Kento Ogawa

Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringers solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Tandospirone, a 5-HT1A agonist, ameliorates movement disorder via non-dopaminergic systems in rats with unilateral 6-hydroxydopamine-generated lesions.

Serotonin 1A (5-HT1A) receptors are distributed throughout the brain with their highest concentrations in the frontal cortex, subthalamic nucleus and entopeduncular nucleus as well as the dorsal and median raphe nucleus. There is growing evidence that 5-HT1A receptor agonists have an antidepressant effect in individuals with major depressive disorders. Recent clinical studies suggest that tandospirone, a highly potent and selective 5-HT1A receptor agonist used clinically as an antidepressant in Japan and China, may act as an antiparkinsonian drug. In the present study, we investigated the effect of tandospirone on contralateral rotational behavior in a unilateral hemiparkinsonian rat model produced with 6-hydroxydopamine (6-OHDA). Tandospirone, as well as 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT), significantly increased contralateral turnings in a dose-dependent manner (0.5-10 mg/kg). Tandospirone also remarkably potentiated the contralateral turning induced by 0.025 mg/kg of apomorphine. Pretreatment with WAY-100635, a 5-HT1A receptor antagonist, almost completely blocked the contralateral turning behavior evoked by tandospirone and 8-OHDPAT, but not that by apomorphine. SCH-23390, a selective dopamine D1 receptor antagonist, did not affect on the tandospirone-induced rotational behavior. These results suggested that tandospirone could act on postsynaptic 5-HT1A receptors and modulate excitatory amino acid pathways in the basal ganglia. Thus, tandospirone could have therapeutic potential for the treatment of Parkinsons disease by modulating neuronal activities of non-dopaminergic pathways.

Norharman, an indoleamine-derived β-carboline, but not Trp-P-2, a γ-carboline, induces apoptotic cell death in human neuroblastoma SH-SY5Y cells

Summary. Carbolines, azaheterocyclic amines derived from indoleamines, have various biological activities, such as neurotoxicity of β-carbolines and potent mutagenicity of γ-carbolines. In this study, structural significance among these carbolines was investigated in relation to the types of cell death, apoptosis and necrosis, using human neuroblastoma SH-SY5Y cells. DNA damage was quantitatively analyzed by a single-cell gel electrophoresis assay. DNA damage was induced by both β-carbolines, harman and norharman, and γ-carbolines, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-4-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), in a dose dependent manner. γ-Carbolines were more potent to damage DNA than β-carbolines. Alkaline lysis of the cells prevented DNA damage induced by β-carboline, and pre-treatment of the cells with cycloheximide, an inhibitor of protein synthesis, reduced DNA damage caused by norharman. Morphological observation showed condensed and fragmented nuclei typical for apoptosis, in the cells treated with norharman. Thus, DNA damage induced by norharman was proved to be apoptotic. However, harman, which had a methyl substitution at the position 1, might induce necrosis in the cells. On the other hand, γ-carbolines, Trp-P-1 and Trp-P-2, directly damaged DNA. Thus, the nitrogen atom at the γ-position and/or an amino group in carboline structure would be required to induce the direct DNA cleavage.

The tissue distribution of lidocaine in acute death due to overdosing

A 78-year-old woman committed double suicide with a bolus injection of 1 g of lidocaine hydrochloride. The tissue distribution of lidocaine in this fatal poisoning was investigated using an improved gas chromatographic method. The blood level was 42 microg/ml and the brain concentration was 70 miccro/g. Thus, the cause of death in this case was clearly diagnosed as lidocaine overdosing. The blood level of 42 microg/ml was higher than that calculated using the standard pharmacokinetic parameters. Lower blood/tissue ratios were also detected compared with the data described in the literature. These results indicated that the cardiac failure should have occurred immediately after the lidocaine injection. The present data show the distribution of lidocaine after acute death due to overdosing.

