Exploration of urine metabolic biomarkers for new-onset, untreated pediatric epilepsy: A gas and liquid chromatography mass spectrometry-based metabolomics study.

OBJECTIVE: The discovery of objective indicators for recent epileptic seizures will help confirm the diagnosis of epilepsy and evaluate therapeutic effects. Past studies had shortcomings such as the inclusion of patients under treatment and those with various etiologies that could confound the analysis results significantly. We aimed to minimize such confounding effects and to explore the small molecule biomarkers associated with the recent occurrence of epileptic seizures using urine metabolomics. METHODS: This is a multicenter prospective study. Subjects included pediatric patients aged 2 to 12 years old with new-onset, untreated epilepsy, who had had the last seizure within 1 month before urine collection. Controls included healthy children aged 2 to 12 years old. Those with underlying or chronic diseases, acute illnesses, or recent administration of medications or supplements were excluded. Targeted metabolome analysis of spot urine samples was conducted using gas chromatography (GC)- and liquid chromatography (LC)-tandem mass spectrometry (MS/MS). RESULTS: We enrolled 17 patients and 21 controls. Among 172 metabolites measured by GC/MS/MS and 41 metabolites measured by LC/MS/MS, only taurine was consistently reduced in the epilepsy group. This finding was subsequently confirmed by the absolute quantification of amino acids. No other metabolites were consistently altered between the two groups. CONCLUSIONS: Urine metabolome analysis, which covers a larger number of metabolites than conventional biochemistry analyses, found no consistently altered small molecule metabolites except for reduced taurine in epilepsy patients compared to healthy controls. Further studies with larger samples, subjects with different ages, expanded target metabolites, and the investigation of plasma samples are required.

Metabolic reprogramming in Nrf2-driven proliferation of normal rat hepatocytes.

BACKGROUND AND AIMS: Cancer cells reprogram their metabolic pathways to support bioenergetic and biosynthetic needs and to maintain their redox balance. In several human tumors, the Keap1-Nrf2 system controls proliferation and metabolic reprogramming by regulating the pentose phosphate pathway (PPP). However, whether this metabolic reprogramming also occurs in normal proliferating cells is unclear. APPROACH AND RESULTS: To define the metabolic phenotype in normal proliferating hepatocytes, we induced cell proliferation in the liver by 3 distinct stimuli: liver regeneration by partial hepatectomy and hepatic hyperplasia induced by 2 direct mitogens: lead nitrate (LN) or triiodothyronine. Following LN treatment, well-established features of cancer metabolic reprogramming, including enhanced glycolysis, oxidative PPP, nucleic acid synthesis, NAD + /NADH synthesis, and altered amino acid content, as well as downregulated oxidative phosphorylation, occurred in normal proliferating hepatocytes displaying Nrf2 activation. Genetic deletion of Nrf2 blunted LN-induced PPP activation and suppressed hepatocyte proliferation. Moreover, Nrf2 activation and following metabolic reprogramming did not occur when hepatocyte proliferation was induced by partial hepatectomy or triiodothyronine. CONCLUSIONS: Many metabolic changes in cancer cells are shared by proliferating normal hepatocytes in response to a hostile environment. Nrf2 activation is essential for bridging metabolic changes with crucial components of cancer metabolic reprogramming, including the activation of oxidative PPP. Our study demonstrates that matured hepatocytes exposed to LN undergo cancer-like metabolic reprogramming and offers a rapid and useful in vivo model to study the molecular alterations underpinning the differences/similarities of metabolic changes in normal and neoplastic hepatocytes.

Identification of predictive biomarkers for endometrial cancer diagnosis and treatment response monitoring using plasma metabolome profiling.

BACKGROUND: Endometrial cancer (EMC) is the most common female genital tract malignancy with an increasing prevalence in many countries including Japan, a fact that renders early detection and treatment necessary to protect health and fertility. Although early detection and treatment are necessary to further improve the prognosis of women with endometrial cancer, biomarkers that accurately reflect the pathophysiology of EMC patients are still unclear. Therefore, it is clinically critical to identify biomarkers to assess diagnosis and treatment efficacy to facilitate appropriate treatment and development of new therapies for EMC. METHODS: In this study, wide-targeted plasma metabolome analysis was performed to identify biomarkers for EMC diagnosis and the prediction of treatment responses. The absolute quantification of 628 metabolites in plasma samples from 142 patients with EMC was performed using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The concentrations of 111 metabolites increased significantly, while the concentrations of 148 metabolites decreased significantly in patients with EMC compared to healthy controls. Specifically, LysoPC and TGs, including unsaturated fatty acids, were reduced in patients with stage IA EMC compared to healthy controls, indicating that these metabolic profiles could be used as early diagnostic markers of EMC. In contrast, blood levels of amino acids such as histidine and tryptophan decreased as the risk of recurrence increased and the stages of EMC advanced. Furthermore, a marked increase in total TG and a decrease in specific TGs and free fatty acids including polyunsaturated fatty acids levels were observed in patients with EMC. These results suggest that the polyunsaturated fatty acids in patients with EMC are crucial for disease progression. CONCLUSIONS: Our data identified specific metabolite profiles that reflect the pathogenesis of EMC and showed that these metabolites correlate with the risk of recurrence and disease stage. Analysis of changes in plasma metabolite profiles could be applied for the early diagnosis and monitoring of the course of treatment of EMC patients.

