Mitochondrial toxins potentiate hydroxyl radical production in rat striatum during carbon monoxide poisoning.

Hydroxyl radical (•OH) production in the rat striatum during carbon monoxide (CO) poisoning, which inhibits complex IV, was enhanced synergistically by malonate, a mitochondrial complex II inhibitor, but not N-methyl-4-phenylpyridinium or NaCN, complex I and IV inhibitors, respectively. No such enhancement appeared in the case of NaCN combined with malonate. Intrastriatal dopamine, which is involved in •OH production by malonate, did not synergistically enhance CO-induced •OH production. Diphenyleneiodonium, a nonselective NADPH oxidase inhibitor, partly suppressed the potentiation of CO-induced •OH production by malonate. Impairment of mitochondrial functions might potentiate oxidative stress and intensify CO toxicity in the brain.

Kurarinone from Sophora Flavescens Roots Triggers ATF4 Activation and Cytostatic Effects Through PERK Phosphorylation.

In response to cellular stresses, activating transcriptional factor 4 (ATF4) regulates the expression of both stress-relieving genes and apoptosis-inducing genes, eliciting cell fate determination. Since pharmacological activation of ATF4 exerts potent anti-tumor effects, modulators of ATF4 activation may have potential in cancer therapy. We herein attempted to identify small molecules that activate ATF4. A cell-based screening to monitor TRB3 promoter activation was performed using crude drugs used in traditional Japanese Kampo medicine. We found that an extract from Sophora flavescens roots exhibited potent TRB3 promoter activation. The activity-guided fractionation revealed that kurarinone was identified as the active ingredient. Intriguingly, ATF4 activation in response to kurarinone required PKR-like endoplasmic reticulum kinase (PERK). Moreover, kurarinone induced the cyclin-dependent kinase inhibitor p21 as well as cytostasis in cancer cells. Importantly, the cytostatic effect of kurarinone was reduced by pharmacological inhibition of PERK. These results indicate that kurarinone triggers ATF4 activation through PERK and exerts cytostatic effects on cancer cells. Taken together, our results suggest that modulation of the PERK-ATF4 pathway with kurarinone has potential as a cancer treatment.

A glucosyltransferase specific for 4-hydroxy-2,5-dimethyl-3(2H)-furanone in strawberry.

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is a key aroma compound in Fragaria × ananassa (strawberry). A considerable amount of HDMF is converted into HDMF ß-D-glucoside and accumulated in mature strawberry fruits. Here we isolated a novel UDP-glucose: HDMF glucosyltransferase, UGT85K16 from Fragaria × ananassa. UGT85K16 preferentially glucosylated the hydroxyl group of HDMF and its structural analogs. Although UGT85K16 also catalyzed the glucosylation of vanillin, its affinity and efficiency toward HDMF was higher. The expression of UGT85K16 mRNA correlated with the accumulation of HDMF and its glucoside in Fragaria × ananassa plants. These results suggest that UGT85K16 might be UDP-glucose: HDMF glucosyltransferase in strawberries. ABBREVIATIONS: DMMF: 2,5-dimethyl-4-methoxy-3(2H)-furanone; EHMF: 2(5)-ethyl-4-hydroxy-5(2)-methyl-3(2H)-furanone; GBV: glycosidically bound volatile; HDMF: 4-hydroxy-2,5-dimethyl-3(2H)-furanone; HMF: 4-hydroxy-5-methyl-3(2H)-furanone; HMMF: 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone; PSPG: Plant secondary product glycosyltransferase; RT-PCR: reverse transcription-PCR; OMT: O-methyltransferase; UGT: UDP-glycosyltransferase.

Anti-Tumorigenic Activity of Chrysin from Oroxylum indicum via Non-Genotoxic p53 Activation through the ATM-Chk2 Pathway.

