Rational Supramolecular Strategy via Halogen Bonding for Effective Halogen Recognition in Molecular Imprinting.

Halogen bonding is a highly directional interaction and a potential tool in functional material design through self-assembly. Herein, we describe two fundamental supramolecular strategies to synthesize molecularly imprinted polymers (MIPs) with halogen bonding-based molecular recognition sites. In the first method, the size of the σ-hole was increased by aromatic fluorine substitution of the template molecule, enhancing the halogen bonding in the supramolecule. The second method involved sandwiching hydrogen atoms of a template molecule between iodo substituents, which suppressed competing hydrogen bonding and enabled multiple recognition patterns, improving the selectivity. The interaction mode between the functional monomer and the templates was elucidated by 1H NMR, 13C NMR, X-ray absorption spectroscopy, and computational simulation. Finally, we succeeded in the effective chromatographic separation of diiodobenzene isomers on the uniformly sized MIPs prepared by multi-step swelling and polymerization. The MIPs selectively recognized halogenated thyroid hormones via halogen bonding and could be applied to screening endocrine disruptors.

Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions.

We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.

Hg isotopic composition and total Hg mass fraction in NIES Certified Reference Material No. 28 Urban Aerosols.

An interlaboratory study on the National Institute for Environmental Studies (NIES) certified reference material (CRM) No. 28 Urban Aerosols, collected on the filters of a central ventilating system in a building in Beijing city center, was performed to obtain informative values of Hg isotopic composition and total Hg (THg) mass fraction. The THg mass fraction was determined by four organizations using atomic absorption spectrometry; it resulted in the mean value of 1.19 ± 0.12 mg/kg (2SD, n = 24). The Hg isotopic composition of the CRM was measured and intercompared at two different institutions by cold vapor generation system coupled to multicollector inductively coupled plasma mass spectrometry. Subsequently, a conventional dissolution method that uses a mixture of HNO3/HCl/H2O2 in Hotblock® and two different dissolution methods that use a mixture of HNO3/HCl with a microwave and a digestion bomb were applied. The Hg isotopic compositions were δ202Hg = - 1.26 ± 0.17‰, Δ199Hg = - 0.23 ± 0.06‰, Δ200Hg = 0.01 ± 0.07‰, and Δ201Hg = - 0.22 ± 0.09‰ (2SD, n = 18) for the conventional method, which agree well with those obtained using microwave and bomb digestion. Our results indicate that, for the quality control of particulate matter analyses, this CRM is appropriate for use in environmental and geochemical studies. Graphical abstract.

DNA methylation changes involved in the tumor increase in F2 males born to gestationally arsenite-exposed F1 male mice.

Multigenerational adverse effects from the environment such as nutrition and chemicals are among important concerns in environmental health issues. Previously, we have found that arsenite exposure of only F0 females during their pregnancy increases hepatic tumors in the F2 males in C3H mice. In the current study, we investigated the association of DNA methylation with the hepatic tumor increase in the F2 males of the arsenite group. Reduced-representation bisulfite sequencing analysis newly identified that DNA methylation levels of regions around the transcriptional start sites of Tmem54 and Cd74 were decreased and the expression of these genes were significantly increased in the hepatic tumors of F2 males of the arsenite group. The associations between DNA methylation in these regions and gene expression changes were confirmed by treatment of murine hepatoma cell lines and hepatic stellate cell line with 5-aza-2'-deoxycytidine. Overexpression of Cd74 in Hepa1c1c7 cells increased Trib3 expression and suppressed the expression of tumor suppressor genes Id3 and Atoh8. Human database analysis using the Cancer Genome Atlas indicated that TMEM54, CD74, and TRIB3 were significantly increased and that ATOH8 was decreased in hepatocellular carcinoma. The data also showed that high expression of TMEM54 and TRIB3 and low expression of ATOH8 were associated with poor survival. These results suggested that an increase in Tmem54 and Cd74 expression via DNA methylation reduction was involved in the tumor increase in the F2 male offspring by gestational arsenite exposure of F0 females. This study also suggested that genes downstream of Cd74 were involved in tumorigenesis.

Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography.

Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.

Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light.

Unique Separation Behavior of a C60 Fullerene-Bonded Silica Monolith Prepared by an Effective Thermal Coupling Agent.

