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With a focus on marine sediment launched by the tsunami accompanying the Great East Japan Earthquake, we examined the vertical (i.e., depths of 0-2, 2-10, and 10-20 mm) profiles of reduced inorganic sulfur species and microbial community using a newly improved sulfur-fractionation method and 16S rRNA gene sequencing. S0 accumulated at the largest quantities at a depth of 2-10 mm, while the reduced forms of sulfur, such as iron(II) sulfide and pyrite, were abundant below 2 mm of the sediment. Operational taxonomic units (OTUs) related to chemolithotrophically sulfur-oxidizing Sulfurimonas denitrificans and Sulfurimonas autotrophica were only predominant at 2-10 mm, suggesting the involvement of these OTUs in the oxidation of sulfide to S0. In addition, Desulfocapsa sulfexigens, which is capable of chemolithotrophically disproportionating S0, prevailed at the same depth, indicating that accumulated S0 was converted to sulfate and sulfide. Although no significant differences were observed in sulfate concentrations across the depths examined, specific species of chemoorganotrophic sulfate reducers, i.e., Desulfotignum toluenicum and Desulfosalsimonas propionicica, showed significantly higher abundance at a depth of 2-10 mm than at the other depths examined. Organic matter potentially generated from sulfur oxidation and disproportionation may have served as the carbon source for the growth of these sulfate reducers. The present results demonstrated that sulfur oxidizers, a sulfur disproportionator, and sulfate reducers played vital roles in sulfur cycling with S0 as the key inorganic sulfur species in the oxic-anoxic boundary layer of the launched marine sediment.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

Several of the procarcinogens inhaled in tobacco smoke, the primary risk factor for bladder cancer, are activated by CYP2A6. The association between the whole-gene deletion of CYP2A6 (CYP2A6*4) and a reduced risk of bladder cancer was suggested in Chinese Han smokers. However, there is no evidence for association between the risk of bladder cancer and CYP2A6 genotypes in the Japanese population. Using genomic DNA from smokers of the Japanese population (163 bladder cancer patients and 116 controls), we conducted a case-control study to assess the association between CYP2A6 polymorphisms and the risk of bladder cancer. Determination of CYP2A6 genotypes was carried out by amplifying each exon of CYP2A6 using polymerase chain reaction (PCR) and Sanger sequencing. The CYP2A6*4 allele was identified by an allele-specific PCR assay. Bladder cancer risk was evaluated using the activity score (AS) system based on CYP2A6 genotypes. The odds ratios (95% confidence interval) for the AS 0, AS 0.5, AS 1.0, and AS 1.5 groups were 0.46 (0.12-1.83), 0.43 (0.15-1.25), 0.86 (0.40-1.86), and 1.36 (0.60-3.06), respectively. In conclusion, although decreased CYP2A6 AS tended to reduce the risk of bladder cancer in Japanese smokers, no significant association was recognized in this population. However, given the relatively small size of the sample, further study is required to conclude the lack of a statistically significant association between CYP2A6 genotypes and the risk of bladder cancer.

Genetic variations in cytochrome P450 4A11 (CYP4A11) contributes to inter-individual variability in the metabolism of fatty acids such as arachidonic acid. CYP4A11 metabolizes arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), which is important for the regulation of blood pressure. Polymorphisms in CYP4A11 are associated with susceptibility to hypertension. In this study, we evaluated the in vitro ω-hydroxylation of arachidonic acid by 10 CYP4A11 allelic variants, which cause amino acid substitutions in the encoded proteins. CYP4A11 variants were heterologously expressed in COS-7 cells and the kinetic parameters of arachidonic acid ω-hydroxylation were estimated. Among 10 CYP4A11 variants, 5 (CYP4A11-v1, CYP4A11-v2, CYP4A11-v3, CYP4A11-v4, and CYP4A11-v7) showed no or markedly lower activity compared to wild-type CYP4A11. This functional analysis of CYP4A11 variants could provide useful information for the effective prevention and treatment of hypertension.

Cytochrome P450 2A13 (CYP2A13) is responsible for the metabolism of chemical compounds such as nicotine, coumarin, and tobacco-specific nitrosamine. Several of these compounds have been recognized as procarcinogens activated by CYP2A13. We recently showed that CYP2A13*2 contributes to inter-individual variations observed in bladder cancer susceptibility because CYP2A13*2 might cause a decrease in enzymatic activity. Other CYP2A13 allelic variants may also affect cancer susceptibility. In this study, we performed an in vitro analysis of the wild-type enzyme (CYP2A13.1) and 8 CYP2A13 allelic variants, using nicotine and coumarin as representative CYP2A13 substrates. These CYP2A13 variant proteins were heterologously expressed in 293FT cells, and the kinetic parameters of nicotine C-oxidation and coumarin 7-hydroxylation were estimated. The quantities of CYP2A13 holoenzymes in microsomal fractions extracted from 293FT cells were determined by measuring reduced carbon monoxide-difference spectra. The kinetic parameters for CYP2A13.3, CYP2A13.4, and CYP2A13.10 could not be determined because of low metabolite concentrations. Five other CYP2A13 variants (CYP2A13.2, CYP2A13.5, CYP2A13.6, CYP2A13.8, and CYP2A13.9) showed markedly reduced enzymatic activity toward both substrates. These findings provide insights into the mechanism underlying inter-individual differences observed in genotoxicity and cancer susceptibility.