The p53 tumor suppressor plays critical roles in cell cycle regulation and apoptotic cell death in response to various cellular stresses, thereby preventing cancer development. Therefore, the activation of p53 through small molecules is an attractive therapeutic strategy for the treatment of cancers retaining wild-type p53. We used a library of 700 Myanmar wild plant extracts to identify small molecules that induce p53 transcriptional activity. A cell-based screening method with a p53-responsive luciferase-reporter assay system revealed that an ethanol extract of Oroxylum indicum bark increased p53 transcriptional activity. Chrysin was isolated and identified as the active ingredient in the O. indicum bark extract. A treatment with chrysin increased p53 protein expression and the p53-mediated expression of downstream target genes, and decreased cell viability in MCF7 cells, but not in p53-knockdown MCF7 cells. We also found that chrysin activated the ATM-Chk2 pathway in the absence of DNA damage. Hence, the inactivation of the ATM-Chk2 pathway suppressed p53 activation induced by chrysin. These results suggest the potential of chrysin as an anti-cancer drug through the activation of p53 without DNA damage.

A 7-deoxyloganetic acid glucosyltransferase contributes a key step in secologanin biosynthesis in Madagascar periwinkle.

Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-sugar glycosyltransferase6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.

Balloon-occluded arterial stump pressure before balloon-occluded transarterial chemoembolization.

OBJECTIVE: To evaluate balloon-occluded arterial stump pressure (BOASP), which is responsible for effective balloon-occluded transarterial chemoembolization (B-TACE), at each hepatic arterial level before B-TACE using a 1.8-French tip microballoon catheter for unresectable hepatocellular carcinoma (HCC). MATERIAL AND METHODS: The BOASP at various embolization portions was retrospectively investigated. "Selective" and "non-targeted" BOASP was defined as the BOASP at the subsegmental or segmental artery and the lobar artery, respectively. RESULTS: The measurement of the BOASP was carried out in 87 arteries in 47 patients. BOASP > 64 mmHg was revealed in the caudate lobe artery (A1) and the left medial segmental (A4), right anterior superior segmental (A8), anterior segmental, right and left hepatic arteries. Significant difference was noted in the incidence of BOASP above 64 mmHg between "non-targeted" and "selective" BOASP (p = 0.01). "Non-targeted" BOASP was significantly greater than "selective" BOASP (p = 0.0147). In addition, the BOASP in A1, 4, 8 and the anterior segmental arteries were significantly greater than in the other subsegmental and segmental arteries (p = 0.0007). CONCLUSION: "Non-targeted" B-TACE should be avoided to perform effective B-TACE and "selective" B-TACE at A1, 4, 8 and the anterior segmental arteries may become less effective than at the other segmental or subsegmental arteries.

The Influence of Drinking Based on Data from Health Check-up.

A survey of 21,493 patients who visited our Health Check-up Center during the 6-year period from 2005 to 2010 was conducted for the endpoints of drinking situation and various lifestyle-related diseases. Males accounted for 57.2% (mean age: 53.2 ± 11.4) and females accounted for 42.8% (mean age: 52.5 ± 11.4) of patients surveyed. Patients with no drinking habit accounted for 24.8% and 62.9% of males and females, respectively, and a large gender difference was seen in drinking frequency. When examined by age group, drinking frequency was found to increase with age in males, but gradually decreased with age in females. An examination of alcohol consumption in males revealed that 23.5% had an ethanol conversion rate of 10 g/day, 19.1% had a rate of < 20 g/day, and 18.2% had a rate of < 40 g/day. Meanwhile, in females, 22.7% had a rate of ≤ 10 g/day, 7.6% had a rate of ≤ 20 g/day and 4.6% had a rate of ≤ 40 g/day. The association between lifestyle-related disease endpoints and alcohol consumption was next examined by multivariate logistic analysis. The association between drinking and body mass index (BMI) revealed an odds ratio of around 0.8 in patients who consumed ≤ 40 g/day and a significantly reduced frequency of obesity. The odds ratio of hypertension increased in a dose-dependent manner from 1.3 to 1.6 in patients who consumed ≥ 40 g/day. The frequency of high low-density lipoprotein cholesterol (LDL-C) was reduced in light drinkers and the odds ratio decreased from 0.77 to about 0.6 as alcohol consumption increased: The frequency of low high-density lipoprotein cholesterol (HDL-C) was similarly reduced in light drinkers, and the odds ratio decreased remarkably in a dose-dependent manner from 0.73 to 0.22 as alcohol consumption increased. The risk of triglycerides was reduced in light drinkers and was conversely significantly enhanced in heavy drinkers. In patients who consumed ≥ 20 g/day, the risk of impaired glucose tolerance increased significantly in a dose-dependent manner. Increased risk of hyperuricemia was seen even in light drinkers. and the odds ratio increased from 1.2 to 1.8 as alcohol consumption increased. The results of this cross-sectional study suggested that light drinking has a positive effect on BMI, LDL-C, HDL-C and triglycerides. On the other hand, heavy drinking was found to have a positive effect on LDL-C and HDL-C, but a negative effect on systolic blood pressure, triglycerides, fasting blood glucose and uric acid.

