The impact of differential lignin S/G ratios on mutagenicity and chicken embryonic toxicity.

Lignin and lignin-based materials have received considerable attention in various fields due to their promise as sustainable feedstocks. Guaiacol (G) and syringol (S) are two primary monolignols that occur in different ratios for different plant species. As methoxyphenols, G and S have been targeted as atmospheric pollutants and their acute toxicity examined. However, there is a rare understanding of the toxicological properties on other endpoints and mixture effects of these monolignols. To fill this knowledge gap, our study investigated the impact of different S/G ratios (0.5, 1, and 2) and three lignin depolymerization samples from poplar, pine, and miscanthus species on mutagenicity and developmental toxicity. A multitiered method consisted of in silico simulation, in vitro Ames test, and in vivo chicken embryonic assay was employed. In the Ames test, syringol showed a sign of mutagenicity, whereas guaiacol did not, which agreed with the T.E.S.T. simulation. For three S and G mixture and lignin monomers, mutagenic activity was related to the proportion of syringol. In addition, both S and G showed developmental toxicity in the chicken embryonic assay and T.E.S.T. simulation, and guaiacol had a severe effect on lipid peroxidation. A similar trend and comparable developmental toxicity levels were detected for S and G mixtures and the three lignin depolymerized monomers. This study provides data and insights on the differential toxicity of varying S/G ratios for some important building blocks for bio-based materials.

Steamed Ginger May Enhance Insulin Secretion through KATP Channel Closure in Pancreatic ß-Cells Potentially by Increasing 1-Dehydro-6-Gingerdione Content.

Ginger (Zingiber officinale Roscoe) and its active compounds (gingerols, shogaols and paradols) have been reported as having beneficial functions for several diseases, including diabetes. In this study, we revealed that the steaming process could enhance the anti-diabetic potential of ginger. To confirm the anti-diabetic effect of steamed ginger extract (GG03), we assessed pancreatic islets impaired by alloxan in zebrafish and demonstrated anti-hyperglycemic efficacy in a mouse model. The EC50 values of ginger extract (GE) and GG03 showed that the efficacy of GG03 was greater than that of GE. In addition, LC50 values demonstrated that GG03 had lower toxicity than GE, and the comparison of the Therapeutic Index (TI) proved that GG03 is a safer functional food. Furthermore, our data showed that GG03 significantly lowered hyperglycemia in a diabetic mouse model. HPLC was performed to confirm the change in the composition of steamed ginger. Interestingly, GG03 showed a 375% increase in 1-dehydro-6-gingerdione (GD) compared with GE. GD has not yet been studied much pharmacologically. Thus, we identified the protective effects of GD in the damaged pancreatic islets of diabetic zebrafish. We further assessed whether the anti-diabetic mechanism of action of GG03 and GD involves insulin secretion. Our results suggest that GG03 and GD might stimulate insulin secretion by the closure of KATP channels in pancreatic ß-cells.

Syntheses, crystal structures and biological evaluation of two new Cu(II) and Co(II) complexes based on (E)-2-(((4H-1,2,4-triazol-4-yl)imino)methyl)-6-methoxyphenol.

Two transition metal complexes of [M(TMP)2(H2O)2] (TMP-Cu, M = Cu; TMP-Co, M = Co) with (E)-2-(((4H-1,2,4-triazol-4-yl)imino)methyl)-6-methoxyphenol (H-TMP) were first synthesized and characterized by infrared analysis, elemental analysis and single crystal X-ray diffraction analysis. Notably, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that TMP-Cu displayed relatively high cytotoxic activity against Hep-G2 cancer cells, and high selectivity between human hepatocellular carcinoma cells and normal HL-7702 cells, in comparison to TMP-Co and cisplatin. Further studies showed that TMP-Cu and TMP-Co caused cell cycle arrest at S phase through regulation of S phase related protein expressions and induced Hep-G2 cell apoptosis via the mitochondrial pathway.

Acute toxicity of emerging atmospheric pollutants from wood lignin due to biomass burning.