Herein, we report a newly developed C60 fullerene-bonded silica monolith in a capillary with unique retention behavior due to the structure of C60 fullerene. N-Hydroxysuccinimide (NHS)-conjugated C60 fullerene was successfully synthesized by a thermal coupling agent, perfluorophenyl azide (PFPA), and assigned by spectroscopic analyses. Then, NHS-PFPA-C60 fullerene was attached onto the surface of a silica monolith in a capillary. The capillary provided specific separation ability for polycyclic aromatic hydrocarbons in liquid chromatography by an effective π-π interaction. Furthermore, corannulene, which has a hemispherical structure, was selectively retained in the capillary based on the specific structural recognition due to the spherical C60 fullerene. This is the first report revealing the spherical recognition ability by C60 fullerene in liquid chromatographic separation.

The effect of a methyl-deficient diet on the global DNA methylation and the DNA methylation regulatory pathways.

Methyl-deficient diets are known to induce various liver disorders, in which DNA methylation changes are implicated. Recent studies have clarified the existence of the active DNA demethylation pathways that start with oxidization of 5-methylcytosine (5meC) to 5-hydroxymethylcytosine by ten-eleven translocation (Tet) enzymes, followed by the action of base-excision-repair pathways. Here, we investigated the effects of a methionine-choline-deficient (MCD) diet on the hepatic DNA methylation of mice by precisely quantifying 5meC using a liquid chromatography-electrospray ionization-mass spectrometry and by investigating the regulatory pathways, including DNA demethylation. Although feeding the MCD diet for 1 week induced hepatic steatosis and lower level of the methyl donor S-adenosylmethionine, it did not cause a significant reduction in the 5meC content. On the other hand, the MCD diet significantly upregulated the gene expression of the Tet enzymes, Tet2 and Tet3, and the base-excision-repair enzymes, thymine DNA glycosylase and apurinic/apyrimidinic-endonuclease 1. At the same time, the gene expression of DNA methyltransferase 1 and a, was also significantly increased by the MCD diet. These results suggest that the DNA methylation level is precisely regulated even when dietary methyl donors are restricted. Methyl-deficient diets are well known to induce oxidative stress and the oxidative-stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine (8OHdG), is reported to inhibit DNA methylation. In this study, we also clarified that the increase in 8OHdG number per DNA by the MCD diet is approximately 10 000 times smaller than the reduction in 5meC number, suggesting the contribution of 8OHdG formation to DNA methylation would not be significant.

In vivo mutagenicity of arsenite in the livers of gpt delta transgenic mice.

While arsenic has been classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC), its mutagenicity has not been fully characterized in experimental animals. The aim of this study was to assess the in vivo mutagenicity of arsenite in C57BL/6J gpt delta mice. Male gpt delta mice were given drinking water containing sodium arsenite for 3 weeks, and the hepatic genome was assayed for mutations 2 weeks later. The gpt mutation assays showed a significant increase in mutation frequency in the liver following arsenite exposure. Sequence analysis revealed that 67% of mutations detected are G:C to A:T transitions and 5% are G:C to T:A transversions in the control group, and arsenite exposure resulted in a markedly higher rate of G:C to T:A transversions (46% of mutations detected). G:C to T:A transversions have been reported to be induced following formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a representative product that results from oxidative DNA damage. We also detected a significant increase in 8-OHdG in the livers of the mice exposed to arsenite. These results demonstrate that arsenite has mutagenicity in vivo and suggest that arsenite induces G:C to T:A transversions through oxidative-stress-induced 8-OHdG formation.

Genome-wide analysis of DNA methylation changes induced by gestational arsenic exposure in liver tumors.

Inorganic arsenic is known to be a human carcinogen. Previous studies have reported that DNA methylation changes are involved in arsenic-induced carcinogenesis, therefore, DNA methylation changes that are specific to arsenic-induced tumors would be useful to distinguish tumors induced by arsenic from tumors caused by other factors and to dissect arsenic carcinogenesis. Previous studies have shown that gestational arsenic exposure of C3H mice, which tend to spontaneously develop liver tumors, increases the incidence of tumors in male offspring. In this study we used the same experimental protocol as in those previous studies and searched for DNA regions where methylation status was specifically altered in the liver tumors of arsenic-exposed offspring by using methylated DNA immunoprecipitation-CpG island microarrays. The methylation levels of the DNA regions selected were measured by quantitative methylation-specific PCR and bisulfite sequencing. The results of this study clarified a number of regions where DNA methylation status was altered in the liver tumors in the C3H mice compared to normal liver tissues. Among such regions, we showed that a gene body region of the oncogene Fosb underwent alteration in DNA methylation by gestational arsenic exposure. We also showed that Fosb expression significantly increased corresponding to the DNA methylation level of the gene body in the arsenic-exposed group. These findings suggest that the DNA methylation status can be used to identify tumors increased by gestational arsenic exposure.

