Toxicological interactions between the mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro

Beauvericin (BEA), deoxynivalenol (DON) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health. In this study, the acute toxicity of individual and combined mycotoxins (BEA, DON and T-2) were tested in immortalized hamster ovarian cells (CHO-K1) at 24, 48 and 72 h of exposure, by the tetrazolium salt (MTT) and neutral red (NR) assays. The IC₅₀ values obtained for all mycotoxins by the MTT and NR assays ranged from 0.017 to 12.08 μM and from 0.042 to 17.22 μM, respectively. Both, individual and combined mycotoxins demonstrated a significant cytotoxic effect in CHO-K1 cells in a dose-dependent manner. When mycotoxins were assayed individually, T-2 showed the strongest IC₅₀ values (from 0.017 to 0.052 μM), by both endpoints tested, followed by DON (0.53-2.30 μM) and BEA, showing this last one, the weakest IC₅₀ values (from 2.77 to 17.22 μM). On the other hand, cytotoxicity interactions were evaluated by the isobologram method. In acute binary tests, DON + BEA (CI = 1.60-25.07) and DON + T-2 (CI = 1.74-7.71) showed antagonism at 24, 48 and 72 h of exposure. By contrast, the binary BEA + T-2 combination (CI = 0.35-0.78) showed synergism at all time of exposure tested. The tertiary BEA + DON + T-2 combination demonstrated synergism effect (CI = 0.47-0.86) after 24 and 48 h of exposure; however moderate antagonistic effect (CI = 1.14-1.60) was presented after 72 h of exposure at the lower doses. These results provide quantitative evidence regarding potentially important interactions between BEA, DON and T-2 depending of the time of exposure. The combination index-isobologram equation method can serve as a useful tool in food risk assessment. Due to the potent toxic effects of BEA, DON and T-2, its combined exposure might be an important trigger for development of several diseases in humans, from the mycotoxicological point of view, especially after long period of exposure time.     

Study of the cytotoxic activity of beauvericin and fusaproliferin and bioavailability in vitro on Caco-2 cells

Beauvericin (BEA) is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and produces cytotoxic effects in mammalian cells. Fusaproliferin (FUS) is a mycotoxin that has toxic activity against brine shrimp, insect cells, and teratogenic effects on chicken embryos. The aim of this study was to determine the cytotoxicity of BEA and FUS in human epithelial colorectal adenocarcinoma HT-29 and Caco-2 cells, the transepithelial transport and the bioavailability using Caco-2 cells as a simulated in vitro gastrointestinal model of the human intestinal epithelium. The inhibitory concentration (IC₅₀) evidenced by BEA in the Caco-2 cells was 24.6 and 12.7 μM at 24 and 48 h exposure, respectively, whereas the IC₅₀ values evidenced in HT-29 cells were 15.0 and 9.7 μM, respectively. FUS was cytotoxic, but no IC₅₀ data were observed in the range of concentration tested. BEA bioavailability was variable from 50.1% to 54.3%, whereas FUS presented a bioavailability variable from 80.2% to 83.2%. Results obtained demonstrated a potential risk for human health.     

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E,CFU-E,and CFU-GM): Species-related susceptibility

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001–50 μg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3–3 mg/kg b.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data.We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC₅₀ values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC₅₀: 2.45 ± 1.08 and 1.82 ± 0.8 μM for humans, and 11.08 ± 2.92 and 1.81 ± 0.20 μM for mice, respectively), than the mature CFU-E (AFB1 IC₅₀: 12.58 ± 5.4 and 40.27 ± 6.05 μM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.     

Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29

Enniatins (ENs) are ionophoric, phytotoxic, antihelminthic, and antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The cytotoxicity effect of the ENs A, A₁, A₂, B, B₁, B₄ and J₃ was compared on three tumor cell lines, the human epithelial colorectal adenocarcinoma (Caco-2), the human colon carcinoma (HT-29), and the human liver carcinoma (Hep-G2). The endpoint evaluated was the mitochondrial integrity by using the MTT assays, after 24 and 48 h of incubation. The IC₅₀ value for EN A₂ on Caco-2 cells, after 24 h exposure, was 18.7 ± 4.5 μM and decrease to 2.6 ± 0.7 μM at 48 h of incubation. However, ENs A, A₁, B₁ and B₄ exert pronounced cytotoxic effects in all the cell lines tested by the MTT assay after 24 and 48 h of incubation. The EN A₁ demonstrated to be the most cytotoxic ENs tested. Moreover, no statistical differences were found between the IC₅₀ values obtained for EN A₁ on Caco-2, HT-29 and Hep-G2, with IC₅₀ values ranging from 9.1 ± 2.2 μM to 12.3 ± 4.3 μM at 24 h and decreasing in a range variable from 1.4 ± 0.7 μM to 2.7 ± 0.8 μM at 48 h. On the other hand, EN A, B₁ and B₄ showed lower cytotoxicity, but in a similar range as the IC₅₀ values reported on HT-29 (IC₅₀ values (24 h): 16.8 ± 4.3-26.2 ± 6.7 μM), Caco-2 (IC₅₀ values (24 h): 19.5 ± 4.1 μM) and Hep-G2 (IC₅₀ values (24 h): 23.4 ± 5.6-26.2 ± 7.6 μM) cells. Cytotoxic effect with a 48 h of incubation revealed also a significant toxicity of ENs A (IC₅₀ values ranged from 8.2 ± 1.8 to 11.4 ± 4.6 μM), B₁ (IC₅₀ values variables from 3.7 ± 0.7 to 11.5 ± 5.3 μM) and B₄ (IC₅₀ of 4.5 ± 2.9-15.0 ± 4.0 μM). In summary, this study demonstrated that ENs can exert toxic activity at low micromolar concentrations in mammalian cells.     

Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(L_n)₂], 1-7}, derived from carboplatin by a substitution of two NH₃ molecules for two 2,6,9-trisubstituted 6-benzylaminopurine-based N-donor ligands (L_n), was studied by the MTT assay for their in vitro cytotoxic activity against seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases even more than cisplatin. The best results were achieved for complexes 1 (IC₅₀ = 17.4 ± 2.0 μM) and 2 (IC₅₀ = 14.8 ± 2.1 μΜ) against HOS cells, 1 (IC₅₀ = 15.1 ± 6.8 μM), 2 (IC₅₀ = 13.6 ± 5.2 μM) and 6 (IC₅₀ = 19.0 ± 6.6 μM) against MCF7, 6 (IC₅₀ = 6.4 ± 0.1 μM) against A2780, and 1-6 (IC₅₀ = 15.6 ± 4.0, 12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 μM, respectively) against A2780cis. Viability of human hepatocytes was not declined by the tested complexes up to the concentration of 50 μM (for 1, 3-7) and 20 μM (for 2; caused by lower solubility of this complex).     

In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450

The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC₅₀) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC₅₀ = 412.68 ± 15.44 μM) and CYP3A4 (IC₅₀ = 343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC₅₀ = 539.04 ± 14.18 μM) and CYP3A4 (IC₅₀ = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (K_i = 385.24 ± 8.75 μM) and CYP3A4 (K_i = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (K_i = 109.62 ± 6.14 μM) and CYP3A4 (K_i = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.     

In vitro cytotoxicity of fungi spoiling maize silage

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC₅₀) of the major P. roqueforti metabolites roquefortine C (48 μg/mL), andrastin A (>50 μg/mL), mycophenolic acid (>100 μg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 μg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC₅₀ of 0.6 μg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 μg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.     

Reactive oxygen species involvement in apoptosis and mitochondrial damage in Caco-2 cells induced by enniatins A,A1, B and B1

The cytotoxic effects, the generation of reactive oxygen species (ROS) and lipid peroxidation (LPO) as well as the cell cycle disruption, the induction of apoptosis and changes in mitochondrial membrane potential (ΔΨm) as a function of increasing time have been determined in human colorectal adenocarcinoma (Caco-2) cells after exposure to enniatins (ENs) A, A₁, B and B₁. IC₅₀ values obtained by the MTT and Neutral Red assay, after 24, 48 and 72 h of exposure ranged from 0.5 ± 0.1 to >15 μM. A significant increase (p ≤ 0.05) in ROS generation and LPO production, as determined by the fluorescent probe H₂-DCFDA and TBARS method respectively, was observed for all mycotoxins tested at 3.0 μM concentration. The highest increase in ROS generation (2.6 fold higher than control) and LPO production (111%, as compared to control) was observed with EN A. Cell cycle was significantly arrested at G2/M phase after 24 h of exposure to EN A, A₁, B₁, whereas after 72 h of exposure an arrest in S phase was observed almost for all mycotoxins tested. Moreover, after 24 and 48 h of exposure, ENs increased the early apoptotic cells, whereas after 72 h of exposure necrosis was observed. In addition the loss of ΔΨm was produced on Caco-2 cells after ENs exposure. ENs A, A₁, B and B₁ cytotoxicity involved early ROS generation that induced LPO oxidative damage, apoptosis and necrosis via the mitochondrial pathway. ENs A, A₁ and B₁ induced DNA damage. However the same effects cannot be proposed for EN B. Further studies on the toxicological effects induced by ENs A, A₁, B and B₁ are needed.     