Guaiacol (2-methoxyphenol) is an important atmospheric pollutant. It is the major component of wood lignin and is essentially emitted to the atmosphere during biomass burning. Its aging in the tropospheric aqueous phase leads to the generation of the following ring-retaining transformation products, also during nighttime: 4-nitroguaiacol, 6-nitroguaiacol, and dinitroguaiacol. This study presents the first toxicological data of guaiacol and its nitro derivatives and reveals their harmful potential for the ecosystem. Applying V. fischeri bioluminescence acute toxicity test, EC50 values range from 16.7 to 103mgL-1 after a 30-min incubation period, which classifies all investigated compounds as 'harmful' according to the European legislation. The investigation of environmentally relevant mixtures did not show significant joint actions between the four studied compounds. Therefore, their concentration addition can be considered for ecotoxicological purposes. However, a synergistic effect between guaiacol and a minor unidentified first-generation product of its aqueous-phase aging was observed and should be taken into account when assessing the reaction mixture toxicity. These results stress the need for further toxicological testing, including organisms of different trophic levels, to better evaluate the environmental hazard of guaiacol and especially its nitro derivatives.

Does toxicity of aromatic pollutants increase under remote atmospheric conditions?

Aromatic compounds contribute significantly to the budget of atmospheric pollutants and represent considerable hazard to living organisms. However, they are only rarely included into atmospheric models which deviate substantially from field measurements. A powerful experimental-simulation tool for the assessment of the impact of low- and semi-volatile aromatic pollutants on the environment due to their atmospheric aqueous phase aging has been developed and introduced for the first time. The case study herein reveals that remote biotopes might be the most damaged by wet urban guaiacol-containing biomass burning aerosols. It is shown that only after the primary pollutant guaiacol has been consumed, its probably most toxic nitroaromatic product is largely formed. Revising the recent understanding of atmospheric aqueous phase chemistry, which is mostly concerned with the radical nitration mechanisms, the observed phenomenon is mainly attributed to the electrophilic nitrogen-containing reactive species. Here, their intriguing role is closely inspected and discussed from the ecological perspective.

Acute larvicidal activity against mosquitoes and oxygen consumption inhibitory activity of dihydroguaiaretic acid derivatives.

(-)-Dihydroguaiaretic acid (DGA) and its derivatives having 3-hydroxyphenyl (3-OH-DGA) and variously substituted phenyl groups instead of 3-hydroxy-4-methoxyphenyl groups were synthesized to measure their larvicidal activity against the mosquito Culex pipiens Linnaeus, 1758 (Diptera: Culicidae). Compared with DGA and 3-OH-DGA (LC50 (M), 3.52 × 10(-5) and 4.57 × 10(-5), respectively), (8R,8'R)-lignan-3-ol (3) and its 3-Me (10), 2-OH (12), 3-OH (13), and 2-OMe (15) derivatives showed low potency (ca. 6-8 × 10(-5) M). The 4-Me derivative (11) showed the lowest potency (12.1 × 10(-5) M), and the 2-F derivative (4) showed the highest (2.01 × 10(-5) M). All of the synthesized compounds induced an acute toxic symptom against mosquito larvae, with potency varying with the type and position of the substituents. The 4-F derivative (6), which killed larvae almost completely within 45 min, suppressed the O2 consumption of the mitochondrial fraction, demonstrating that this compound inhibited mitochondrial O2 consumption contributing to a respiratory inhibitory activity.

3-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a novel combretastatin A-4 analog, induces G2/M arrest and apoptosis by disrupting tubulin polymerization in human cervical HeLa cells and fibrosarcoma HT-1080 cells.

