Neuroprotective effect of oral S/B remedy (Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd) on iron-induced neurodegeneration in the nigrostriatal dopaminergic system of rat brain.

AIM OF THE STUDY: S/B remedy prepared from Scutellaria baicalensis Georgi and Bupleurum scorzonerifolfium Willd, two herbals of Xiao-Tsai-Hu-Tang or Sho-Saiko-To (TJ-9), contains active flavonoids. In this study, the protective effect of S/B remedy on iron-induced neurodegeneration was investigated in the nigrostriatal dopaminergic system of rat brain. MATERIALS AND METHODS: The antioxidative activity of S/B remedy was studied using brain homogenates incubated with ferrous citrate (iron, 1M), S/B remedy, Trolox and melatonin. Furthermore, a Parkinsonian animal model by an intranigral infusion of iron in the anesthetized rats was employed to investigate the protective effect of S/B remedy in the nigrostriatal dopaminergic system. RESULTS: Our in vitro studies showed that S/B remedy was more potent than melatonin and equal to trolox in inhibiting iron-induced lipid peroxidation of brain homogenates. Our in vivo studies found that oral administration of S/B remedy dose-dependently attenuated iron-elevated lipid peroxidation in the infused substantia nigra (SN) and iron-depleted dopamine levels in the ipsilateral striatum. Furthermore, iron-induced reductions in glutathione (GSH) content and increases in GSSG (oxidized GSH)/GSH ratio in the infused SN were inhibited in S/B remedy-treated rats. Systemic S/B remedy attenuated the iron-induced increases in heme-oxygenase-1 levels and α-synuclein aggregation in the infused SN. Moreover, S/B remedy reduced iron-induced apoptosis via attenuating mitochondrial and endoplasmic reticulum stress. In addition, S/B remedy was anti-inflammatory as indicated by the attenuation of iron-induced elevations in inducible nitric oxide synthase and cyclo-oxygenase II levels as well as glial fibrillary acidic protein (a biological marker of astrocytes) and ED-1 (a protein indicative of activated microglia) levels in the infused SN of S/B remedy-treated rats. CONCLUSIONS: These findings suggest that oral administration of S/B remedy is protective against iron-induced neurodegeneration in the nigrostriatal dopaminergic system of rat brain. Therefore, S/B remedy may be therapeutically useful for the treatment of CNS neurodegenerative diseases.

Catechin stimulates osteogenesis by enhancing PP2A activity in human mesenchymal stem cells.

SUMMARY: Using human mesenchymal stem cells, we identified catechin from a panel of herbal ingredients and Chinese traditional compounds with the strongest osteogenic effects. Catechin increased alkaline phosphatase activity, calcium deposition, and mRNA expression of Runx2 and osteocalcin. We further clarified the signaling pathway that catechin mediated to stimulate osteogenesis. INTRODUCTION: Human mesenchymal stem cells (hMSCs), useful as a species specific cell culture system for studying cell lineage differentiation, were examined as a tool to identify novel herbal ingredients and Chinese traditional compounds for enhancing osteogenesis. METHODS: Immortalized and primary hMSCs were induced in osteogenic induction medium in the presence of a variety of herbal ingredients and Chinese traditional compounds and osteogenic differentiation was evaluated by histochemical assays and quantitative RT-PCR. RESULTS: Using immortalized hMSCs, we first identified catechin, 18ß-glycyrrhetinic acid, baishao, and danggui with osteogenic properties, which enhanced calcium deposition at the dose without significant cytotoxic effects. Primary hMSCs were then applied for confirming the osteogenic effects of catechin, which increased alkaline phosphatase activity, calcium deposition, and mRNA expression of Runx2 and osteocalcin. We further found the extracellular signal-regulated kinase (ERK) pathway was downregulated upon stimulation with catechin. Catechin increased the level and activity of protein phosphatases 2A (PP2A) that dephosphorylates ERK kinase (MEK) and ERK. Further, PP2A inhibitor, okadaic acid, abolished the effect of catechin-mediated inactivation of ERK and stimulation of osteogenesis. The blocking effect of okadaic acid on osteogenesis was further reversed by PD98059, a specific inhibitor of MEK. Co-immunoprecipitation revealed the association of PP2A to both MEK and ERK. CONCLUSIONS: These studies propose catechin enhanced osteogenesis by increasing the PP2A level that inhibits the MEK and ERK signaling in hMSCs. These results prove the concept of using hMSCs as a convenient tool for rapid and consistent screening of the osteogenic herbal ingredients and traditional Chinese compounds.