Identification of predictive biomarkers for diagnosis and radiation sensitivity of uterine cervical cancer using wide-targeted metabolomics.

AIM: Uterine cervical cancer (UCC) is the fourth most common cancer in women, responsible for more than 300 000 deaths worldwide. Its early detection, by cervical cytology, and prevention, by vaccinating against human papilloma virus, greatly contribute to reducing cervical cancer mortality in women. However, penetration of the effective prevention of UCC in Japan remains low. Plasma metabolome analysis is widely used for biomarker discovery and the identification of cancer-specific metabolic pathways. Here, we aimed to identify predictive biomarkers for the diagnosis and radiation sensitivity of UCC using wide-targeted plasma metabolomics. METHODS: We analyzed 628 metabolites in plasma samples obtained from 45 patients with UCC using ultra-high-performance liquid chromatography with tandem mass spectrometry. RESULTS: The levels of 47 metabolites were significantly increased and those of 75 metabolites were significantly decreased in patients with UCC relative to healthy controls. Increased levels of arginine and ceramides, and decreased levels of tryptophan, ornithine, glycosylceramides, lysophosphatidylcholine, and phosphatidylcholine were characteristic of patients with UCC. Comparison of metabolite profiles in groups susceptible and non-susceptible to radiation therapy, a treatment for UCC, revealed marked variations in polyunsaturated fatty acid, nucleic acid, and arginine metabolism in the group not susceptible to treatment. CONCLUSIONS: Our findings suggest that the metabolite profile of patients with UCC may be an important indicator for distinguishing these patients from healthy cohorts, and may also be useful for predicting sensitivity to radiotherapy.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Nrf2 deficiency deteriorates diabetic kidney disease in Akita model mice.

Oxidative stress is an essential component in the progression of diabetic kidney disease (DKD), and the transcription factor NF-E2-related factor-2 (Nrf2) plays critical roles in protecting the body against oxidative stress. To clarify the roles of Nrf2 in protecting against DKD, in this study we prepared compound mutant mice with diabetes and loss of antioxidative defense. Specifically, we prepared compound Ins2Akita/+ (Akita) and Nrf2 knockout (Akita::Nrf2-/-) or Akita and Nrf2 induction (Akita::Keap1FA/FA) mutant mice. Eighteen-week-old Akita::Nrf2-/- mice showed more severe diabetic symptoms than Akita mice. In the Akita::Nrf2-/- mouse kidneys, the glomeruli showed distended capillary loops, suggesting enhanced mesangiolysis. Distal tubules showed dilation and an increase in 8-hydroxydeoxyguanosine-positive staining. In the Akita::Nrf2-/- mouse kidneys, the expression of glutathione (GSH) synthesis-related genes was decreased, and the actual GSH level was decreased in matrix-assisted laser desorption/ionization mass spectrometry imaging analysis. Akita::Nrf2-/- mice exhibited severe inflammation and enhancement of infiltrated macrophages in the kidney. To further examine the progression of DKD, we compared forty-week-old Akita mouse kidney compounds with Nrf2-knockout or Nrf2 mildly induced (Akita::Keap1FA/FA) mice. Nrf2-knockout Akita (Akita::Nrf2-/-) mice displayed severe medullary cast formation, but the formation was ameliorated in Akita::Keap1FA/FA mice. Moreover, in Akita::Keap1FA/FA mice, tubule injury and inflammation-related gene expression were significantly suppressed, which was evident in Akita::Nrf2-/- mouse kidneys. These results demonstrate that Nrf2 contributes to the protection of the kidneys against DKD by suppressing oxidative stress and inflammation.

Wide-Targeted Metabolome Analysis Identifies Potential Biomarkers for Prognosis Prediction of Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is a fatal gynecologic cancer, and its poor prognosis is mainly due to delayed diagnosis. Therefore, biomarker identification and prognosis prediction are crucial in EOC. Altered cell metabolism is a characteristic feature of cancers, and metabolomics reflects an individual's current phenotype. In particular, plasma metabolome analyses can be useful for biomarker identification. In this study, we analyzed 624 metabolites, including uremic toxins (UTx) in plasma derived from 80 patients with EOC using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Compared with the healthy control, we detected 77 significantly increased metabolites and 114 significantly decreased metabolites in EOC patients. Especially, decreased concentrations of lysophosphatidylcholines and phosphatidylcholines and increased concentrations of triglycerides were observed, indicating a metabolic profile characteristic of EOC patients. After calculating the parameters of each metabolic index, we found that higher ratios of kynurenine to tryptophan correlates with worse prognosis in EOC patients. Kynurenine, one of the UTx, can affect the prognosis of EOC. Our results demonstrated that plasma metabolome analysis is useful not only for the diagnosis of EOC, but also for predicting prognosis with the variation of UTx and evaluating response to chemotherapy.