The identification and biochemical characterization of drug-like compounds that inhibit botulinum neurotoxin serotype A endopeptidase activity

A robust, high-throughput, two-tiered assay for screening small molecule inhibitors against botulinum neurotoxin serotype A was developed and employed to screen 16,544 compounds. Thirty-four compounds were identified as potent hits employing the first-tier assay. Subsequently, nine were confirmed as actives by our second-tier confirmatory assay. Of these, one displayed potent inhibitory efficacy, possessing an IC₅₀ = 16 μM (±1.6 μM) in our in vitro assay. This inhibitor (0831-1035) is highly water-soluble, and possesses an IC₅₀ = 47 μM (±7.0 μM) in our primary cell culture assay (with virtually no cytotoxicity up to 500 μM), suggesting that this inhibitor is a good candidate for further development as a therapeutic countermeasure to treat botulism resulting from botulinum neurotoxin serotype A intoxication. An enzyme kinetics study indicated that this inhibitor exhibits mixed non-competitive inhibition, with a K_I = 9 μM.     

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K_i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0 μM) and OATP2B1 (8.7 μM) followed by kaempferol (OATP1A2: 25.2 μM, OATP2B1: 15.1 μM) and apigenin (OATP1A2: 32.4 μM OATP2B1: 20.8 μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC₅₀ values of 4.3 μM, 12.0 μM, and 12.6 μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC₅₀ for OATP1A2: 9.3 μM, OATP2B1: 13.9 μM), kaempferol (IC₅₀ for OATP1A2: 37.3 μM, OATP2B1: 20.7 μM) and quercetin (IC₅₀ for OATP1A2: 13.5 μM, OATP2B1: 14.1 μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food-drug or drug-drug interactions in humans.     

The endoperoxide ascaridol shows strong differential cytotoxicity in nucleotide excision repair-deficient cells

Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC₅₀ values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of > 1000-fold in resistance between normal and NER-deficient cells (IC₅₀ values for cells with deficiency in ERCC6: 0.15 μM, XPC: 0.18 μM, and normal cells: > 180 μM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes.     

A COX-2 inhibitor nimesulide analog selectively induces apoptosis in Her2 overexpressing breast cancer cells via cytochrome c dependent mechanisms

Epidemiological and animal model studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anti-cancer effect in different type of cancers. In the current study, five breast carcinoma cell lines were used to explore the anti-cancer mechanisms of a nimesulide derivative compound 76. The compound dose dependently suppressed SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC₅₀ of 0.9 μM, 2.2 μM and 4.0 μM, respectively. However, it needs much higher concentrations to inhibit MCF-7 and MDA-MB-231 breast cancer cell growth with IC₅₀ at 22.1 μM and 19.6 μM, respectively. Further investigation reveals that compound 76 induced apoptosis in SKBR-3 and BT474 cells. Since these cells are Her2 overexpressing cells, the Her2 intracellular signaling pathways were examined after the treatment. There was no significant changing of kinase activity. However, the cytochrome c release assay indicated that the apoptosis induced by the compound was mediated by the mitochondria. These results suggest that compound 76 selectively induce apoptosis in Her2 overexpressing breast cancer cells through the mitochondria, and could be used as a lead to design more potent derivatives.     

Mechanism of action of AZD0865, a K+-competitive inhibitor of gastric H+,K+-ATPase

AZD0865 is a member of a drug class that inhibits gastric H⁺,K⁺-ATPase by K⁺-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K⁺-stimulated H⁺,K⁺-ATPase activity (IC₅₀ 1.0 ± 0.2 μM) but was more potent at pH 6.4 (IC₅₀ 0.13 ± 0.01 μM). The IC₅₀ values for a permanent cation analogue, AR-H070091, were 11 ± 1.2 μM at pH 7.4 and 16 ± 1.8 μM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H⁺,K⁺-ATPase. In ion-tight vesicles, AZD0865 inhibited H⁺,K⁺-ATPase more potently (IC₅₀ 6.9 ± 0.4 nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC₅₀ 0.28 ± 0.01 and 0.26 ± 0.003 μM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3 μM) immediately inhibited H⁺,K⁺-ATPase activity by 88 ± 1%. Immediately after a 10-fold dilution H⁺,K⁺-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H⁺,K⁺-ATPase. In contrast to omeprazole, AZD0865 inhibited H⁺,K⁺-ATPase activity in a K⁺-competitive manner (K_i 46 ± 3 nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC₅₀ 1.7 ± 0.06 μM). At 100 μM, AZD0865 reduced porcine renal Na⁺,K⁺-ATPase activity by 9 ± 2%, demonstrating a high selectivity for H⁺,K⁺-ATPase. Thus, AZD0865 potently, K⁺-competitively, and selectively inhibits gastric H⁺,K⁺-ATPase activity and acid formation in vitro, with a fast onset of effect.     