Nonsteroidal anti-inflammatory drugs for treatment of advanced gastric cancer: cyclooxygenase-2 is involved in hepatocyte growth factor mediated tumor development and progression.

Surgical treatment of gastric cancer patients is dismal because advanced tumor is often noted at diagnosis. In order to obtain better adjuvant therapy for gastric cancer patients after operation, it is important to understand the mechanism of invasion and metastasis. It is well known that binding of hepatocyte growth factor (HGF) to its receptor (c-Met) regulates gastric cancer progression and metastasis. Recently, HGF was found to up-regulate the expression of cyclooxygenase-2 (COX-2) gene and increase prostaglandin (PG)synthesis in gastric mucosa cells. Over-expression of COX-2 and increased PG secretion have also been found to be involved in the growth and metastasis of gastric cancer. These results together suggest that the signaling pathway of HGF and c-Met may be mediated through ERK2 activation, up-regulation of COX-2 and increased production of PGE(2)in gastric cancer cells. In view of the fact that c-Met is over-expressed in the majority of gastric cancer patients with poor prognosis, COX-2 specific inhibitors may provide beneficial effects in these patients.

Accumulation of mitochondrial DNA deletions in human oral tissues -- effects of betel quid chewing and oral cancer.

Accumulation of mitochondrial DNA (mtDNA) mutations in human tissues has been associated with intrinsic aging and environmental insult. Recently, mtDNA mutations have been detected in various tumors, including head and neck tumors. However, the factors affecting the occurrence and accumulation of mtDNA deletions in tumor tissues are poorly understood. In Taiwan, betel quid chewing is a major risk factor for oral cancer. Using polymerase chain reaction (PCR) techniques, we examined large-scale deletions of mtDNA in 53 pairs of tumor and non-tumor oral tissues from the patients with or without betel quid chewing history. The results revealed that irrespective of the history of betel quid chewing, the incidences of the 4977bp deletion and other deletions of mtDNA were lower in the tumor portion as compared with the non-tumor portion. The average proportions of the 4977bp deleted mtDNA in the tumor tissues of the betel quid chewers and non-betel quid chewers were 13- and 5-fold, respectively, lower than those in the corresponding non-tumor tissues. Moreover, the average proportion of 4977bp deleted mtDNA was significantly higher (P<0.05) in the non-tumor oral tissues of the patients with betel quid chewing history than that of the patients without the history of betel quid chewing. These results suggest that betel quid chewing may increase mtDNA mutation in human oral tissues and that accumulation of mtDNA deletions and subsequent cytoplasmic segregation of these mutations during cell division could be an important contributor to the early phase of oral carcinogenesis.

Molecular characterization of an anti-epilepsy peptide from the scorpion Buthus martensi Karsch.

For a long time Asian scorpion Buthus martensi Karsch (BmK) has been used in Chinese traditional medicine to cure many diseases of nervous system. Here we report the purification and characterization of a pharmacologically active neurotoxin from the scorpion BmK. This toxin had little toxicity in mice and insects but was found to have an anti-epilepsy effect in rats, and is thus named as BmK anti-epilepsy peptide (BmK AEP). Its amino-acid sequence was determined by lysylendopeptidase digestion, Edman degradation and mass spectrographic analysis. Based on the determined sequence, the gene coding for this peptide was also cloned and sequenced by the 3' and 5' RACE methods. It encodes a precursor of 85 amino-acid residues including a signal peptide of 21 residues, a mature peptide of 61 residues and three additional residues Gly-Lys-Lys at the C-terminus. The additional Gly sometimes followed by one or two basic residues is prerequisite for the amidation of its C-terminus. C-terminal amidation was also verified by the molecular-mass determination of BmK AEP. This anti-epilepsy peptide toxin shares homology with other depressant insect toxins. The remarkable difference between them was mainly focused at residues 6, 7 and 39; these residues might relate to the unique action of BmK AEP.

