Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Simultaneous removal of nitrate and heavy metals in a continuous flow nitrate-dependent ferrous iron oxidation (NDFO) bioreactor.

Nitrogen and heavy metals can co-occur in various industrial wastewaters such as coke-oven wastewater. Removal of these contaminants is important, but cost-efficient removal technology is limited. In this study, we examined the usefulness of nitrate-dependent ferrous iron oxidation (NDFO) for the simultaneous removal of nitrate and heavy metals (iron and zinc), by using an NDFO strain Pseudogulbenkiania sp. NH8B. Based on the batch culture assays, nitrate, Fe, and Zn were successfully removed from a basal medium as well as coke-oven wastewater containing 5 mM nitrate, 10 mM Fe(II), and 10 mg/L Zn. Zinc in the water was most likely co-precipitated with Fe(III) oxides produced during the NDFO reaction. Simultaneous removal of nitrate, Fe, and Zn was also achieved in a continuous-flow reactor fed with a basal medium containing 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L Zn. However, when the reactor is fed with coke-oven wastewater supplemented with 10 mM nitrate, 5 mM Fe(II), 4 mM acetate, and 10 mg/L ZnCl2, the reactor performance significantly decreased, most likely due to the inhibition of bacterial growth by thiocyanate or organic contaminants present in the coke-oven wastewater. Use of mixed culture of NDFO bacteria and thiocyanate/organic-degrading denitrifiers should help improve the reactor performance.

Classification of heavy-metal toxicity by human DNA microarray analysis.

Microarray technology is proving to be a useful tool to classify undefined environmental toxicants, to investigate underlying mechanisms of toxicity, and to identify candidate toxicant-specific genetic markers by examining global effects of putative toxicants on gene expression profiles. The aim of this study was to evaluate the toxicities of six heavy metals through the comparison with gene expression patterns induced by well-known chemicals. For this purpose, we first identified the genes altered specifically in HepG2 under the exposure of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), phenol, and N-nitrosodimethylamine (DMN), which were selected as the model chemicals, using DNA microarray. On the basis of the expression profiles of these genes, toxicities of six heavy metals, arsenic, cadmium, nickel, antimony, mercury, and chromium, were evaluated. The specific gene alteration and hierarchical clustering revealed that biological action of six heavy metals was clearly related to that of DMNQ which has been reported to be a reactive oxygen species (ROS) generating chemical and which induced the genes associated with cell proliferative responses. These results suggest that cell proliferative responses which are probably caused by ROS are a major apparent biological action of high-dose heavy metals, supporting the previous reports. Overall, a mechanism-based classification by DNA microarray would be an efficient method for evaluation of toxicities of environmental samples.

Randomized comparative study of surgical adjuvant chemotherapy using 5-fluorouracil and dl-leucovorin with CDDP, 5-FU and dl-leucovorin for advanced colorectal cancer.

BACKGROUND: 5-fluorouracil (5-FU) is the most useful chemotherapeutic agent for colorectal carcinoma. Recently, the effectiveness of multidrug therapy in modifying the anticancer activity of 5-FU for advanced cases was clarified. We conducted a randomized comparative study of surgical adjuvant chemotherapy comparing cis-diamminedichloroplatinum (CDDP), 5-FU and dl-leucovorin (LV) (PFL therapy) with dl-LV and 5-FU (FL therapy) in patients who underwent curative resection of stage II and III colorectal carcinoma. METHODS: Thirty-one patients, 13 ungergoing PFL therapy and 18 undergoing FL therapy, completed the scheduled administration, while 20 dropped out. Administration schedules were as follows: PFL therapy consisted of 13 mg/m2 CDDP, 300 mg/m2 5-FU and 30 mg/body weight LV for 5 consecutive days. The FL therapy consisted of 300 mg/m2 5-FU and 30 mg/body weight LV for 5 consecutive days in hospital. Both regimens were followed by biweekly administration of the same dosages of LV and 5-FU in an outpatient setting. RESULTS: Seven of 23 patients in the PFL therapy group and five of 28 patients in the FL therapy group experienced recurrence of colorectal carcinomas. Regarding prognosis of stage II patients, both groups showed similar results in 5-year disease specific survival and disease-free survival. However, stage III patients obtained better prognostic results in the FL therapy group than in the PFL therapy group. Major toxicities in these regimens were vomiting and leukopenia, both being more frequent in the PFL therapy group. Symptoms of toxicity resolved completely in all patients. CONCLUSION: From the present study we found that PFL therapy brought no benefit for stage II and III colorectal carcinoma compared with FL therapy. We should search for more appropriate adjuvant therapy, increasing the therapeutic effect of 5-FU + LV for stage II and III colorectal carcinoma.

Investigation into the usefulness and adverse events of CDDP, 5-fU and dl-leucovorin (PFL-therapy) for advanced colorectal cancer.

Biochemical modulation of 5-fluorouracil (5-FU) has been verified the evidence of significant improvement of survival and quality of life in patients with advanced colorectal carcinoma. We investigated the therapeutic and adverse drug reaction of intensive chemotherapy using cisplatin (CDDP), 5-FU and dl-leucovorin (LV) (PFL-therapy), which may be producing dual biochemical modulation effect of 5-FU for advanced colorectal carcinoma. Administration schedule was 13 mg/m2 of CDDP, 300 mg/m2 of 5-FU, and 30 mg/body of dl-LV for 5 consecutive days. This regimen was repeated at 3-week intervals in hospital. Sixteen patients were enrolled in this study, most of whom had a history of previous chemotherapy as adjuvant treatment, and the response rate was 25%, with four patients having "partial response" and eight "no change". In respect to performance status, 46% of patients who completed the protocol were markedly improved in spite of their poor performance status before treatment. Moreover, when patients were classified into two groups based on changes of the serum level of CEA, "responder in CEA level" showed better prognosis than "non-responder in CEA level". Major toxicities were nausea, hyperglycemia and neutropenia. Three patients experienced Grade 4 hematological side effect, but these complications resolved quickly in all patients except for one patient. PFL-therapy is effective for advanced colorectal cancer with large tumor burden and showed the same prognostic result as the American and European trials in spite of smaller number of treatment cycles and a history of previous chemotherapy. We will be able to demonstrate the usefulness of this regimen for Japanese patients with advanced colorectal cancers after adding new cases to the present report.