Involvement of cardiac glycosides targeting Na/K-ATPase in their inhibitory effects on c-Myc expression via its transcription, translation and proteasomal degradation.

Cardiac glycosides (CGs) have been used for decades to treat heart failure and arrhythmic diseases. Recent non-clinical and epidemiological findings have suggested that CGs exhibit anti-tumor activities. Therefore, CGs may be repositioned as drugs for the treatment of cancer. A detailed understanding of the anti-cancer mechanisms of CGs is essential for their application to the treatment of targetable cancer types. To elucidate the factors associated with the anti-tumor effects of CGs, we performed transcriptome profiling on human multiple myeloma AMO1 cells treated with periplocin, one of the CGs. Periplocin significantly down-regulated the transcription of MYC (c-Myc), a well-established oncogene. Periplocin also suppressed c-Myc expression at the protein levels. This repression of c-Myc was also observed in several cell lines. To identify target proteins for the inhibition of c-Myc, we generated CG-resistant (C9) cells using a sustained treatment with digoxin. We confirmed that C9 cells acquired resistance to the inhibition of c-Myc expression and cell proliferation by CGs. Moreover, the sequencing of genomic DNA in C9 cells revealed the mutation of D128N in α1-Na/K-ATPase, indicating the target protein. These results suggest that CGs suppress c-Myc expression in cancer cells via α1-Na/K-ATPase, which provides further support for the anti-tumor activities of CGs.

Quantitative Determination of Carthamin in Carthamus Red by 1H-NMR Spectroscopy.

Carthamus Red is a food colorant prepared from the petals of Carthamus tinctorius (Asteraceae) whose major pigment is carthamin. Since an authentic carthamin standard is difficult to obtain commercially for the preparation of calibration curves in HPLC assays, we applied (1)H-NMR spectroscopy to the quantitative determination of carthamin in commercial preparations of Carthamus Red. Carthamus Red was repeatedly extracted in methanol and the extract was dissolved in pyridine-d(5) containing hexamethyldisilane (HMD) prior to (1)H-NMR spectroscopic analysis. The carthamin contents were calculated from the ratios of singlet signal intensities at approximately σ: 9.3 derived from H-16 of carthamin to those of the HMD signal at σ: 0. The integral ratios exhibited good repeatability among NMR spectroscopic analyses. Both the intra-day and inter-day assay variations had coefficients of variation of <5%. Based on the coefficient of absorption, the carthamin contents of commercial preparations determined by (1)H-NMR spectroscopy correlated well with those determined by colorimetry, although the latter were always approximately 1.3-fold higher than the former, irrespective of the Carthamus Red preparations. In conclusion, the quantitative (1)H-NMR spectroscopy used in the present study is simple and rapid, requiring no carthamin standard for calibration. After HMD concentration has been corrected using certified reference materials, the carthamin contents determined by (1)H-NMR spectroscopy are System of Units (SI)-traceable.

Prenylated-coumarins from Gmelina arborea and evaluation for neurotrophic activity.

A MeOH extract of the stem of Gmelina arborea Roxb. ex Sm. (Lamiaceae) exhibited neurite outgrowth-promoting activity in NGF-mediated PC12 cells. Bioassay-guided fractionation resulted in the isolation of eight previously undescribed prenylated coumarin compounds along with nine known compounds. Structural elucidation of these compounds was accomplished by analysis of extensive spectroscopic data, comparison with the literature, and chemical reactions. It was the first time to find prenylated coumarin compounds from G. arborea. Among the isolated compounds, N-methylflindersine and artanin showed neurite outgrowth-promoting activity in NGF-mediated PC12 cells.

