incorporation into CO2 were reduced. The radioactivity in ureides (allantoin and allantoic acid) from [8-14C]guanine and [8-14C]inosine was increased by caffeine. For pyrimidines, caffeine enhanced the incorporation of radioactivity from [2-14C]uridine into nucleotides, which was accompanied by a decrease in pyrimidine catabolism. Such difference was not found in the metabolism of [2-14C]uracil. Caffeine did not influence the pyridine metabolism of [carbonyl-14C]-nicotinamide and [2-14C]nicotinic acid. The possible control steps of caffeine on nucleotide metabolism in rice are discussed.

Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals NewURCGenes Involved in Pyrimidine Catabolism

ABSTRACTPyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, theURCpathway, has been initially discovered in our laboratory in the yeastLachancea kluyveri. Here, we present the global changes in gene expression inL. kluyveriin response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the knownURCgenes,URC1-6, helped to identify nine putative novelURCgenes with a similar expression pattern. The microarray analysis provided evidence that both theURCandPYDgenes are under nitrogen catabolite repression inL. kluyveriand are induced by uracil or dihydrouracil, respectively. We determined the function ofURC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that theL. kluyveriFui1p protein transported uridine, just like its homolog inSaccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter inL. kluyveri. We also showed that theL. kluyverihomologs ofDUR3andFUR4do not have the same function that they have inS. cerevisiae, where they transport urea and uracil, respectively. InL. kluyveri, both of these deletion strains grew normally on uracil and urea.

13C-uracil breath test to predict 5-fluorouracil toxicity in gastrointestinal cancer patients.

e13008 Background: Up to 30% of patients on 5-fluorouracil (5FU) experience severe toxicity. Dihydropyrimidine-dehydrogenase (DPD) deficiency explains 36-61% of cases. Predicting toxicity is an unmet challenge. Uracil breath test (UraBT) consists of measuring 13CO2 in exhaled breath after ingestion of 2-13C-uracil to evaluate pyrimidine (and 5FU) catabolism. Methods: We studied 33 gastrointestinal cancer patients previously exposed to 5FU: thirteen had grade 3-4 and 20, grade 0-1 toxicity. Groups were well-balanced regarding: age (median, 57 years); gender (males, 35%); primary (colorectal, 90%); ethnicity (Caucasians, 55%); chemotherapy (Mayo clinic regimen, 75%). Main toxicities were febrile neutropenia, diarrhea and stomatitis. Tests used to evaluate pyrimidine catabolism: (1) sequencing of three exons of DPYD; (2) plasma dihydrouracil/uracil ratio (UH2/U); (3) UraBT. We tested the performance of UraBT to discriminate patients who had grade 0-1 toxicity versus grade 3-4 toxicity and patients with and without proven DPD-deficiency. DPD-deficients were defined as having had grade 3-4 toxicity plus either UH2/U < 1.8 or deleterious mutation. Results: 4/13 grade 3-4 toxicity patients proved to be DPD-deficient: three had deleterious mutations (IVS14+1G>A in one; SNP 2846A>T in two), and one had low UH2/U ratio. Mean delta over baseline in 50 minutes (DOB50) significantly differed between groups. DOB50 ≤ 161.4 discriminated individuals with grade 3-4 versus grade 0-1 toxicity (sensitivity= 61.5%; specificity= 85%) and DPD-deficient versus non DPD-deficient (sensitivity= 75%; specificity= 85%). Conclusions: UraBT is a non-invasive and easy to perform method with promising accuracy in discriminating individuals with severe toxicity to 5FU, comparing favorably to most tests available to predict 5FU toxicity. [Table: see text]