Tobacco-specific nitrosamines including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN), which can be activated by the metabolic enzyme CYP2A13, are potent procarcinogens. Smoking plays a role in carcinogenesis in the human bladder, which expresses CYP2A13 at a relatively high level. Numerous genetic polymorphisms of CYP2A13 causing amino acid substitution might reduce CYP2A13 metabolic activity toward NNK and NNN, resulting in decreased susceptibility to bladder cancer. The aim of this study was to reveal any association between bladder cancer development and CYP2A13 genetic polymorphisms in Japanese smokers. The CYP2A13 genotype of each subject (163 bladder cancer patients and 161 controls) was determined by next-generation sequencing (NGS) of the full CYP2A13 gene. All samples were genotyped for five CYP2A13 variant alleles (CYP2A13*2, *3, *4, *6, *7). Based on biological logistic regression, the odds ratio (95% confidence interval) for the CYP2A13*1/*2 genotype was 0.34 (0.17-0.69). Thus, CYP2A13 genetic polymorphisms might play important roles in the development of bladder cancer in Japanese smokers.

CYP2A6, a member of the cytochrome P450 (P450) family, is one of the enzymes responsible for the metabolism of therapeutic drugs and such tobacco components as nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and N-nitrosodiethylamine. Genetic polymorphisms in CYP2A6 are associated with individual variation in smoking behavior, drug toxicities, and the risk of developing several cancers. In this study, we conducted an in vitro analysis of 34 allelic variants of CYP2A6 using nicotine and coumarin as representative CYP2A6 substrates. These variant CYP2A6 proteins were heterologously expressed in 293FT cells, and their enzymatic activities were assessed on the basis of nicotine C-oxidation and coumarin 7-hydroxylation activities. Among the 34 CYP2A6 variants, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.10, CYP2A6.26, CYP2A6.36, and CYP2A6.37 exhibited no enzymatic activity, whereas 14 other variants exhibited markedly reduced activity toward both nicotine and coumarin. These comprehensive in vitro findings may provide useful insight into individual differences in smoking behavior, drug efficacy, and cancer susceptibility.

A microanalysis system with functions including automatic valve operation for mixing solutions, pH regulation, and detection based on electrochemiluminescence (ECL) was fabricated. Automatic valve operation was achieved by means of direct electrowetting using one of two solutions as a part of an electronic circuit. A valve formed with a gold electrode was opened when the second solution was filled in a compartment, causing the mixing of the two solutions. The pH regulation was achieved by using a nonstandard three-electrode system. The pH of the solution could be changed as desired by changing the working electrode potential, and the negative feedback function maintained the pH at a constant level. The ECL was generated on a platinum working electrode. Amino acids were detected as model analytes. A reagent solution containing tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy) 3 2+ ) and a sample solution containing an amino acid were mixed, and the pH was adjusted using the pH regulator. When +1.1 V (vs. Ag/AgCl) was applied to the electrode, red luminescence was observed. An increase in the ECL intensity was observed with an increase in the concentration. Amino acids of concentrations ranging from sub nM to 1 mM could be determined on the integrated device.

An integrated device that carries out the timely transport of solutions and conducts electroanalysis was constructed. The transport of solutions was based on capillary action in overall hydrophilic flow channels and control by valves that operate on the basis of electrowetting. Electrochemical sensors including glucose, lactate, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), pH, ammonia, urea, and creatinine were integrated. An air gap structure was used for the ammonia, urea, and creatinine sensors to realize a rapid response. To enhance the transport of ammonia that existed or was produced by the enzymatic reactions, the pH of the solution was elevated by mixing it with a NaOH solution using a valve based on electrowetting. The sensors for GOT and GPT used a freeze-dried substrate matrix to realize rapid mixing. The sample solution was transported to required sensing sites at desired times. The integrated sensors showed distinct responses when a sample solution reached the respective sensing sites. Linear relationships were observed between the output signals and the concentration or the logarithm of the concentration of the analytes. An interferent, L-ascorbic acid, could be eliminated electrochemically in the sample injection port.