A case of breast cancer with extensive colon metastasis.

Breast cancer is one of the most common malignancies in women worldwide. Although most breast cancers are curable, in cases of metastasis, many are often found in the lungs, bones, liver, and central nervous system; however, metastasis to the gastrointestinal tract is rare. Invasive lobular carcinoma, which represents only 5%-10% of breast cancers, has a higher risk of metastasis to the gastrointestinal tract than invasive ductal carcinoma. Here, we report a rare case of gastrointestinal metastasis of invasive lobular carcinoma that spread extensively to the colonic mucosa. Given the improved survival rates of breast cancer patients with current treatments, many rarer metastatic diseases, including gastrointestinal metastases, are likely to be increased in the future.

Iridoid-specific glucosyltransferase from Gardenia jasminoides.

Iridoids are one of the most widely distributed secondary metabolites in higher plants. They are pharmacologically active principles in various medicinal plants and key intermediates in the biosynthesis of monoterpenoid indole alkaloids as well as quinoline alkaloids. Although most iridoids are present as 1-O-glucosides, the glucosylation step in the biosynthetic pathway has remained obscure. We isolated a cDNA coding for UDP-glucose:iridoid glucosyltransferase (UGT85A24) from Gardenia jasminoides. UGT85A24 preferentially glucosylated the 1-O-hydroxyl group of 7-deoxyloganetin and genipin but exhibited only weak activity toward loganetin and no activity toward 7-deoxyloganetic acid. This suggests that, in the biosynthetic pathway of geniposide, a major iridoid compound in G. jasminoides, glucosylation occurs after methylation of 7-deoxyloganetic acid. UGT85A24 showed negligible activity toward any acceptor substrates other than iridoid aglycones. Thus, UGT85A24 has a remarkable specificity for iridoid aglycones. The mRNA level of UGT85A24 overlaps with the marked increase in genipin glucosylation activity in the methyl jasmonate-treated cell cultures of G. jasminoides and is related to iridoid accumulation in G. jasminoides fruits.

Antitubercular activity of disulfiram, an antialcoholism drug, against multidrug- and extensively drug-resistant Mycobacterium tuberculosis isolates.

The antimycobacterial activities of disulfiram (DSF) and diethyldithiocarbamate (DDC) against multidrug- and extensively drug-resistant tuberculosis (MDR/XDR-TB) clinical isolates were evaluated in vitro. Both DSF and DDC exhibited potent antitubercular activities against 42 clinical isolates of M. tuberculosis, including MDR/XDR-TB strains. Moreover, DSF showed remarkable bactericidal activity ex vivo and in vivo. Therefore, DSF might be a drug repurposed for the treatment of MDR/XDR-TB.

3-Monoglucuronyl-glycyrrhretinic acid is a substrate of organic anion transporters expressed in tubular epithelial cells and plays important roles in licorice-induced pseudoaldosteronism by inhibiting 11ß-hydroxysteroid dehydrogenase 2.

Licorice (glycyrrhiza root) has been used as a herbal medicine worldwide with its main active constituent being glycyrrhizin (GL). Licorice sometimes causes adverse effects such as inducing pseudoaldosteronism by inhibiting type 2 11ß-hydroxysteroid dehydrogenase (11ß-HSD2) caused by glycyrrhetinic acid (GA), a major metabolite of GL. In this study we compared the inhibitory effects of GA, GL, and 3-monoglucuronyl-glycyrrhetinic acid (3MGA), another metabolite of GL, on 11ß-HSD2 activity by using microsomes and rat kidney tissue slices. GA, 3MGA, and GL inhibited 11ß-HSD2 in rat kidney microsomes, with IC(50) values of 0.32, 0.26, and 2.2 µM, respectively. However, the inhibitory activity of these compounds was reduced markedly, in the slices, in a medium containing 5% bovine serum albumin. Assays using human embryonic kidney 293 cells with transient transformation in transporter genes showed that 3MGA is a substrate of human organic anion transporter (OAT) 1, human OAT3, and human organic anion-transporting peptide 4C1, whereas GA is not. When GA (100 mg/kg/day) was administered orally for 16 days to Eisai hyperbilirubinemic rats, plasma concentrations and urinary excretion of 3MGA were significantly higher, whereas the activity of 11ß-HSD2 in kidney microsomes was significantly lower compared with Sprague Dawley rats. These results suggest that 3MGA is actively transported into tubules through OATs, resulting in the inhibition of 11ß-HSD2. Because the plasma level of 3MGA depends on the function of hepatic transporters, monitoring 3MGA levels in plasma or urine may be useful for preventing pseudoaldosteronism when licorice or GL is prescribed to patients.