A novel retinoic acid analogue, 7-hydroxy retinoic acid, isolated from cyanobacteria.

BACKGROUND: All-trans retinoic acid (RA) is a low-molecular compound derived from vitamin A. It induces events in various ways by binding with the retinoic acid receptor (RAR), a nuclear receptor, in animal cells. RA and its metabolites have been found in animal tissues. In this paper, we report a novel RA analogue found in cyanobacterial cells, describe the method for its isolation, and compare its photo-stability with that of all-trans RA. METHODS: The new A analogue was extracted from cells of Microcystis aeruginosa and Spirulina sp. and fractionated by high-performance liquid chromatography. The analogue was analysed using a yeast two-hybrid assay method to measure in vitro RAR-agonistic activity. Liquid chromatography-mass spectrometry/mass spectrometry analyses was performed to elucidate the chemical structure of this RA analogue. RESULTS: The results of the analysis of the fragments revealed that the novel RA analogue was 7-hydroxy RA. The yields from 3.5 µg (4.5% of the total RAR-agonistic activity of Spirulina sp. cells) of 7-hydroxy RA was a mixture of 4 isomers due to cis-trans isomerisation coupled with keto-enol tautomerism; its relative RAR agonistic activity was 0.49 ± 0.01 (n=3) when the activity of all trans RA was set up to 1.00. Under fluorescent light, the mixture of 7-hydroxy RA isomers was more stable than all- trans RA. CONCLUSIONS: We isolated a novel RAR-activating compound, 7-hydroxy RA, from cyanobacteria. GENERAL SIGNIFICANCE: 7-hydroxy RA is more stable than all-trans RA under UV-A.

Molecular basis and phylogenetic implications of deoxycylindrospermopsin biosynthesis in the cyanobacterium Raphidiopsis curvata.

New insights into the distribution and biochemistry of the cyanotoxin cylindrospermopsin (CYN) have been provided by the recent determination of its biosynthesis gene cluster (cyr) in several cyanobacterial species. Raphidiopsis curvata CHAB1150 isolated from China was analyzed for CYN analogues. Only 7-deoxy-CYN was detected in the cell extracts. The cyr gene cluster of R. curvata CHAB1150 was sequenced, and the cyr genes of this strain were found to have extremely high similarities (96% to 100%) to those from other nostocalean species. These species include Cylindrospermopsis raciborskii AWT205, Aphanizomenon sp. strain 10E6, and Aphanizomenon ovalisporum ILC-146. Insertion mutation was identified within the cyrI gene, and transcripts of cyrI and another functional gene cyrJ were detected in R. curvata CHAB1150. General congruence between the phylogenetic trees based on both cyr and 16S rrn was displayed. Neutral evolution was found on the whole sequences of the cyr genes, and 0 to 89 negative selected codons were detected in each gene. Therefore, the function of CyrI is to catalyze the oxygenation of 7-deoxy-CYN in CYN biosynthesis. The transcripts of the mutated cyrI gene may result from polycistronic transcription. The high conservation of the cyr genes may be ascribed to purifying selection and horizontal gene transfer.

Biogenic phosphorus compounds in sediment and suspended particles in a shallow eutrophic lake: a ³¹P-nuclear magnetic resonance (³¹P NMR) study.

Differences in biogenic phosphorus (P) compounds between sediment and suspended particles in aquatic environments are important for understanding the mechanisms of internal P loading, but these differences are still unknown. We used solution-state (31)P-nuclear magnetic resonance spectroscopy ((31)P NMR) with NaOH-ethylenediaminetetraacetic extraction to detect the multiple P compounds in suspended particles and sediment in the eutrophic Lake Kasumigaura, including orthophosphate monoesters, orthophosphate diesters, pyrophosphate, and polyphosphate. We tested the hypothesis that there is a significant difference between these groups in suspended particles and sediment. Biogenic P other than orthophosphate was found in significantly higher proportions in suspended particles (74.3% of total P) than in sediment (25.6%). Orthophosphate monoesters were comparatively more abundant in suspended particles, as indicated by the ratio of orthophosphate diesters to monoesters (average, 0.31 for suspended particles; 1.05 for sediment). The compounds identified as orthophosphate monoesters by (31)P NMR spectroscopy originated mainly from phospholipids (α-glycerophosphate and ß-glycerophosphate) and ribonucleic acid (RNA-P), whereas the orthophosphate diesters included mostly DNA (DNA-P). These results suggest that the dynamics of orthophosphate diesters, the production of DNA-P, or the degradation of phospholipids, play an important role in P cycling in Lake Kasumigaura.

Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins.

Per-(15)N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography-mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in (15)NO(3)-containing TS-15 medium. To change from the incorporation of (14)N to (15)N into all cell components, cells of Microcystis aeruginosa were precultured in Na(15)NO(3)-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-(15)N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-(15)N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-(15)N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98-106%) was obtained, with the m/z of M(+), [M+1](+), and [M+2](+) of both microcystins and the per-(15)N-labeled microcystins as surrogates being completely separated. In conclusion, per-(15)N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography-mass spectrometry.

Global DNA methylation in the mouse liver is affected by methyl deficiency and arsenic in a sex-dependent manner.

Arsenic, a carcinogen, is assumed to induce global DNA hypomethylation by consuming the universal methyl donor S-adenosylmethionine (SAM) in the body. A previous study reported that a methyl-deficient diet (MDD) with arsenic intake greatly reduced global DNA methylation (the content of 5-methylcytosine) in the liver of male C57BL/6 mice. In the present study, we investigated the DNA methylation level, SAM content, and expression of DNA methyltransferases (DNMTs) in the liver of male and female C57BL/6 mice fed a methyl-sufficient diet (MSD), an MDD, or an MDD + arsenic. The DNA methylation level was accurately determined by measuring the content of genomic 5-methyldeoxycytidine (5medC) by high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) using stable-isotope-labeled 5medC and deoxycytidine (dC) as internal standards. The results of this study revealed that while the MDD and arsenic tended to reduce the genomic 5meC content in the male mice livers, the MDD + arsenic significantly increased the 5meC content in the female mice livers. Another unexpected finding was the small differences in 5meC content among the groups. The MDD and MDD + arsenic suppressed DNMT1 expression only in the male mice livers. In contrast, SAM content was reduced by the MDD and MDD + arsenic only in the livers of female mice, showing that the changes in 5meC content were not attributable to SAM content. The sex-dependent changes in 5meC content induced by methyl deficiency and arsenic may be involved in differences in male and female susceptibility to diseases via epigenetic modification of physiological functions.

l-Menthol increases extracellular dopamine and c-Fos-like immunoreactivity in the dorsal striatum, and promotes ambulatory activity in mice.

Similar to psychostimulants, the peripheral administration of menthol promotes mouse motor activity, and the neurotransmitter dopamine has been suggested to be involved in this effect. The present study aimed to elucidate the effects of l-menthol on parts of the central nervous system that are involved in motor effects. The subcutaneous administration of l-menthol significantly increased the number of c-Fos-like immunoreactive nuclei in the dorsal striatum of the mice, and motor activity was promoted. It also increased the extracellular dopamine level in the dorsal striatum of the mice. These observations indicated that after subcutaneous administration, l-menthol enhances dopamine-mediated neurotransmission, and activates neuronal activity in the dorsal striatum, thereby promoting motor activity in mice.

An acidic morpholine derivative containing glyceride from thraustochytrid, Aurantiochytrium.

A novel acidic morpholine-derivative containing glyceride (M-glyceride) was isolated from the cells of two strains of the thraustochytrid, Aurantiochytrium. The glyceride accounted for approximately 0.1 -0.4% of the lyophilized cells. The glyceride consisted of peaks I (85%) and II (15%). The structures of the intact and acetylated glycerides were elucidated by liquid chromatography-quadrupole time-of-flight chromatograph mass spectrometer (LC-Q/TOF) and NMR spectroscopy. The hydrate type of M-glyceride was detected as a minor component by LC-MS/MS. By 2D-NMR experiments, peaks I of the intact M-glyceride were elucidated as 1,2-didocosapentaenoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid, and peak II was estimated 1,2-palmitoyldocosapentaenoyl- and/or 1,2-docosapentaenoylpalmitoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid. The double bond position of docosapentaenoic acid was of the ω - 6 type (C22 = 5.ω - 6). The M-glyceride content varied by the cell cycle. The content was 0.4% of lyophilized cells at the mid logarithmic phase, and decreased to 0.1% at the mid stationary phase. When cells were grown in 1.0 µM M-glyceride-containing growth media, cell growth was stimulated to 110% of the control. With 0.1 µM acetyl M-glyceride, stimulation of 113% of the control was observed. Finding morpholine derivatives in biological components is rare, and 2-hydroxy-3-oxomorpholino propionic acid (auranic acid) is a novel morpholine derivative.