The neuroprotective effects of phytoestrogens on amyloid beta protein-induced toxicity are mediated by abrogating the activation of caspase cascade in rat cortical neurons.

Amyloid beta protein (Abeta) elicits a toxic effect on neurons in vitro and in vivo. In present study we attempt to elucidate the mechanism by which Abeta confers its neurotoxicity. The neuroprotective effects of phytoestrogens on Abeta-mediated toxicity were also investigated. Cortical neurons treated with 5 microm Abeta-(25-35) for 40 h decreased the cell viability by 45.5 +/- 4.6% concomitant with the appearance of apoptotic morphology. 50 microm kaempferol and apigenin decreased the Abeta-induced cell death by 81.5 +/- 9.4% and 49.2 +/- 9.9%, respectively. Abeta increased the activity of caspase 3 by 10.6-fold and to a lesser extent for caspase 2, 8, and 9. The Abeta-induced activation of caspase 3 and release of cytochrome c showed a biphasic pattern. Apigenin abrogated Abeta-induced cytochrome c release, and the activation of caspase cascade. Kaempferol showed a similar effect but to a less extent. Kaempferol was also capable of eliminating Abeta-induced accumulation of reactive oxygen species. These two events accounted for the remarkable effect of kaempferol on neuroprotection. Quercetin and probucol did not affect the Abeta-mediated neurotoxicity. However, they potentiated the protective effect of apigenin. Therefore, these results demonstrate that Abeta elicited activation of caspase cascades and reactive oxygen species accumulation, thereby causing neuronal death. The blockade of caspase activation conferred the major neuroprotective effect of phytoestrogens. The antioxidative activity of phytoestrogens also modulated their neuroprotective effects on Abeta-mediated toxicity.

Male predominance in hepatocellular carcinoma: new insight and a possible therapeutic alternative.

Hepatocellular carcinoma is one of the most common cancers in the world. The male to female ratio is 3-6 to 1 in patients with hepatocellular carcinoma. Although steroid hormones and receptors have been examined extensively for their role in the growth regulation of hepatocellular carcinoma, the direct stimulation of hepatocellular carcinoma by steroid hormones still awaits elucidation. On the other hand, clinical trials using antagonists for steroid hormones to treat hepatocellular carcinoma were found to be mostly ineffective. Recently it has been found that 2-methoxyestradiol - an estrogen metabolite - is effective in growth inhibition of various tumor cells as well as in angiogenesis inhibition. Since estrogen is metabolized in the liver, it is conceivable that females with menstruation cycles have more estrogen metabolized in their liver, consequently more 2-methoxyestradiol produced which could inhibit tumor growth in situ. We propose that the low incidence and mortality of hepatocellular carcinoma found in females may have resulted from the high levels of 2-methoxyestradiol produced in the liver during their reproductive years. Consequently, the growth of hepatocellular carcinoma in females is delayed significantly as compared to males. The potential of using 2-methoxyestradiol for treatment of patients with hepatocellular carcinoma after resection of tumor should be explored.

Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels.

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K(+) channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3' and 5' rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.

Safrole-like DNA adducts in oral tissue from oral cancer patients with a betel quid chewing history.

Betel quid (BQ) chewing has been associated with an increased risk of oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). Piper betel inflorescence, which contains 15 mg/g safrole, is a unique ingredient of BQ in Taiwan. Chewing such prepared BQ may contribute to safrole exposure in human beings (420 microM safrole in saliva). Safrole is a known rodent hepatocarcinogen, yet its carcinogenicity in human beings is largely undetermined. In this study, using a (32)P-post-labeling method, we have found a high frequency of safrole-like DNA adducts in BQ-associated OSCC (77%, 23/30) and non-cancerous matched tissue (NCMT) (97%, 29/30). This was in contrast to the absence (< 1/10(9) nucleotides) of such adducts in all of non-BQ-associated OSCC and their paired NCMT (P < 0.001). Six of seven OSF also exhibited the same safrole-like DNA adduct. The DNA adduct levels in OSF and NCMT were significantly higher than in OSCC (P < 0.05). Using co-chromatography and rechromatography techniques, we further demonstrated that these adducts were identical to synthetic safrole-dGMP adducts as well as DNA adducts from 1'-hydroxysafrole-treated HepG2 cells. These results suggest that safrole forms stable safrole-DNA adducts in human oral tissue following BQ chewing, which may contribute to oral carcinogenesis.