Microtubule is a popular target for anticancer drugs. In this study, we describe the effect 3-(3-hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1,2,5-selenadiazole (G-1103), a newly synthesized analog of combretastatin A-4 (CA-4), showing a strong time- and dose-dependent anti-proliferative effect on human cervical cancer HeLa cells and human fibrosarcoma HT-1080 cells. We demonstrated that the growth inhibitory effects of G-1103 in HeLa and HT-1080 cells were associated with microtubule depolymerization and proved that G-1103 acted as microtubule destabilizing agent. Furthermore, cell cycle analysis revealed that G-1103 treatment resulted in cell cycle arrest at the G2/M phase in a time-dependent manner with subsequent apoptosis induction. Western blot analysis revealed that down-regulation of cdc25c and up-regulation of cyclin B1 was related with G2/M arrest in HeLa and HT-1080 cells treatment with G-1103. In addition, G-1103 induced HeLa cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8 expression, which indicated that G-1103 induced HeLa cell apoptosis was mainly associated with death receptor pathway. However, G-1103 induced HT-1080 cell apoptosis by up-regulating cleaved caspase-3, Fas, cleaved caspase-8, Bax and cleaved caspase-9 expression and down-regulating anti-apoptotic protein Bcl-2 expression, which indicated that G-1103 induced HT-1080 cell apoptosis was associated with both mitochondrial and death receptor pathway. Taken together, all the data demonstrated that G-1103 exhibited its antitumor activity through disrupting the microtubule assembly, causing cell cycle arrest and consequently inducing apoptosis in HeLa and HT-1080 cells. Therefore, the novel compound G-1103 is a promising microtubule inhibitor that has great potentials for therapeutic treatment of various malignancies.

[Chemical constituents of Kadsura oblongifolia and evaluation of their toxicity].

To study the chemical constituents of K. oblongifolia, silica gel column chromatography, MCI and Sephadex LH-20 were used to separate the 70% acetone extract of the stems of K. oblongifolia. The structures of the isolated compounds have been established on the basis of physicochemical and NMR spectroscopic evidence as well as ESI-MS in some cases. Twenty compounds were obtained and identified as heteroclitalignan A (1), kadsulignan F (2), kadoblongifolin C (3), schizanrin F (4), heteroclitalignan C (5), kadsurarin (6), kadsulignan O (7), eburicol (8), meso-dihydroguaiaretic acid (9), kadsufolin A (10), tiegusanin M (11), heteroclitin B (12), (7'S)-parabenzlactone (13), angeloylbinankadsurin B (14), propinquain H (15), quercetin (16), kadsulignan P (17), schizanrin G (18), micrandilactone C (19) and (-)-shikimic acid (20). Compouds 1, 5, 8, 11-15, 18 and 20 were isolated from this plant for the first time. Toxicity of compounds 1-10 were evaluated with zebrafish model to observe the effect on its embryonic development and heart function. The results showed that compounds 7, 9 and 10 caused edema of zebrafish embryo and decreased the heart rate of zebrafish, which exhibited interference effect on heart development of zebrafish.

Cytotoxic activity of dietary lignan and its derivatives: structure-cytotoxic activity relationship of dihydroguaiaretic acid.

Cytotoxic activities of synthesized lignan derivatives were estimated by WST-8 reduction assay against HL-60 and HeLa cells to show the structure-activity relationship. The activities of some effective compounds were examined against Colon 26 and Vero cells. Dietary secoisolariciresinol (SECO, 1) and its metabolite, 9,9'-anhydrosecoisolariciresinol (2), did not show the cytotoxic activity. On the other hand, all stereoisomers of dihydroguaiaretic acid (DGA, 9,9'-dehydroxysecoisolariciresinol, 3-5) exhibited the activity (IC50: around 30 µM). The IC50 value of (8R,8'R)-9-butyl DGA derivative 13 was around 6 µM. This fact means that the hydrophobic group was advantageous for higher activity at 9- and 9'-positions. By the evaluation of the effect of 7and 7'-aryl group on the activity, we discovered the highest activity of (8R,8'R)-7-(3-hydroxy-4-methoxyphenyl)-7'-(2-ethoxyphenyl) DGA derivative 47 showing around 1 µM of IC50 value, which is about 24-fold higher activity than that of natural (8R,8'R)-DGA. The derivative of dietary lignan showed the high cytotoxic activity.

The impact of Triton WR-1339 induced hyperlipidemia on the effects of benzo(a)pyrene or guaiacol on α- and γ-tocopherol pools and selected markers of pro-/antioxidative balance in rat plasma and erythrocytes.