Several of the procarcinogens inhaled in tobacco smoke, the primary risk factor for bladder cancer, are activated by CYP2A6. The association between the whole-gene deletion of CYP2A6 (CYP2A6*4) and a reduced risk of bladder cancer was suggested in Chinese Han smokers. However, there is no evidence for association between the risk of bladder cancer and CYP2A6 genotypes in the Japanese population. Using genomic DNA from smokers of the Japanese population (163 bladder cancer patients and 116 controls), we conducted a case-control study to assess the association between CYP2A6 polymorphisms and the risk of bladder cancer. Determination of CYP2A6 genotypes was carried out by amplifying each exon of CYP2A6 using polymerase chain reaction (PCR) and Sanger sequencing. The CYP2A6*4 allele was identified by an allele-specific PCR assay. Bladder cancer risk was evaluated using the activity score (AS) system based on CYP2A6 genotypes. The odds ratios (95% confidence interval) for the AS 0, AS 0.5, AS 1.0, and AS 1.5 groups were 0.46 (0.12-1.83), 0.43 (0.15-1.25), 0.86 (0.40-1.86), and 1.36 (0.60-3.06), respectively. In conclusion, although decreased CYP2A6 AS tended to reduce the risk of bladder cancer in Japanese smokers, no significant association was recognized in this population. However, given the relatively small size of the sample, further study is required to conclude the lack of a statistically significant association between CYP2A6 genotypes and the risk of bladder cancer.

On-chip determination of amino acids was conducted by electrochemiluminescence (ECL) following microfluidic transport, mixing of solutions, and generation of ECL. The fabricated system consisted of a polydimethylsilixane (PDMS) substrate with a flow channel structure and three-electrode systems formed on a glass substrate that resulted in the microfluidic transport and the ECL. An amino acid solution and a reagent solution containing tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+) were transported by electrowetting by applying a negative potential to gold working electrodes formed along the flow channels. The two solutions were then mixed in the mixing channel using an additional gold working electrode formed between the electrodes for transport. ECL was generated by applying a potential to a platinum working electrode formed in the mixing channel. The ECL from l -proline, l -lysine, l -leucine, l -valine, and l -histidine was examined. Clear ECL was observed, and its intensity depended on the concentration of the amino acids. By increasing the pH by mixing solutions and generating the oxidized form, Ru(bpy)33+, on the chip, the detection sensitivity could be improved. l -proline showed the strongest ECL, and concentrations of the nM order could be detected.

An integrated system was developed that performs microfluidic transport, mixing, and sensing on a single chip. The operation principle for the microfluidic transport was based on electrowetting. A solution to be transported was confined in a space between a row of gold working electrodes and a protruding poly(dimethylsiloxane) (PDMS) structure. When a negative potential was applied to one of the gold working electrodes, it became hydrophilic, and the solution was transported through the flow channel. The solution could be transported in any desired direction in a network of flow channels by switching on necessary electrodes one by one. Furthermore, two solutions transported through two flow channels could be mixed using a mixing electrode based on the same principle. To demonstrate the applicability of a lab-on-a-chip, an air gap ammonia electrode was integrated by taking advantage of the open structure of the flow channel. Gaseous ammonia that was produced after pH adjustment and diffused through an air gap caused a pH change in the electrolyte layer, which was measured with an iridium oxide pH indicator electrode. The 90% response time was less than 1 min for the millimolar order of ammonia. The calibration curve was linear down to 10 microM. The ammonia-sensing system was also applied to construct biosensing systems for urea and creatinine. A linear relationship was observed between the potential and the logarithm of the concentration of the analytes down to 50 microM for both urea and creatinine. The developed microfluidic system can be a basic building block for future systems.

Cytochrome P450 2A6 (CYP2A6) is an enzyme involved in the metabolism of tobacco carcinogens, which are important risk factors in lung cancer. We and others have previously reported that CYP2A6*4 , a whole-gene deletion polymorphism, is associated with lower risk of lung cancer than the wild-type allele. However, the genotyping method used in these previous studies considered only the CYP2A6*4 allele; this lead to insufficient classification of the CYP2A6 genotype, thereby underestimating the frequencies of the deficient alleles. In this study, CYP2A6 genotypes of Japanese smokers (110 individuals with squamous cell lung cancer (SQCC) and 132 sex-matched cancer-free controls) were determined using a sequencing-based approach to determine CYP2A6 haplotypes. The risk of SQCC was evaluated using the activity score (AS) system to predict CYP2A6 phenotype from its genotype. The risk of developing SQCC was significantly lower in the poor metabolizers assigned as AS 0.5 (adjusted odds ratio [OR] = 0.13, 95% CI = 0.04–0.45, P = 0.001) and AS 0 (adjusted OR = 0.15, 95% CI = 0.03–0.82, P = 0.028) than in the extensive metabolizers assigned as AS 2.0. In conclusion, CYP2A6 genetic polymorphisms may play important roles in the development of SQCC in Japanese smokers.