Berberine modulates expression of mdr1 gene product and the responses of digestive track cancer cells to Paclitaxel.

Berberine is the major constituent of Coptis chinese and is commonly used in Chinese herbal medicine to treat patients with gastrointestinal disorders. In this study, using flow cytometry, we have found that a 24-h berberine treatment up-regulated the multidrug-resistant transporter (pgp-170) expression in two oral (KB, OC2), two gastric (SC-M1, NUGC-3) and two colon (COLO 205, CT 26) cancer cell lines. Decreased retention of rhodamine 123 was observed in berberine-treated cells as compared to vehicle control. To examine whether the berberine modulated pgp-170 expression in cancer cells is associated with changes in drug resistance, we determined the cytotoxicity, cell cycle progression and cell morphology of Paclitaxel-treated cells. Paclitaxel (1 nM-10 microM) treatment for 24 h induced cytotoxicity in OC2, SC-M1 and COLO 205 cells in a dose-dependent manner. Pretreatment of cells with 32 microM berberine for 24 h prior to Paclitaxel treatment resulted in increased viability as compared to that of Paclitaxel-treated cells. In addition, Paclitaxel-induced apoptosis and/or G2/M arrest in these three cancer cell lines. Pretreatment of cells with berberine prior to Paclitaxel blocked the Paclitaxel-induced cell cycle responses and morphological changes. These results together suggest that berberine modulated the expression and function of pgp-170 that leads to reduced response to Paclitaxel in digestive track cancer cells.

Preparation and in vitro evaluation of B-lipiodol as a boron delivery agent for neutron capture therapy of hepatoma.

BACKGROUND: We prepared boron containing lipiodol (B-lipiodol), elucidated the retention of B-lipiodol in hepatoma cells and evaluated the in vitro cellular toxicity of B-lipiodol for neutron capture therapy. MATERIALS AND METHODS: Human hepatoma HepG2 cells were used to examine the uptake and retention of B-lipiodol. Light microscopes were used to examine the interaction and retention of B-lipiodol globules in individual hepatoma cells. Boron and lipiodol concentrations were determined by inductively coupled plasma-atomic emission spectroscopy and neutron activation analysis, respectively. RESULTS: The boron concentration in B-lipiodol drug could reach 2500 ppm. B-lipiodol could be stably retained in serum and culture medium. HepG2 cells appeared proficiently at internalization and persistent retention of B-lipiodol. The boron concentration reached 3.5 micrograms/10(6) cells without approaching saturation at 48 h treatment. CONCLUSION: Hepatoma cells could actively uptake B-lipiodol and a sufficient amount of boron was retained inside the HepG2 cells which could be used for neutron capture therapy.

Genomic organization of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch.

According to the known primary sequences of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3'- and 5'-RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C-terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same -10 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively.

Up-regulation of multidrug resistance transporter expression by berberine in human and murine hepatoma cells.

BACKGROUND: Berberine, one of the major constitutents of alkaloids of Coptis chinensis is frequently utilized in the treatment of inflammation and liver-related diseases. In Chinese herbal medicine, Coptis chinensis is used as a prophylactic drug to treat gastrointestinal disorders. In a previous study, the authors found that berberine reduced cell proliferation and alpha-fetoprotein expression in human hepatoma HepG2 cells. Multidrug resistance transporter (pgp-170) is known to be overexpressed in HepG2 cells. Whether berberine regulates the expression of pgp-170 in HepG2 and other hepatoma cell lines is unknown and worthy of investigation. METHODS: Human and murine hepatoma cells were treated with berberine (0.32, 3.2, 32, and 320 microM), tamoxifen (1 microM), or verapamil (10 microM) for 24 hours. Flow cytometry was used to measure retention of a fluorescence dye, rhodamine 123, and the level of immunoreactive pgp-170 in berberine-treated hepatoma cells. RESULTS: Berberine up-regulated the expression of pgp-170 in three human hepatoma cell lines. The function of pgp-170 was blocked by tamoxifen and verapamil, resulting in increased retention of rhodamine 123. Retention of rhodamine 123 was significantly reduced in berberine-treated hepatoma cells. CONCLUSIONS: Berberine modulates the expression and function of pgp-170 in hepatoma cells. These results suggest that treatment of tumor cells with berberine may result in reduced retention of chemotherapeutic agents.