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC₅₀ values of approximately 105 μM, 65 μM, and 54 μM were found for respiration, acidification, and adhesion, respectively. An IC₅₀ value of approximately 36 μM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC₅₀ values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.     

Identification of dual DNA-PK MDR1 inhibitors for the potentiation of cytotoxic drug activity

Inhibition of DNA repair is an attractive therapeutic approach to enhance the activity of DNA-damaging anticancer chemotherapeutic agents. Similarly, blockade of the multidrug-resistance protein 1 (MDR1) can overcome efflux-mediated resistance. DNA-dependent protein kinase (DNA-PK) is essential for the non-homologous end-joining DNA repair pathway. NU7441 is a potent DNA-PK inhibitor (IC₅₀ = 14 nM) that is used widely to study the effects of DNA-PK inhibition in vitro. In growth inhibition studies, 1 μM NU7441 sensitised vincristine-resistant CCRF-CEM VCR/R leukaemia cells (1200-fold resistant) to a range of MDR1 substrates, including doxorubicin (8-fold, p = 0.03), vincristine (14-fold, p = 0.01) and etoposide (63-fold, p = 0.02), compared with 1.4-fold (p = 0.02), 2.2-fold (p = 0.04) and 3.6-fold (p = 0.01) sensitisation, respectively, in parental CCRF-CEM cells. This difference in NU7441 sensitivity was confirmed in another two parental and MDR1-overexpressing cell line pairs. A doxorubicin fluorescence assay showed that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1 μM NU7441 increased doxorubicin nuclear fluorescence 16-fold. NU7441 and 3 structurally related compounds (NU7742 (an NU7441 analogue that does not inhibit DNA-PK – IC₅₀ > 10 μM), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative – IC₅₀ = 2 nM) and DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative - IC₅₀ = 7 μM)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC–MS. This paper demonstrates that NU7441 is a dual DNA-PK and MDR1 inhibitor, and this extends the therapeutic potential of the compound when used in combination with MDR substrates.     

The supplementation of zinc increased the apoptosis of airway smooth muscle cells by increasing p38 phosphorylation

Proliferation of airway smooth muscle cells (ASMCs) is believed to play an important role in causing airway hyperresponsiveness (AHR). It has also been reported that platelet-derived growth factor (PDGF) can stimulate proliferation of ASMCs. We hypothesize that the concentration of zinc in the bodies of asthmatic patients may play a role in PDGF activity and therefore may be related to the variations in severity of airway inflammation and narrowing seen in asthmatic patients. We investigated the effects and mechanisms of zinc supplementation in PDGF-treated ASMCs.In this study, PDGF-treated primary ASMCs were cultured with 3, 12, 24, or 96 μM ZnSO₄. We found that the highest concentration of ZnSO₄ (96 μM) was cytotoxic for ASMCs. PDGF was used to induce ASMCs proliferation under different zinc concentrations. Neither 3 μM nor 12 μM ZnSO₄ inhibited proliferation of PDGF-treated ASMCs, although 24 μM ZnSO₄ caused treatment-induced apoptosis in PDGF-treated ASMCs. Supplementation with 24 μM ZnSO₄ may therefore increase p38 activation and reduce Akt phosphorylation. Zinc supplementation may reduce proliferation of PDGF-treated ASMCs through the activation of p38 mitogen-activated protein kinase (MAP kinase) and suppression of Akt phosphorylation, which both drive the induction of cellular apoptosis, subsequently reducing the proliferation of ASMCs.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Metasequoia glyptostroboides Miki ex Hu

This study was undertaken to assess the antioxidant and antidermatophytic potential of the essential oil and extracts (hexane, chloroform, ethyl acetate and methanol) of Metasequoia glyptostroboides Miki ex Hu. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ = 9.1 and 14.24 μg/ml, respectively) as compared to butylatedhydroxyanisole (BHA), (IC₅₀ = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (93.26 mg/g of dry wt) as compared to the other extracts. Further, oil (1250 μg/disc) and extracts (1750 μg/disc) revealed 35.33–67.66 and 18.0–53.3% antidermatophytic effect, respectively, along with their respective MIC values (62.5–500 and 250–4000 μg/ml) against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349. The oil also had a strong detrimental effect on spore germination as well as concentration and time-dependent kinetic inhibition of M. canis KCTC 6591.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.