Determination of novel CYP2D6 haplotype using the targeted sequencing followed by the long-read sequencing and the functional characterization in the Japanese population.

Next-generation sequencing (NGS) has identified variations in cytochrome P450 (CYP) 2D6 associated with drug responses. However, determination of novel haplotypes is difficult because of the short reads generated by NGS. We aimed to identify novel CYP2D6 variants in the Japanese population and predict the CYP2D6 phenotype based on in vitro metabolic studies. Using a targeted NGS panel (PKSeq), 990 Japanese genomes were sequenced, and then novel CYP2D6 haplotypes were determined. Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl-tamoxifen 4-hydroxylation were calculated by in vitro metabolic studies using cDNA-expressed CYP2D6 proteins. After determination of the CYP2D6 diplotypes, phenotypes of the individuals were predicted based on the in vitro metabolic activities. Targeted NGS identified 14 CYP2D6 variants not registered in the Pharmacogene Variation Consortium (PharmVar) database. Ten novel haplotypes were registered as CYP2D6*128 to *137 alleles in the PharmVar database. Based on the Vmax/Km value of each allele, *128, *129, *130, *131, *132, and *133 were predicted to be nonfunctional alleles. According to the results of the present study, six normal metabolizers (NM) and one intermediate (IM) metabolizers were designated as IM and poor metabolizers (PM), respectively. Our findings provide important insights into novel haplotypes and haplotypes of CYP2D6 and the effects on in vitro metabolic activities.

Geldanamycin-Derived HSP90 Inhibitors Are Synthetic Lethal with NRF2.

Activating mutations in KEAP1-NRF2 are frequently found in tumors of the lung, esophagus, and liver, where they are associated with aggressive growth, resistance to cancer therapies, and low overall survival. Despite the fact that NRF2 is a validated driver of tumorigenesis and chemotherapeutic resistance, there are currently no approved drugs which can inhibit its activity. Therefore, there is an urgent clinical need to identify NRF2-selective cancer therapies. To this end, we developed a novel synthetic lethal assay, based on fluorescently labeled isogenic wild-type and Keap1 knockout cell lines, in order to screen for compounds which selectively kill cells in an NRF2-dependent manner. Through this approach, we identified three compounds based on the geldanamycin scaffold which display synthetic lethality with NRF2. Mechanistically, we show that products of NRF2 target genes metabolize the quinone-containing geldanamycin compounds into more potent HSP90 inhibitors, which enhances their cytotoxicity while simultaneously restricting the synthetic lethal effect to cells with aberrant NRF2 activity. As all three of the geldanamycin-derived compounds have been used in clinical trials, they represent ideal candidates for drug repositioning to target the currently untreatable NRF2 activity in cancer.

In Vitro Assessment of Fluoropyrimidine-Metabolizing Enzymes: Dihydropyrimidine Dehydrogenase, Dihydropyrimidinase, and ß-Ureidopropionase.

Fluoropyrimidine drugs (FPs), including 5-fluorouracil, tegafur, capecitabine, and doxifluridine, are among the most widely used anticancer agents in the treatment of solid tumors. However, severe toxicity occurs in approximately 30% of patients following FP administration, emphasizing the importance of predicting the risk of acute toxicity before treatment. Three metabolic enzymes, dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP), and ß-ureidopropionase (ß-UP), degrade FPs; hence, deficiencies in these enzymes, arising from genetic polymorphisms, are involved in severe FP-related toxicity, although the effect of these polymorphisms on in vivo enzymatic activity has not been clarified. Furthermore, the clinical usefulness of current methods for predicting in vivo activity, such as pyrimidine concentrations in blood or urine, is unknown. In vitro tests have been established as advantageous for predicting the in vivo activity of enzyme variants. This is due to several studies that evaluated FP activities after enzyme metabolism using transient expression systems in Escherichia coli or mammalian cells; however, there are no comparative reports of these results. Thus, in this review, we summarized the results of in vitro analyses involving DPD, DHP, and ß-UP in an attempt to encourage further comparative studies using these drug types and to aid in the elucidation of their underlying mechanisms.

Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals.