Genetic polymorphisms of the dihydropyrimidinase gene ( DPYS ) may be associated with the development of severe toxicity to 5-fluorouracil, a drug used to treat solid tumors. In this study, we analyzed the nine coding exons and exon–intron junctions of DPYS in 183 Japanese individuals. We detected two novel single nucleotide polymorphisms (SNPs)–285C > T (Thr95Thr) and 349T > C (Trp117Arg)–in exon 2. The nonsynonymous SNP 349T > C was analyzed in 208 Japanese individuals. Although the allele frequency of the SNP in the Japanese population was found to be extremely low (0.13%), the enzymatic activity of the variant protein might be reduced compared with that of the wild-type protein.

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.

In conducting a bio/chemical analysis on a chip, several different procedures are usually required. In this study, we demonstrate the possibility to conduct necessary procedures on a single chip. Our fabricated device has functions such as automatic mixing of solutions, pH-regulation, and detection based on electrochemiluminescence (ECL). A reagent solution containing Ru(bpy)3 2+ and a sample solution containing an amino acid were mixed and the solution pH was adjusted using the above- mentioned mechanisms. When a potential was applied to the electrode for ECL, red luminescence was observed. The ECL intensity increased with the increase in the concentration of amino acids.

An integrated device which can conduct timely transport of a sample solution and analyze its components was constructed. The transport of solutions was based on capillary action on hydrophilic glass areas and the control by valves which operate based on electrowetting. Electrochemical sensors including glucose, lactate, GOT, GPT, pH, ammonia, urea, and creatinine were integrated. Sensors for the former four are amperometric whereas the detection of the latter four are based on potentiometry. The ammonia, urea, and creatinine sensors had an air gap structure to realize a fast response. The sensors for GOT and GPT had a freeze-dried substrate matrix to realize rapid mixing. The sample solution was transported to required sensing sites at desired times. The integrated sensors showed distinct responses upon the introduction of a sample solution. Linear relationships were observed between the output signals and the concentration or the logarithm of the concentration of the analytes.

Electrowetting was used to fabricate an integrated highly efficient microfluidic transport system. As an option, a row of elongated gold working electrodes were used to generate a driving force to mobilize a solution in a flow channel. Flow velocity could be changed by changing the potential of the working electrode. The solutions could be transported to any desired direction without using any valves. In another option, a working electrode was formed in a hydrophilic flow channel and used as a valve. When the valve was switched on, a solution in the flow channel passed the area and went forward by capillary action. Furthermore, two solutions transported through two flow channels could be mixed based on the same principle. Integrated biosensing systems could easily be constructed using these microfluidic components. In this study, the concentration of L-amino acids was determined by generating electrochemical luminescence (ECL) on an integrated platinum working electrode. Also, the activity of protease could be determined using an integrated potentiometric pH-sensing system

An integrated micro analysis system was fabricated using a microfluidic transport system driven by electrowetting and an air-gap ammonia sensor. The basic element in the system was a row of elongated gold working electrodes and a protruding polydimethylsiloxane (PDMS) structure which form an open channel structure. The wettability of the gold electrode was changed by applying a negative potential with respect to a Ag/AgCl electrode, and a solution introduced from an inlet was mobilized through the gap between the working electrode and the protruding structure. Also, a solution could be transported to any desired directions without using any valves. Furthermore, two solutions could be mixed based on the same principle. The open structure of the flow channel facilitated the integration of an air-gap ammonia sensor. Ammonia diffused from the mixing area was detected as the potential change of a pH-indicator electrode. The 90% response time was 45 s for 10 mM ammonia. The relation between the potential of the pH-indicator electrode and the logarithm of ammonia concentration was linear. Furthermore, a biosensing system was constructed by using immobilized urease or creatinine deiminase and the ammonia sensor. The concentration of urea and creatinine could be determined by measuring ammonia produced enzymatically from a sample solution. Linear calibration plot was obtained for urea concentrations down to 100 /spl mu/M and creatinine concentration down to 50 /spl mu/M.

The behavior of a microfluidic device based on transport by capillary action and control by direct electrowetting was examined by changing the device parameters. The device was constructed with a polydimethylsiloxane (PDMS) substrate with a flow channel and a glass substrate with electrodes to control electrowetting. The microfluidic transport and valve action could be explained by Washburn’s model, which suggests that the model can be used for designing such a device. The device could be used for the timely delivery of a solution into many flow channels as well as for the mixing of solutions.