The toxicity of carcinogenic benzo(a)pyrene (BaP) can be intensified by the pro-oxidative effects of metabolic activation. The oxidatively active products can be formed during enzymatic biotransformation or in the process of co-oxygenation with lipid peroxidation. This study assesses if the acute hyperlipidemia can increase pro-oxidative effects of BaP as a factor intensifying processes of lipid peroxidation and co-oxygenation. After three days of i.p. administration of BaP or guaiacol (equimolar dose 10mg/kg b.w.) without or with the hyperlipidemia inducer-Triton WR-1339 to male Wistar rats, the levels of α- and γ-tocopherol were measured in erythrocytes and plasma together with the level of lipid peroxidation as malonyldialdehyde (MDA) concentration. Guaiacol was chosen as a reference substance due to its high ability to co-oxygenate. Additionally, the activity of superoxide dismutase (Cu,ZnSOD) in erythrocytes and plasma was monitored. In normolipaemic groups the significant decrease in erythrocyte α-tocopherol pool and the increase in lipid peroxidation level were observed after BaP or guaiacol administration. In hyperlipaemic groups, despite the increase in the level of lipid peroxidation, there were no additional effects in tocopherol pools compared to the normolipaemic groups which could be attributed to co-oxygenation. Decrease of α-tocopherol in erythrocytes was proportional to the reduction in normolipemic subjects when accounting for the migration to hyperlipemic plasma. There was no co-oxygenation effect on the activity of superoxide dismutase (Cu,ZnSOD) in blood.

4-fluoro-2-methoxyphenol, an apocynin analog with enhanced inhibitory effect on leukocyte oxidant production and phagocytosis.

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNFα) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNFα release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases.

Analysis of annual fluctuations in the content of phenol, chlorophenols and their derivatives in chlorinated drinking waters.

PURPOSE: Chlorophenols are widely represented, toxic, and persistent environmental pollutants. In this work, we analyzed annual fluctuations in the content of phenol, guaiacol, chlorophenols, chlorocatechols, and chlorinated methoxyphenols in drinking water collected in Warsaw and Tomaszów Mazowiecki (Poland). Moreover, the effect of dissolved organic matter content on the occurrence of phenolic compounds in drinking water was studied. METHODS: The compounds were adsorbed on octadecyl C18 solid-phase discs, separated by the use of gas chromatography, and analyzed using mass spectrometry. The content of organic matter was evaluated by the analysis of UV absorption at 254 nm by water samples. In Warsaw, raw water (derived from infiltration intakes situated in the Vistula River) and treated water (subjected to coagulation, filtration, and disinfection with chlorine dioxide) were collected in order to analyze phenols. In Tomaszów Mazowiecki, raw water (taken directly form the river) and treated water (subjected to coagulation, sand filtration, ozonation, and disinfection with gaseous chlorine) were taken to determine phenolic substances. RESULTS AND CONCLUSIONS: The obtained results showed the occurrence of phenol, guaiacol, 2,4,6-trichlorophenol (2,4,6-TCP), tetrachlorophenol (TeCP), and pentachlorophenol in drinking water of both cities. Occasionally, in the waters studied, the appearance of chloroguaiacols, 3-chlorosyringol, and some chlorocatechols were noted. It was also observed that the content of dissolved organic matter in river waters may have contributed to the formation of some phenols, e.g., phenol, guaiacol, 2,4,6-TCP, and TeCP in drinking water. Finally, it was found that there were no annual (seasonal) fluctuations in phenolic compounds contents in drinking waters examined.

Chlorophenols, chlorocatechols and chloroguaiacols induce DNA base oxidation in human lymphocytes (in vitro).

Phenolic compounds are strong environmental toxicants, which are found in food, drinking water as well as in the indoor and outdoor air environment. In this work we investigated the effect of low concentrations of 0.2, 1 and 5 microg/ml of 2,4,5-trichlorophenol (2,4,5-TCP), pentachlorophenol (PCP), 4,6-dichloroguaiacol (4,6-DCG), tetrachloroguaiacol (TeCG), 4,5-dichlorocatechol (4,5-DCC) and tetrachlorocatechol (TeCC) on DNA bases oxidation in human peripheral blood lymphocytes. The analysis was performed using alkaline single cell gel electrophoresis (the comet assay). To detect oxidized pyrimidynes and purines we used the repair enzymes such as endonuclease III and formamidopyrimidine-DNA glycosylase. DNA oxidation was expressed as a percentage of comet tail, which was formed after the xenobiotics treatment. The obtained results showed that all the compounds examined were able to oxidize DNA bases in human lymphocytes. It was also observed that pyrimidine bases were more strongly oxidized in comparison to purine ones. Finally, it was found that chlorinated catechols and TeCC in particular, revealed a higher oxidative potential in comparison to chlorophenols and chloroguaiacols, and a rise in the number of chlorine atoms in the compound from each group examined led to an increase in DNA bases damage.