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2), encoded by the DPYD gene, is the rate-limiting enzyme in the degradation pathway of endogenous pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil (5-FU). DPD catalyzes the reduction of uracil, thymine, and 5-FU. In Caucasians, DPYD mutations, including DPYD*2A, DPYD*13, c.2846A>T, and c.1129-5923C>G/hapB3, are known to contribute to interindividual variations in the toxicity of 5-FU; however, none of these DPYD polymorphisms has been identified in the Asian population. Recently, 21 DPYD allelic variants, including some novel single-nucleotide variants (SNVs), were identified in 1070 healthy Japanese individuals by analyzing their whole-genome sequences (WGSs), but the functional alterations caused by these variants remain unknown. In this study, in vitro analysis was performed on 22 DPD allelic variants by transiently expressing wild-type DPD and 21 DPD variants in 293FT cells and characterizing their enzymatic activities using 5-FU as a substrate. DPD expression levels and dimeric forms were determined using immunoblotting and blue-native PAGE, respectively. Additionally, the values of three kinetic parameters-the Michaelis constant (Km ), maximum velocity (Vmax ), and intrinsic clearance (CLint = Vmax/Km )-were determined for the reduction of 5-FU. Eleven variants exhibited significantly decreased intrinsic clearance compared with wild-type DPD. Moreover, the band patterns observed in the immunoblots of blue-native gels indicated that DPD dimerization is required for enzymatic activity in DPD. Thus, the detection of rare DPYD variants might facilitate severe adverse effect prediction of 5-FU-based chemotherapy in the Japanese population.

Functional characterization of 21 allelic variants of dihydropyrimidinase.

Dihydropyrimidinase (DHP, EC 3.5.2.2), encoded by the gene DPYS, is the second enzyme in the catabolic pathway of pyrimidine and of fluoropyrimidine drugs such as 5-fluorouracil, which are commonly used in anticancer treatment; DHP catalyzes the hydrolytic ring opening of dihydrouracil and dihydro-5-fluorouracil. DPYS mutations are known to contribute to interindividual variations in the toxicity of fluoropyrimidine drugs, but the functional characterization of DHP allelic variants remains inadequate. In this study, in vitro analysis was performed on 22 allelic variants of DHP by transiently expressing wild-type DHP and 21 DHP variants in 293FT cells and characterizing their enzymatic activities by using dihydrouracil and dihydro-5-fluorouracil as substrates. DHP expression levels and oligomeric forms were determined using immunoblotting and blue native PAGE, respectively, and the stability of the DHP variants was assessed by examining the proteins in variant-transfected cells treated with cycloheximide or bortezomib. Moreover, three kinetic parameters, Km, Vmax, and intrinsic clearance (Vmax/Km), for the hydrolysis of dihydrouracil and dihydro-5-fluorouracil were determined. We found that 5/21 variants showed significantly decreased intrinsic clearance as compared to wild-type DHP, and that 9/21 variants were expressed at low levels and were inactive due to proteasome-mediated degradation. The band patterns observed in the immunoblotting of blue native gels corresponded to DHP activity, and, notably, 18/21 DHP variants exhibited decreased or null enzymatic activity and these variants also showed a drastically reduced ability to form large oligomers. Thus, detection of DPYS genetic polymorphisms might facilitate the prediction severe adverse effects of fluoropyrimidine-based treatments.

Genetic polymorphisms of dihydropyrimidinase in a Japanese patient with capecitabine-induced toxicity.

Dihydropyrimidinase (DHP) is the second enzyme in the catabolic pathway of uracil, thymine, and chemotherapeutic fluoropyrimidine agents such as 5-fluorouracil (5-FU). Thus, DHP deficiency might be associated with 5-FU toxicity during fluoropyrimidine chemotherapy. We performed genetic analyses of the family of a patient with advanced colon cancer who underwent radical colectomy followed by treatment with 5-FU prodrug capecitabine and developed severe toxicity attributable to a lack of DHP. We measured urinary uracil and dihydrouracil, and genotyped DPYS in the patient and her family. We also measured the allele frequency of DPYS polymorphisms in 391 unrelated Japanese subjects. The patient had compound heterozygous missense and nonsense polymorphisms comprising c.1001A>G (p.Gln334Arg) in exon 6 and c.1393C>T (p.Arg465Ter) in exon 8, which are known to result in a DHP enzyme with little or no activity. The urinary dihydrouracil/uracil ratio in the patient was 17.08, while the mean ± SD urinary dihydrouracil/uracil ratio in family members who were heterozygous or homozygous for wild-type DPYS was 0.25 ± 0.06. In unrelated subjects, 8 of 391 individuals were heterozygous for the c.1001A>G mutation, while the c.1393C>T mutation was not identified. This is the first report of a DHP-deficient patient with DPYS compound heterozygous polymorphisms who was treated with a fluoropyrimidine, and our findings suggest that polymorphisms in the DPYS gene are pharmacogenomic markers associated with severe 5-FU toxicity in Japanese patients.