Effect of genetic polymorphism of human CYP2B6 on the metabolic activation of chlorpyrifos.

Chlorpyrifos (CPS) is a broad-spectrum organophosphate insecticide that is neurotoxic in humans. Chlorpyrifos oxon (CPO) is a toxic metabolite of CPS that is produced by CYP2B6. In this study, we examined the variability of CPS metabolism resulting from single-nucleotide polymorphisms in CYP2B6. Wild-type CYP2B6 (CYP2B6.1) and two variants each with a single amino acid substitution: CYP2B6.5 (R487C) and CYP2B6.8 (K139E) were co-expressed together with human NADPH-dependent cytochrome P450 reductase in Escherichia coli (E. coli). Both of the CYP2B6 variants were successfully expressed in E. coli. The conversion of CPS to CPO by the CYP2B6 variants was analyzed with high-performance liquid chromatography. Km and Vmax of the reaction by CYP2B6.1 were 18.50±2.94µM and 17.07±1.15mol/min/mol P450, respectively. The CYP2B6 variants produced CPO with the following kinetic parameters: Km for CYP2B6.5 and CYP2B6.8 were 20.44±6.43 and 44.69±9.97µM, respectively; and Vmax were 1.10±0.10 and 1.77±0.26mol/min/mol P450, respectively. These results indicate that the amino acid substitutions in the CYP2B6 variants suppressed the metabolic activation of CPS. CYP2B6 variants have altered capacity to bioactivate CPF and may affect individual susceptibility of CPF.

The synthesis and evaluation of polymer prodrug/collagen hybrid gels for delivery into metastatic cancer cells.

Metastatic cancer cells degrade extracellular matrix containing collagen. In this study, a variety of different polymer prodrugs have been synthesized and embedded in collagen gels for application in a metastasis-associated drug delivery system (DDS). Dendrimer-doxorubicin (Dox) prodrugs were prepared with different surfaces, including collagen peptides and polyethylene glycol. Furthermore, Dox was conjugated to linear poly(glutamic acid) (poly-Glu) instead of the dendrimer. The cytotoxicities of each of these polymer prodrug systems against the poorly invasive MCF-7 and highly invasive MDA-MB-231 cells were similar. The highly invasive MDA-MB-231 cells, however, were more sensitive than the MCF-7 cells to the polymer prodrugs-embedded collagen gels, suggesting that these polymer prodrugs/collagen hybrid gels would be useful for the development of metastasis-associated DDSs. The cytotoxicities of the polymer prodrugs were dependent on their chemical compositions. The collagen peptide-conjugated dendrimer prodrug/collagen hybrid gel demonstrated in vivo anticancer effects in an orthotopic metastatic mouse model. FROM THE CLINICAL EDITOR: In this study, a variety of polymer prodrugs have been synthesized and embedded in collagen gels to be used in a metastasis-associated drug delivery system, demonstrating in vivo anticancer effects in an orthotopic metastatic mouse model.

Metabolism of 7-ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants.

CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.

Hematological- and Neurological-Expressed Sequence 1 Gene Products in Progenitor Cells during Newt Retinal Development.

Urodele amphibians such as Japanese common newts have a remarkable ability to regenerate their injured neural retina, even as adults. We found that hematological- and neurological-expressed sequence 1 (Hn1) gene was induced in depigmented retinal pigment epithelial (RPE) cells, and its expression was maintained at later stages of newt retinal regeneration. In this study, we investigated the distribution of the HN1 protein, the product of the Hn1 gene, in the developing retinas. Our immunohistochemical analyses suggested that the HN1 protein was highly expressed in an immature retina, and the subcellular localization changed during this retinogenesis as observed in newt retinal regeneration. We also found that the expression of Hn1 gene was not induced in mouse after retinal removal. Our results showed that Hn1 gene can be useful for detection of undifferentiated and dedifferentiated cells during both newt retinal development and regeneration.

Microarray of human P450 with an integrated oxygen sensing film for high-throughput detection of metabolic activities.

A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.

Vertically integrated human P450 and oxygen sensing film for the assays of P450 metabolic activities.

An assaying method of cytochrome P450 (P450 or CYP) monooxygenase activities for toxicological evaluation of drugs and environmental pollutants was developed by immobilizing P450 on an oxygen sensoring layer. Membrane fractions from E. coli expressing human P450 were entrapped in agarose or silica-based gels and immobilized on 96-well microarrays having an oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). P450 activity toward the substrates was monitored through the fluorescence intensity enhancement due to the oxygen consumption by the metabolic reactions. For the metabolism of chlortoluron, a selective herbicide used to control grass weeds, CYP1A1 immobilized in agarose gel showed a higher activity and stability compared with those in silica gels and free suspensions. The luminescence changing rate evaluated by the dynamic transient method (DTM) could be correlated with the substrate concentration. We also compared the metabolic responses of human P450s (CYP1A1,CYP2C8, CYP2E1, CYP3A4) toward various substances. The use of immobilized P450 on an oxygen sensing layer provides a versatile assaying platform owing to the following features. First, the oxygen sensor can detect metabolic reactions of any P450 species, in contrast with assays using fluorogenic substrates. Second, vertical integration of the oxygen sensor and immobilized P450 enhanced the sensitivity because of the effective depletion of oxygen in the vicinity of the oxygen sensing layer. Third, immobilization enables repeated use of P450 by replacing the substrate solutions using a flow cell. Furthermore, the activity of immobilized P450 was retained at least for 3 weeks at 4 °C, suggesting its long-term stability, which is an additional attractive feature.

Isokinetic trunk and knee muscle strengths and gait performance in walking patients with T-cell lymphotropic virus type 1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP).