Sequence polymorphism of the group 1 allergen of Bermuda grass pollen.

BACKGROUND: Cyn d 1, the major allergen of Bermuda grass pollen, consists of a number of isoforms. OBJECTIVE: To examine the extent of sequence variation of Cyn d 1 isoforms at the molecular level. METHODS: A Bermuda grass pollen lambdaZAP II cDNA expression library was immunoscreened with anti-Cyn d 1 monoclonal antibodies. The reactive clones were isolated, subcloned into Escherichia coli, and sequenced. Some of them were expressed in the yeast Pichia pastoris to obtain recombinant Cyn d 1 proteins. RESULTS: Ten cDNA clones were obtained, all these clones encode the full length of Cyn d 1 protein. Their deduced mature proteins can be grouped into: the long ones with 246 amino acids, and the short ones with 244 amino acids. The last two amino acids (AG) of the long Cyn d 1 are deleted in the short Cyn d 1. The remaining amino acid sequences share more than 98% identity; a total of nine amino acid variations were observed. Two recombinant Cyn d 1 proteins (rCyn d 3-2 and rCyn d 5-4) with three amino acid substitutions showed differential IgE-binding profiles. CONCLUSION: The present study extended our understanding of the primary structure of isoforms of Cyn d 1.

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase.

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H- 1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.

Using monoclonal antibodies to characterize a sequential epitope on the group I allergen of Bermuda grass pollen.

BACKGROUND: Cyn d 1, the group I allergen of Bermuda grass pollen, had been purified and characterized. METHODS: A sequential B cell epitope on Cyn d 1 was studied with monoclonal antibodies (MoAbs). Cyn d 1 was cleaved by Achromobacter protease I into fragments, and the resulting peptides were fractionated on reversed-phase columns before being reacted with anti-Cyn d 1 MoAbs in a radioimmunoassay. A Cyn d 1 fragment recognized by its MoAb was selected for Edman degradation. A synthetic peptide was constructed according to the determined sequence. RESULTS: The epitope on Cyn d 1 recognized by MoAb 18-53 was found to be conformation independent, since its activity was not changed after sodium periodate, guanidine or urea treatment. The enzyme-cleaved fragment containing this epitope was determined to be DVDKPPFDGMTACGNEPIF which corresponds to the N-terminal 46-64 residues of Cyn d 1. The presence of this sequence in the epitope recognized by MoAb 18-53 was demonstrated by enzyme immunoassay and further confirmed by inhibition of binding enzyme immunoassay with synthetic peptides. Some cross-reactivity with the N-terminal 45-63 residues of Lol p 1 was also found. CONCLUSIONS: The primary structure of a sequential epitope on Cyn d 1 was determined, and its activity was confirmed with peptides synthesized according to the determined sequence.

[Effect of intrathecal or intracerebroventricular administrition of OFQ on pain threshold and acpuncture analgesia in rats].

Orphanin FQ (OFQ) is a newly discovered 17-amino-acid peptide capable of inducing hyperalgesia. In the present study, the effects of OFQ on basal pain threshold and acupuncture anlgesia (AA) in rats were observed using the tail-flick test. It was found that intrathecal (i.t.) or intracerebroventricular (i.c.v.) administrition of 0.1 microgram OFQ had no effect on basal pain threshold of rats, while 1 microgram OFQ could lower the threshold. However, OFQ at both the doses (0.1 or 1.0 microgram) administered by either i.t. or i.c.v. injection could antagonize AA with that occuring in the brain being more prominent then in the spinal cord. When the rats were repeatedly treated with antisense oligonucleotide to block synthesis of OFQ receptor, pain threshold increased significantly. At such instance, when the OFQ was combined with acupuncture, the effect of AA showed no obvious change. The above results show that the OFQ at small dose has no effect on pain threshold but can lower it at larger dose; while in both cases OFQ can antagonize AA.