Radiomodifying and anticlastogenic effect of Zingerone on Swiss albino mice exposed to whole body gamma radiation.

The radioprotective effect and antigenotoxic potential of phenolic alkanone, Zingerone (ZO) were investigated in Swiss albino mice exposed to gamma radiation. To study the optimum dose for radiation protection, mice were administered with ZO (10-100mg/kgb.wt.), once daily for five consecutive days. One hour after the last administration of ZO on the fifth day, animals were whole body exposed to 10 Gy gamma radiations. The radioprotective potential was assessed using animal survival at an optimal ZO dose of 20mg/kgb.wt., administered prior to 7-11 Gy. Further, the radioprotective potential of ZO was also analyzed by haemopoietic stem cell survival (CFU) assay, mouse bone marrow micronucleus test and histological observations of intestinal and bone marrow damage. Effect of ZO pretreatment on radiation-induced changes in glutathione (GSH), glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPx) levels was also analyzed. ZO treatment resulted increase in the LD(50/30) by 1.8 Gy (dose reduction factor = 1.2). The number of spleen colonies after whole body irradiation of mice (4.5 or 7.5 Gy) was increased when ZO was administered 1h prior to irradiation. The histological observations indicated a decline in the villus height and crypt number with an increase in goblet and dead cell population in the irradiated group, which was normalized by pretreatment with ZO. A significant (p < 0.001) reduction in micronucleated polychromatic, normochromatic erythrocytes, increased PCE/NCE ratio, increase in the GSH, GST, SOD, CAT and decreased LPx levels were observed in ZO pretreated group when compared to the irradiated animals. Our findings demonstrate the potential of ZO in mitigating radiation-induced mortality and cytogenetic damage, which may be attributed to inhibition radiation-induced decline in the endogenous antioxidant levels and scavenging of radiation-induced free radicals.

Isolation, structure elucidation and cytotoxic evaluation of endiandrin B from the Australian rainforest plant Endiandra anthropophagorum.

Chemical investigations of the DCM extract from the roots of Endiandra anthropophagorum resulted in the isolation of a new cyclobutane lignan endiandrin B (1), together with the known natural products, endiandrin A (2), and (-)-dihydroguaiaretic acid (3). The structure of 1 was determined by extensive spectroscopic analyses, and confirmed by single crystal X-ray crystallography. Methylation of 1 using diazomethane afforded the previously reported natural product, cinbalansan (4). All compounds were evaluated for their cytotoxicity towards human lung carcinoma cells (A549) using high-content screening. (-)-Dihydroguaiaretic acid (3) was found to be the most potent cytotoxin against the A549 lung carcinoma cell line, with an IC(50) value of 7.49 microM.

Acute toxicity of guaiacol administered subcutaneously in the mouse.

Guaiacol is a compound used as expectorant. In Mexico City, this product is being illegally used for aesthetic treatment with fatal results. The aim of this study is to confirm the lethal toxicity documented in humans. Male Swiss Webster mice (CFW) 30-45g were employed. Dose-response curves to guaiacol were performed by subcutaneous administration (6.25-400 microl/40g). Basal temperature was recorded 30-120 min following administration of guaiacol. Animals were continuously observed for 120 min after guaiacol administration, lethality and toxicity manifestations were recorded. Depending of the dose, high toxicity was observed; sub lethal doses (6.25-12.5 microl/40 g) produced tachycardia and hyperactivity, follow by sedation, hypnosis, high hypothermic effect (loss of 6 degrees C) dyspnea, myoclonus, hematuria, blindness, abdominal distension and in higher doses (25-400 microl/40 g) lethal effect. Necropsy showed hepatic and renal necrosis, pulmonary edema, hemorrhages and bladder clotting. We concluded that guaiacol is an extremely toxic product (toxic rating class 5) whose use should be restricted or banned.