The aim of this study was to investigate the isokinetic trunk and knee muscle strengths, and examine the clinical relevance of dynamic muscle strengths and gait performance in walking patients with human T-cell lymphotropic virus type 1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP). Thirteen patients with HAM/TSP (8 females and 5 males, aged 38-76) and 13 sex- and age-matched healthy control subjects participated in the study. We assessed gait speed, stride length, cadence; and maximal isokinetic torque of trunk and knee extensors and flexors at 30°/s, 60°/s and 90°/s using a Biodex System 3 dynamometer. Furthermore, we calculated the isokinetic trunk extensor/flexor (E/F) and hamstrings/quadriceps (H/Q) strength ratios (parameter of the muscle strength balance about the trunk and knee joint). Compared with the age-matched controls, the patients with HAM/TSP had significantly reduced gait speed, stride length and cadence (P < 0.05). Peak torque values related to body weight (PTBW) were significantly reduced, especially for the knee flexors (P < 0.05). For the knee extensors, the PTBW values were significantly reduced at an increased angular velocity (P < 0.05). The PTBW of knee flexors was positively correlated with gait speed and cadence in the patients with HAM/TSP. The H/Q ratio but not E/F ratio was significantly decreased compared with the control. Our results indicated that the isokinetic trunk and knee muscle performance had reduced from the ambulatory stage, and suggested the deterioration in knee muscle performance to be associated with gait disturbance in walking HAM/TSP patients.

Glucose concentration determination based on silica sol-gel encapsulated glucose oxidase optical biosensor arrays.

Optical biosensor arrays for rapidly determining the glucose concentrations in a large number of beverage and blood samples were developed by immobilizing glucose oxidase (GOD) on oxygen sensor layer. Glucose oxidase was first encapsulated in silica based gels through sol-gel approach and then immobilized on 96-well microarrays integrated with oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). The oxidation reaction of glucose by glucose oxidase could be monitored through fluorescence intensity enhancement due to the oxygen consumption in the reaction. The luminescence changing rate evaluated by the dynamic transient method (DTM) was correlated with the glucose concentration with the wide linear range from 0.1 to 5.0mM (Y=13.28X-0.128, R=0.9968) and low detection limit (0.06 mM). The effects of pH and coexisting ions were systemically studied. The results showed that the optical biosensor arrays worked under a wide range of pH value, and normal interfering species such as Na(+), K(+), Cl(-), PO(4)(3-), and ascorbic acid did not cause apparent interference on the measurement. The activity of glucose oxidase was mostly retained even after 2-month storage, indicating their long-term stability.

The effects of single nucleotide polymorphisms in CYP2A13 on metabolism of 5-methoxypsoralen.

A number of studies have demonstrated that cytochrome P450 (P450) converts furanocoumarin derivatives into reactive molecules, which form covalent bonds to biomolecules. 5-Methoxypsoralen (5-MOP) is a natural furanocoumarin from apiaceous plants. In this study, we examined the effect on 5-MOP metabolism of single nucleotide polymorphisms (SNPs) in CYP2A13. We used Escherichia coli-generated recombinant enzymes of wild-type CYP2A13*1 and five variants, CYP2A13*4 (R101Q), CYP2A13*5 (F453Y), CYP2A13*6 (R494C), CYP2A13*8 (D158E), and CYP2A13*9 (V323L). In high-performance liquid chromatography analyses of 5-MOP metabolic products, CYP2A13*1 converted 5-MOP into 5-MOP dihydrodiol; K(m) and V(max) values of the reaction were 1.44 ± 0.17 µM and 4.23 ± 0.36 nmol/(min · nmol P450), respectively. The generation of a dihydrodiol from 5-MOP implies that conversion by CYP2A13 causes toxicity due to the formation of covalent bonds with DNA or proteins. Most of the CYP2A13 variants could metabolize 5-MOP; K(m) values for CYP2A13*5, *6, *8, and *9 were 1.63 ± 0.12, 1.36 ± 0.10, 0.85 ± 0.09, and 0.58 ± 0.06 µM, respectively, and V(max) values were 3.20 ± 0.13, 4.69 ± 0.13, 2.34 ± 0.07, and 1.84 ± 0.09 nmol/(min · nmol P450), respectively. However, the processing of 5-MOP by CYP2A13*4 was not detectable. Based on this data, we hypothesize that SNPs within the CYP2A13 gene affect metabolism of 5-MOP in humans.

Induced expression of hematopoietic- and neurologic-expressed sequence 1 in retinal pigment epithelial cells during newt retina regeneration.

Newts can regenerate their organs even as adults. For instance, when their neural retinas are completely removed by operation, the remaining retinal pigment epithelial (RPE) cells dedifferentiate to reconstruct neural retinas. To elucidate the molecular mechanisms of newt retina regeneration, we investigated genes upregulated in dedifferentiating RPE cells using differential display methods. We observed that a cDNA fragment of hematopoietic- and neurologic-expressed sequence 1 (Hn1) appeared to be induced within a few days of surgical removal of newt neural retina. Using an anti-HN1 antiserum against the recombinant HN1 protein, we carried out immunohistochemical analyses. The anti-HN1 antiserum recognized the plexiform layers and ganglion cell layer (GCL) but not the RPE cell layer in unoperated (normal) newt retinas. Using a glial fibrillary acidic protein antibody, Hn1 was shown to be possibly expressed in glial cells in normal neural retina. During retina regeneration, immunoreactivity for HN1 appeared in dedifferentiating RPE cells 10 days post-operation, and in retinal progenitor cells 18 days post-operation. Twenty seven days post-operation, HN1 immunoreactivity was localized in the plexiform layers and GCL as in the normal retina. Therefore, HN1 possibly plays an undefined role in dedifferentiating RPE cells and retinal progenitor cells during newt retina regeneration.