Role of oxidative DNA damage in hydroxychavicol-induced genotoxicity.

Chewing betel quid has been linked to the development of oral cancer. In Taiwan, fresh Piper betle inflorescence is uniquely added to betel quid, and hydroxychavicol is the major phenolic components of P.betle inflorescence. In this study, we tested the mutagenic potential of hydroxychavicol in Salmonella typhimurium TA97, TA98, TA100 and TA102 with and without Aroclor-1254 induced S9 fraction. The results showed that hydroxychavicol was positive in S.typhimurium TA102 without metabolic activation. This increase in revertants was partially inhibited by catalase and superoxide dismutase. In Chinese hamster ovary (CHO-K1) cells, hydroxychavicol induced chromosome aberrations in a dose-dependent manner (10-50 microM) and the majority were chromosome-type aberrations. Hydroxychavicol also significantly increased the frequency of micronuclei in CHO-K1 cells up to 3-fold at a concentration of 40 microM. In addition, hydroxychavicol dose-dependently (0.1-20 microM) induced copper-dependent strand breaks in plasmid DNA. We further tested the oxidative DNA damage potential of hydroxychavicol by measuring 8-hydroxydeoxyguanosine (8-OH-dG) formation in CHO-K1 cells following an 18-h incubation and found that hydroxychavicol (6.25-100 microM) induced 8-OH-dG levels dose-dependently. The increase of 8-OH-dG formation was positively correlated (r = 0.79) with the hydroxychavicol-induced cytotoxicity. In conclusion, hydroxychavicol may exert its genotoxic potential through oxidative DNA damage.

Antagonistic action of orphanin FQ on acupuncture analgesia in rat brain.

The influence of orphanin FQ (OFQ) (a newly discovered 17-amino acid peptide) on acupuncture analgesia (AA) was assessed in rat tail-flick model. Intracerebroventricular (i.c.v.) injection of OFQ (1 microgram) elicited a significant decrement of pain threshold which was abolished by the repeated pretreatment with antisense oligonucleotide (ASO) to OFQ receptor. Electroacupuncture (EA) induced an obvious analgesic effect; when OFQ was used combined with EA, it showed a dose-dependent effect on antagonizing the EA analgesia. When rat was repeatedly i.c.v. injected with ASO to block the synthesis of OFQ receptor, the EA analgesia was enhanced markedly. In this instance, the OFQ did not show antagonistic effect on EA analgesia any more. The results suggest that the OFQ play its antagonistic role on EA analgesia via activating OFQ receptor.

Oxidative damage to DNA induced by areca nut extract.

In Taiwan, people chew betel quid which contains tender areca nut with husk. In other countries, people prefer ripe and dried areca nut without husk. In this study, we compared the reactive oxygen species-induced oxidative DNA damage in isolated DNA and CHO-K1 cells between treatments with tender areca nut extract (ANE) and ripe ANE. Incubation of these two ANE preparations with isolated DNA generated 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in an alkaline environment in a dose-dependent manner. Ripe ANE generated higher levels of 8-OH-dG compared to tender ANE. The addition of iron(II) (100 microM) resulted in 1.4- and 3.1-fold increases of 8-OH-dG when incubated with 1 mg/ml each of tender and ripe ANE. In testing the effect of ANE to cellular DNA, CHO-K1 cells were used for its documented sensitivity to reactive oxygen species. In CHO-K1 cells, ripe ANE was more cytotoxic than tender ANE following an 18-h incubation. The cytotoxicity to CHO-K1 cells was positively correlated with the formation of 8-OH-dG following tender (r=0.97) and ripe (r=0.91) ANE treatment. Addition of the iron chelating agent o-phenanthroline (10 and 20 microM) to cells prior to ri ANE exposure significantly increased (p<0.05) the survival of CHO-K1 cells. In addition, ripe ANE induced dichlorofluorescein-mediated fluorescence which indicated the formation of hydrogen peroxide in CHO-K1 cells. In conclusion, this study demonstrated that ANE-induced oxidative damage to isolated and cellular DNA which may result from the generation of hydrogen peroxide, and iron may serve as a catalyst in this process. Furthermore, ripe ANE generated higher oxidative DNA damage levels compared to tender ANE.