A naturally occurring rexinoid, honokiol, can serve as a regulator of various retinoid x receptor heterodimers.

We investigated the properties of honokiol, a natural rexinoid, as a regulator of retinoid X receptor (RXR) heterodimers with various partner nuclear receptors (NRs), in comparison with those of the synthetic rexinoid bexarotene. Honokiol alone was hardly capable of activating peroxisome proliferator-activated receptor (PPAR) γ/RXR, RXR/liver X receptor (LXR), and RXR/vitamin D receptor (VDR) heterodimers, whereas it effectively potentiated their activation by agonists for the partner NRs of the RXR heterodimers. These findings were further supported by increased mRNA and protein levels for the respective NR target genes. Bexarotene alone activated PPARγ/RXR and RXR/LXR heterodimers, but not RXR/VDR heterodimers, and facilitated the activation of all three RXR heterodimers by the respective PPARγ, LXR, and VDR agonists. When the potencies of honokiol and bexarotene were compared, honokiol was able to serve as a subsidiary agonist in the activation of RXR heterodimers in a similar manner to bexarotene. However, it seemed to potentiate the activation of PPARγ/RXR heterodimers by the PPARγ agonist rosiglitazone more efficiently than bexarotene, and was a less potent RXR agonist than bexarotene. In conclusion, we have demonstrated that honokiol is a rexinoid that possesses distinct properties from bexarotene, and mainly has subsidiary roles in the activation of RXR heterodimers by potentiating the activation of RXR heterodimers by agonists for the partner NRs.

Characterization of Triterpene Saponin Glycyrrhizin Transport by Glycyrrhiza glabra.

Glycyrrhizin (GL), a triterpene compound produced by Glycyrrhiza species, is a crucial pharmacologically active component of crude drugs. In contrast to the biosynthesis of GL in plants, little is known about GL transport and accumulation in plants. The transport mechanism of GL was characterized using cultured cells of Glycyrrhiza glabra. Cultured cells of G. glabra efficiently incorporated exogenously supplied GL. Proton pump inhibitors, such as probenecid and niflumic acid, as well as a protonophore (carbonylcyanide m-chlorophenylhydrazone), markedly inhibited GL uptake by cultured cells, whereas vanadate exhibited a moderate inhibition. Furthermore, GL transport by G. glabra tonoplast vesicles is dependent not on a H+-electrochemical gradient but MgATP and is markedly inhibited by vanadate. These results suggest that GL uptake by cultured cells is mediated by a H+-symporter in the plasma membrane and an ATP-binding cassette transporter, which has high specificity for the aglycone structure of GL on the tonoplast.

Chukranoids A-I, isopimarane diterpenoids from Chukrasia velutina.

Bioactivity guided separation of Chukrasia velutina root methanolic extract led to the isolation of nine new isopimarane diterpenoids, chukranoids A-I (1-9). The absolute configuration was then assigned by comparing the experimental CD spectra and the calculated CD spectra. Chukranoids A-I (1-9) showed moderate antimalarial activity against Plasmodium falciparum 3D7 strain. It seems that conjugate system in the isopimarane skeleton may influence their antimalarial activity.

Differential gene expression in rat vascular smooth muscle cells following treatment with coptisine exerts a selective antiproliferative effect.