4-ethylphenol and 4-ethylguaiacol in wines: estimating non-microbial sourced contributions and toxicological considerations.

Analyses of commercially available wines suggested non-Brettanomyces sources of 4-ethylphenol and 4-ethylguaiacol. Grapes, enological additions, exposure to plastics, and oak-barrel aging were potential inputs considered. Investigations of whole grape bunch samples from two major red wine Vitis vinifera cultivars (L. cv. Cabernet Franc and Pinot Noir), a commercial mannoprotein additive, and three commercial enological tannin additions indicated they are not likely significant sources of these compounds. Studies on 15 commercial oak barrelled red wines from six Vitis vinifera cultivars (L. cv. Cabernet Franc, Cabernet Sauvignon, Dunkelfelder, Merlot, Pinot Meunier, and Pinot Noir), and a review of volatile phenol extraction from toasted oak wood, suggested that oak-aging may produce concentrations of up to 50 microg L(-1) 4-ethylphenol and 4-ethylguaiacol. Thus, following potential Brettanomyces-sourced aroma impacts in wine using 4-ethylphenol and/or 4-ethylguaiacol concentrations as proxies should only be considered reliable at analyte levels>100 microg L(-1). A review of worldwide 4-ethylphenol and 4-ethylguaiacol concentrations in wine, consumption patterns, and available toxicological data also suggested that levels of 4-ethylphenol being observed in wines worldwide do not warrant concerns about acute or long-term effects. While little is known about the toxicology of 4-ethylguaiacol, it is unlikely that elevated concentrations will pose any health-related concerns.

Temporal interactions between oral irritants: piperine, zingerone, and capsaicin.

Sequential presentation of 2 irritants may produce cross-sensitization or cross-adaptation effects upon introduction of the second irritant. In Experiment 1, subjects were given either 34 min of stimulation with zingerone, capsaicin, or piperine or one of those irritants for 23 min followed by blanks for 23 min. In Experiment 2, subjects received one irritant for 23-min irritants, followed immediately by another for 23 min (piperine --> zingerone, piperine --> capsaicin, zingerone --> piperine, or zingerone --> capsaicin). Cross-sensitization was observed for the piperine --> zingerone, zingerone --> piperine, and piperine --> capsaicin groups; cross-adaptation was observed for the zingerone --> capsaicin group. Cross-adaptation and cross-sensitization were predicted by adding the independent time courses of the respective irritants, starting the second at the offset of the first. These responses were also predicted by a mathematical model of central processing of primary afferent responses.

Reversal of multidrug resistance by 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide through the reversible inhibition of P-glycoprotein.

Overexpression of P-glycoprotein (P-gp) is one of the major obstacles to successful cancer chemotherapy. In this study, we examined the ability of 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide (C-4) to reverse multidrug resistance (MDR) in P-gp expressing KBV20C cells. Treatment of KBV20C cells with C-4 led to a dramatic increase in paclitaxel- or vincristine-induced cytotoxicity without any cytotoxicity by itself. In parallel, C-4 treatment caused an increase in apoptotic cell death by paclitaxel or vincristine. Furthermore, C-4 treatment significantly increases in intracellular accumulation of fluorescent P-gp substrate rhodamine 123, indicating that C-4 treatment leads to reversal of the MDR phenotype resulting from an increased accumulation of anticancer drugs by inhibiting drug efflux function of P-gp. This notion is further supported by the observation that C-4 treatment potentiates paclitaxel-induced G(2)/M arrest of the cell cycle. In addition, the drug efflux function of P-gp was reversibly inhibited by C-4 treatment, while the expression level of P-gp was not affected. Collectively, our results describe the potential of C-4 to reverse the P-gp-mediated MDR phenotype through reversible inhibition of P-gp function, which may make it an attractive new agent for the chemosensitization of cancer cells.