In Vitro Human Monoamine Oxidase Inhibition and Human Dopamine D4 Receptor Antagonist Effect of Natural Flavonoids for Neuroprotection.

Natural flavone and isoflavone analogs such as 3',4',7-trihydroxyflavone (1), 3',4',7-trihydroxyisoflavone (2), and calycosin (3) possess significant neuroprotective activity in Alzheimer's and Parkinson's disease. This study highlights the in vitro human monoamine oxidase (hMAO) inhibitory potential and functional effect of those natural flavonoids at dopamine and serotonin receptors for their possible role in neuroprotection. In vitro hMAO inhibition and enzyme kinetics studies were performed using a chemiluminescent assay. The functional effect of three natural flavonoids on dopamine and serotonin receptors was tested via cell-based functional assays followed by a molecular docking simulation to predict interactions between a compound and the binding site of the target protein. A forced swimming test was performed in the male C57BL/6 mouse model. Results of in vitro chemiluminescent assays and enzyme kinetics depicted 1 as a competitive inhibitor of hMAO-A with promising potency (IC50 value: 7.57 ± 0.14 µM) and 3 as a competitive inhibitor of hMAO-B with an IC50 value of 7.19 ± 0.32 µM. Likewise, GPCR functional assays in transfected cells showed 1 as a good hD4R antagonist. In docking analysis, these active flavonoids interacted with a determinant-interacting residue via hydrophilic and hydrophobic interactions, with low docking scores comparable to reference ligands. The post-oral administration of 1 to male C57BL/6 mice did not reduce the immobility time in the forced swimming test. The results of this study suggest that 1 and 3 may serve as effective regulators of the aminergic system via hMAO inhibition and the hD4R antagonist effect, respectively, for neuroprotection. The route of administration should be considered.

A novel gas production bioassay of thiosulfate utilizing denitrifying bacteria (TUDB) for the toxicity assessment of heavy metals contaminated water.

This study reports for the first-time the possibility of deploying gas production by thiosulfate utilizing denitrifying bacteria (TUDB) as a proxy to evaluate water toxicity. The test relies on gas production by TUDB due to inhibited metabolic activity in the presence of toxicants. Gas production was measured using a bubble-type respirometer. Optimization studies indicated that 300 mg NO3--N/L, 0.5 mL acclimated culture, and 2100 mg S2O32-/L were the ideal conditions facilitating the necessary volume of gas production for sensitive data generation. Determined EC50 values of the selected heavy metals were: Cr6+, 0.51 mg/L; Ag+, 2.90 mg/L; Cu2+, 2.90 mg/L; Ni2+, 3.60 mg/L; As3+, 4.10 mg/L; Cd2+, 5.56 mg/L; Hg2+, 8.06 mg/L; and Pb2+, 19.3 mg/L. The advantages of this method include operational simplicity through the elimination of cumbersome preprocessing procedures which are used to eliminate interferences caused by turbidity when the toxicity of turbid samples is determined via spectrophotometry.

Improved toxicity analysis of heavy metal-contaminated water via a novel fermentative bacteria-based test kit.

The objective of this study was development of a simple and reliable microbial toxicity test based on fermentative bacteria to assess heavy metal (Hg2+, Cu2+, Cr6+, Ni2+, As5+, or Pb2+)-contaminated water. The dominant species of test organisms used in this study was a spore-forming fermentative bacterium, Clostridium guangxiense. Toxicity of water was assessed based on inhibition of fermentative gas production of the test organisms, which was analyzed via a syringe method. Overall, the fermentative bacteria-based test kits satisfactorily identified increased toxicity of water as water was contaminated with high amounts of heavy metals; however, levels of inhibition were dissimilar depending on the species of metals. Inhibitory effects of Hg2+, Cu2+, Cr6+, and Ni2+ were considerably greater than those of As5+ and Pb2+. The 24 h half-maximum effective concentrations (EC50) for Hg2+, Cu2+, Cr6+, Ni2+, As5+, and Pb2+ were analyzed to be 0.10, 0.51, 1.09, 3.61, 101.33, and 243.45 mg/L, respectively, confirming that Hg2+, Cu2+, Cr6+, and Ni2+ are more toxic to fermentative gas production than As5+ and Pb2+. The fermentative bacteria-based toxicity test represents an improvement over other existing toxicity tests because of ease of end-point measurement, high reproducibility, and favorable on-site field applicability. These advantages make the fermentative bacteria-based test suitable for simple and reliable toxicity screening for heavy metal-contaminated water.

Real-time monitoring of water quality of stream water using sulfur-oxidizing bacteria as bio-indicator.

In aquatic ecosystems, real-time water-quality (WQ) biomonitoring has become the most effective technology for monitoring toxic events by using living organisms as a biosensor. In this study, an online WQ monitoring system using sulfur oxidizing bacteria (SOB) was tested to monitor WQ changes in real-time in natural stream water. The WQ monitoring system consisted of three SOB reactors (one continuous and two semi-continuous mode reactors). The SOB system did not detect any toxicity in relatively-unpolluted, natural stream water when operated for more than six months. When diluted swine wastewater (50:1) was added to the influent of the reactors, the system detected toxic conditions in both the continuous and semi-continuous operational modes, showing 90% inhibition of SOB activity within 1 h of operation. The addition of 30 mg/L NO2--N or 2 mg/L of Cr6+ to the influents of SOB reactors resulted in the complete inhibition of the SOB activity within 1-2 h. The results demonstrated the successful application of an SOB bioassay as an online toxicity monitoring system for detecting pollutants from stream or river waters.

Development of an online sulfur-oxidizing bacteria biosensor for the monitoring of water toxicity.

A toxicity monitoring system based on the metabolic properties of sulfur-oxidizing bacteria (SOB) in continuous and fed-batch modes has been applied for the detection of nitrite (NO2 (-)-N). In this study, the effects of different concentrations of NO2 (-)-N (0.1 to 5 mg/L) on the SOB bioreactors were tested. We found that 5 mg/L NO2 (-)-N was very toxic to the SOB bioreactors in both continuous (R1) and fed-batch (R2) modes, showing complete inhibition of SOB activity within 2 h of operation. R1 and R2 were operated in different ways; however, the EC inhibition and recovery patterns were very similar. The EC rate increased with an increasing NO2 (-)-N concentration in both continuous and fed-batch modes. The addition of 5 mg/L NO2 (-)-N in continuous mode decreased the average EC rate by 14.38 ± 2.1 µS/cm/min; while in fed-batch mode, the EC rate decreased by 23 µS/cm/min. Although the toxicity monitoring system could detect 0.5-5 mg/L NO2 (-)-N, it could not detect 0.1 mg/L NO2 (-)-N in either continuous or fed-batch operation. Thus, the SOB biosensor method presented is useful to detect toxic agents such as NO2 (-)-N within a few minutes or hours.