Genotyping of cytochrome P450 isoform genes is useful for forensic identification of cadaver

Tailor-made medical treatment based on the polymorphism of genes encoding drug-metabolizing enzymes has been advocated and is being tried on an experimental basis at numerous centers. If DNA polymorphism analysis becomes routine in tailor-made medical treatment, it will be very useful in forensic identification. In this study, we determined the genotype frequencies of five p450 (CYP) isoform genes, CYP1A2, CYP2D6, CYP2E1, CYP2C9 and CYP2C19 in 196 Japanese individuals to evaluate their forensic usefulness. These genes encode the most important enzymes among the CYP superfamily that metabolize clinically used drug. The frequency of each allele agreed well with those reported previously and their genotype frequencies did not deviate from those expected from Hardy-Weinberg equilibrium. CYP2C subfamilies such as CYP2C9 and CYP2C19 on chromosome 10 showed high sequence homology, as high as over 95% in the regions flanking polymorphic sites. Although 3240 genotype combinations of these five CYP isoform genes are theoretically possible, 101 combinations were detected in this study. The genotype frequencies of these five isoform genes excluded their linkage. The following two genotype combinations showed the highest frequency of 0.036: CYP1A2*1A/*1A, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1 and CYP1A2*1A/*1C, CYP2D6*1/*10, CYP2E1*1/*1, CYP2C9*1/*1 and CYP2C19*1/*1. Thus, genotyping of CYP isoform genes should be useful in forensic identification.

Simultaneous analyses of hypoglycemic agents and C-peptide are essential in a homicide case with the combined dosing insulin and insulin-releasing drug

A 5-year-old girl was given a sulfonylurea hypoglycemic agent, 25 mg of glibenclamide (ten tablets of Euglucon) with two benzodiazepine drugs, 2 mg of estazoram and 0.75 mg of triazolam (one tablet of Eurodin and three tablets of Halcion), by her 37-year-old pharmacist father and then injected with 70 units of insulin (NovoLet 40R). She died several hours after the injection of insulin. Autopsy was carried out 12 h after the death. A glibenclamide level of 103 ng/ml was detected in the serum collected from the heart at autopsy. The serum insulin and C-peptide concentrations were 295 microU/ml and 0.5 ng/ml, respectively. The high level of insulin and the low level of C-peptide indicated that most of the serum insulin was exogenous. The determination of the serum C-peptide concentration was useful to the diagnosis of hypoglycemia caused by exogenous insulin even in the case of co-administration with an endogenous-insulin-releasing agent.

Glutamate is not involved in the MPP+-induced dopamine overflow in the striatum of freely moving C57BL/6 mice.

Summary. The role of glutamate in the N-methyl-4-phenyl-dihydropyridinium (MPP+) toxicity has been argued in the past decade. However, the effects of glutamate efflux and NMDA antagonist on MPP+-induced dopamine overflow have not been documented. To clarify this, we perfused MPP+ through a microdialysis probe in the striatum of freely moving mature C57BL/6 mice. The 60-min perfusion of 10 and 100 μM MPP+ strikingly increased dopamine levels to 28- and 93-fold of the basal values, respectively. In contrast, an administration of MPP+ did not induce marked glutamate release: the MPP+-perfusion slightly increased the glutamate level at 100 μM, but not at 10 μM. The addition of 100 μM (+)-MK-801 or 200 μM (±)-AP-7 to the perfusate did not attenuate MPP+-induced dopamine overflow. The extent of dopamine release only depended on the amount of MPP+ accumulation into the cells. These results indicated that, at least in the striatum, neither glutamate release nor the NMDA antagonist, including (+)-MK-801, could regulate MPP+-evoked dopamine overflow.

Molecular evolution of alleles of the glycophorin A gene

Highly-homologous Glycophorin A (GPA), B and E genes are triplicate genes, and involve many subtypes and minor antigens constructing the Miltenberger subsystem. These genes and most of the variants are hypothesized to arise by recombination, because hot spots are located in the gene sequences. By sequencing exons 1-7 and introns 1-3 of standard alleles of GPA gene, M and N alleles were classified into six variations: provisionally called MN*M101, M102, M201, M202, N101 and N102 in our previous study. Here we further investigated the sequences of introns 4-6 using GPA gene-specific primers and by DNA sequencing, and found eight, five and nine new nucleotide substitutions or deletions in introns 4, 5 and 6, respectively. Using the computer program PHYLIP 3.5, the phylogenetic trees were reconstructed. Phylogenetic analysis of the allele sequences revealed that M200s alleles arose from M101 after the separation of M101 and N101 and branched to M201 and M202 via the accumulation of point mutations. M102 and N102 alleles were estimated to generate via recombination between M101 and N101 occurred around the hot spot. The findings also suggested the existence of other GPA variants with normal antigenicity, and are quite useful in the forensic and anthropological fields.