It is known that coptisine (1), an isoquinoline alkaloid, selectively inhibits proliferation of rat primary vascular smooth muscle cells (VSMCs). In the present study, the characteristics of its antiproliferative effect on several types of smooth muscle-like cells were investigated and compared to the effects of berberine (2) and palmatine (3). To clarify further the mechanism underlying the VSMC-selective antiproliferative effect of 1, the genes responsible were investigated by determining which mRNAs showed expression regulated by 1. Coptisine (1) showed a greater antiproliferative effect on smooth muscle cells derived from the aorta than on those derived from other organs. Analysis of the mRNA expression revealed that 1 upregulated two genes, growth arrest and DNA-damage-inducible alpha (Gadd45a) and response gene to complement32 (Rgc32). Both genes remained unchanged in 3Y1 fibroblasts and were not affected by 2 and 3. Coptisine (1) was found to induce the mRNA of the Gadd45a and Rgc32 genes, specifically in VSMC. Activation of these genes by 1 may mediate inhibition of cell-cycle progression. However, as these genes are commonly expressed in various cell types, a selective target for 1 activity is likely to exist upstream of these genes.

Roles of coagulation pathway and factor Xa in the progression of diabetic nephropathy in db/db mice.

The active type of coagulation factor X (factor Xa) activates various cell-types through protease-activated receptor 2 (PAR2). We previously reported that a factor Xa inhibitor could suppress Thy-1 nephritis. Considering that fibrin deposition is observed in diabetic nephropathy as well as in glomerulonephritis, this study examined the roles of the coagulation pathway and factor Xa in the development of diabetic nephropathy using type 2 diabetic model mice. Diabetic (db/db) and normoglycemic (m+/m+) mice were immunohistochemically evaluated for their expression/deposition of PAR2, transforming growth factor (TGF)-ß, fibrin, extracellular matrix (ECM) proteins, and CD31 at week 20. Significantly greater numbers of PAR2-positive cells and larger amounts of fibronectin, and collagen IV depositions were observed in the glomeruli of db/db mice than those in m+/m+ mice. Next, expression of PAR2 versus deposition of collagen IV and fibronectin was compared between week 20 and week 30, and the number of PAR2-positive cells in the glomeruli decreased in contrast with the increased accumulation of ECM proteins. In an intervention study, fondaparinux, a factor Xa inhibitor, was subcutaneously administered for ten weeks from week 10 to 20. Fondaparinux treatment significantly suppressed urinary protein, glomerular hypertrophy, fibrin deposition, expression of connective tissue growth factor, and ECM proteins deposition together with CD31-positive capillaries. These results suggest that coagulation pathway and glomerular PAR2 expression are upregulated in the early phase of diabetes, together with the increase of profibrotic cytokines expression, ECM proteins deposition and CD-31-positive vessels. Factor Xa inhibition may ameliorate glomerular neoangiogenesis and ECM accumulation in diabetic nephropathy.

Existence of a threshold for hydroxyl radical generation independent of hypoxia in rat striatum during carbon monoxide poisoning.

We examined the role of hypoxia in the carbon monoxide (CO)-induced generation of the hydroxyl radical (•OH) in the striatum, which could contribute to brain damage due to CO poisoning. Exposure of free-moving rats to 1,000 and 3,000 ppm CO or 8 and 5% O2 for 40 min caused concentration-dependent hypoxic conditions in terms of carboxyhemoglobin (COHb), deoxyhemoglobin, oxyhemoglobin, and O2 contents in arterial blood. The hypoxic conditions seemed comparable between 3,000 ppm CO and 5% O2, although alterations of pH and partial O2 pressure (PO2) were complex and concentration independent. In the striatum, CO and low O2 decreased tissue PO2 levels in a concentration-dependent and concentration-independent manner, respectively, but levels at the end of exposure were comparable among all groups. This was also the case for the increase in striatal blood flow. Although the increases in extracellular glutamate (excitatory), taurine (inhibitory), and alanine (non-neurotransmitter), in the striatum in response to CO and low O2 were complex, 3,000 ppm CO and 5% O2 had comparable effects. Thus, 3,000 ppm CO and 5% O2 seemed to induce comparable hypoxic conditions. Nevertheless, the former more strongly stimulated (•OH generation in the striatum than the latter. In addition, in contrast to low O2 which caused a concentration-dependent increase in (•OH, 1,000 ppm CO had no effect. The findings suggest that striatal •OH generation due to CO poisoning may be independent of hypoxia per se and that a threshold might exist.