Fluorescent detection of target proteins via a molecularly imprinted hydrogel.

Proteins are typically separated by an immune reaction, such as an enzyme-linked immunosorbent assay, and are detected by selective fluorescent labeling. This has potential for complicated procedures and the denaturation of proteins by labeling, and is cost consuming. In this study, we propose a technique for the selective separation and detection of a target protein using a molecularly imprinted hydrogel (PI gel) with fluorescent monomers. We focused on 8-anilino-1-naphthalenesulfonic acid (ANS), where the fluorescence intensity is easily changed by the interaction with proteins, and successfully synthesized the ANS monomer and a poly(ethylene glycol) (PEG) conjugated ANS monomer. The PI gel with the ANS monomers using bovine serum albumin (BSA) as a template showed the selective adsorption of BSA and the fluorescence intensity increased due to the adsorption of BSA.

Prohydrojasmon Promotes the Accumulation of Phenolic Compounds in Red Leaf Lettuce.

Prohydrojasmon (PDJ) is a synthetic jasmonate derivative that is primarily used as a growth regulator, but its mechanism of action is unclear. In this study, we elucidated the effects of PDJ on phytochemical production in red leaf lettuce. The PDJ treatments promoted the accumulation of phenolic compounds in aerial plant parts. An LC-MS analysis revealed that these accumulated compounds were identified as cyanidin-3-O-glucoside, cyanidin-3-O-(6″-O-malonyl)-glucoside and cyanidin-3-O-(6″-O-malonyl)-glucoside methyl ester. The abundance of these compounds in lettuce extracts increased significantly in response to the PDJ treatment. Additionally, the LC-MS analysis also identified the accumulated phenolic compounds in the extracts of PDJ-treated lettuce, including caffeoyltartaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, and dicaffeoylquinic acid. Gene expression analyses indicated the PDJ treatments upregulated the expression of PAL, F3H, and ANS genes in lettuce. These results suggest that PDJ treatments enhance the expression of genes involved in the synthesis of anthocyanins and phenolic compounds, resulting in an increase in the quantities of these compounds, which reportedly have various functions affecting human physiology.

Simultaneous blood and brain microdialysis in a free-moving mouse to test blood-brain barrier permeability of chemicals.

Neurotoxic chemicals that pass through the blood-brain barrier (BBB) can influence brain function. Efficient methods to test the permeability of the BBB to specific chemicals would facilitate identification of potentially neurotoxic agents. We report here a simultaneous blood and brain microdialysis in a free-moving mouse to test BBB permeability of different chemicals. Microdialysis sampling was conducted in mice at 3-5 days after implantation of a brain microdialysis probe and 1 day after implantation of a blood microdialysis probe. Therefore, mice were under almost physiological conditions. Results of an intravenous injection of lucifer yellow or uranine showed that the BBB was functioning in the mice under the experimental conditions. Mice were given phenyl arsenic compounds orally, and concentration-time profiles for phenyl arsenic compounds such as diphenylarsinic acid, phenylarsonic acid, and phenylmethylarsinic acid in the blood and brain dialysate samples were obtained using simultaneous blood and brain microdialysis coupled with liquid chromatography-tandem mass spectrometry. Peak area-time profiles for linalool and 2-phenethyl alcohol (fragrance compounds or plant-derived volatile organic chemicals) were obtained using simultaneous blood and brain microdialysis coupled with gas chromatography-mass spectrometry in mice given lavender or rose essential oils intraperitoneally. BBB function was confirmed using lucifer yellow in these mice, and results indicated that the phenyl arsenic compounds, linalool and 2-phenethyl alcohol, passed through the BBB. The present study demonstrates that simultaneous blood and brain microdialysis in a free-moving mouse makes it possible to test the BBB permeability of chemicals when coupled with appropriate chemical analysis methods.