Perspective: repurposed drugs for COVID-19.

Introduction: The article aims to emphasize the necessity of proper research design, both scientifically and ethically, in order to provide good evidence for physicians to base their decisions on when prescribing drug treatment. Methods: Research articles and guidelines related to therapy of COVID-19 were searched from the PubMed database. Results: Only remdesivir and tocilizumab are medicines that have been approved by the US FDA's decision to approve their clinical use in moderate and severe COVID-19. Conclusions: Favipiravir, ivermectin and andrographolide need further well-conducted research to confirm the efficacy and safety against COVID-19 at different stages.

Assessment of ochratoxin A exposure risk from the consumption of coffee beans in Phnom Penh, Cambodia.

This survey aimed to determine OTA contamination in roasted coffee samples commercialised in Phnom Penh, Cambodia and to assess the potential health risk from OTA exposure. Forty locally grown and imported coffee samples were collected and analysed. Analytical validation methods were fully performed. In 3 of 40 samples (7.5%), the results showed detectable levels of OTA, ranging from 0.19 to 1.12 µg kg-1, with an overall average of 0.26 µg kg-1 and an average over the LOQ (n = 3) at 0.81 µg kg-1. OTA estimated daily intake (EDI) of both values were 0.05 (overall average) and 0.17 ng/kg bw/day (the worst-case scenario) with the calculated risk of OTA exposure expressed as a Hazard Quotient at 0.003 and 0.01, respectively. This result could imply a low health risk to Cambodian coffee consumers.

The effect of green tea catechins on breast cancer resistance protein activity and intestinal efflux of aflatoxin B1 via breast cancer resistance protein in Caco-2 cells.

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus spp., was proved as one of the major causes of human hepatocellular carcinoma (HCC) when chronically consumed. An efflux of AFB1 was reported to be associated with breast cancer resistance protein (BCRP) whose activity could also be modulated by green tea catechins. The purpose of this study was, therefore, to examine the impacts of green tea catechins on BCRP activity in Caco-2 cells by H33342 (bis-benzamide, BCRP substrate) accumulation and AFB1 efflux. Results showed a significant decrease (p < 0.05) of AFB1 in the efflux ratio following the incubation with Ko143, a specific BCRP inhibitor, and sodium fluoride, confirming the association of BCRP in AFB1 efflux transport across the cells. Pre-incubation with green tea and gallate catechins (ECG and EGCG) significantly reduced the efflux ratio of AFB1 (p < 0.05) and significantly increased the intracellular H33342 substrate (p < 0.05) in Caco-2 cells, clearly indicating the inhibitory effects of green tea and gallate catechins on BCRP function. Further study on H33342 accumulation revealed a dose-dependent increment of intracellular H33342 when co-administered with increasing concentrations of AFB1. This result implied a possible role of AFB1 as a BCRP competitive inhibitor. The findings from this study concluded the roles of BCRP as an efflux transporter for AFB1 and could be modulated by the exposure of green tea catechins. Owing to a reduction of its efflux, an inhibitory effect of BCRP when pre-exposed with green tea catechins could be crucial for AFB1 cellular accumulation.

Oxidative stress and genotoxicity of co-exposure to chlorpyrifos and aflatoxin B1 in HepG2 cells.

Chlorpyrifos (CPF) and aflatoxin B1 (AFB1) are each known to adversely affect hepatic tissue individually, but their combined hepatic effects have never been previously investigated. HepG2 cell viability, oxidative status, and genetic impairment were examined after exposing HepG2 cells to: (1) CPF alone, (2) AFB1 alone, and (3) CPF and AFB1 combined (20:1). CPF exposure decreased cell viability, reduced glutathione (GSH) content, and superoxide dismutase (SOD) activity but increased both glutathione peroxidase (GPx) and paraoxonase 1 activity. AFB1 exposure decreased cell viability and GSH content but increased reactive oxygen species (ROS) production. CPF and AFB1 combined exposure decreased GSH content (p < 0.05) further over individual CPF and AFB1 exposures. Induction of micronucleus formation was detected in AFB1-treated cells but undetected in both CPF and combination-treated cells. In conclusion, cytotoxic effects caused by combined exposure were antagonistic, as shown by a combination index value of 1.67. Although no change in ROS production was observed in CPF groups, the overall results confirmed the occurrence of oxidative stress through the alterations of GSH content, GPx, and SOD activity. Only intracellular GSH was evidently changed upon exposure to CPF and AFB1 combined. Thus, this study suggested cellular GSH as a potential indicator for detecting the combined effects of CPF and AFB1 in HepG2 cells, the detection of which could be adapted to estimate the potential toxicity of additional multiple toxicant exposures.

Toxicity evaluation of cordycepin and its delivery system for sustained in vitro anti-lung cancer activity.

In the previous study, we have found that the cordycepin which was extracted from Cordyceps mycelia produced by growing Cordyceps militaris on the dead larva of Bombyx mori silkworms showed the anti-proliferative effect toward lung cancer cells without toxicity to non-cancer cells. In this work, the cordycepin was tested for its in vitro mutagenicity and in vivo toxicity. From the Ames test and subacute toxicity test using oral administration in a rat model, the cordycepin was proved to be a non-mutagenic and non-toxic compound. The hematology and blood chemistry as well as the microanatomical characteristic of the tissues of rats fed with cordycepin every day for consecutive 30 days were comparable to those of the normal ones. Then, the cordycepin was incorporated in gelatin type A (GA) and gelatin type B (GB) nanoparticles aimed to sustain its release and activity. The cordycepin incorporated in both GA and GB nanoparticles showed the sustained release profiles. GA nanoparticles could encapsulate cordycepin at higher encapsulation efficiency due to the attractive electrostatic interaction between the positive-charged GA and the negative-charged cordycepin. However, GA nanoparticles released cordycepin at the higher amount possibly because of the large surface area of small size nanoparticles. Comparing to GB nanoparticles, the higher amount of cordycepin released from GA nanoparticles showed the higher anti-proliferative and anti-migratory effects on A549 lung cancer cells. In conclusion, GA nanoparticles were suggested as a suitable carrier for the sustained release of cordycepin. The GA nanoparticles releasing cordycepin could be an effective and non-invasive material for the treatment of lung cancer cells.

Melatonin in patients with cancer receiving chemotherapy: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The MIRCIT trial was a randomized, double-blind, placebo-controlled study of advanced Non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients were randomized to receive 10 mg or 20 mg of melatonin or placebo. Assessment of health-related quality of life (HRQoL) was completed at baseline, and at 2, 3 and 7 months. Survival and adverse events were collected. DNA damage marker 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was measured during the first three months of chemotherapy. RESULTS: Patients in the melatonin-treated group had better adjusted HRQoL scores, with a slightly significantly better score (2.69 points, 95% confidence interval (CI)=0.01-5.38, p=0.049) being found in social well-being. Median survival was 7.3 months (95% CI=3.42-11.14) without significant difference. A great amont of DNA damage marker was observed in the placebo-treated group, and this was associated with lower survival (r(2)=-0.656, p=0.02), implying the protective effect of melatonin in healthy cells. CONCLUSION: Melatonin in combination with chemotherapy did not affect survival and adverse events of advanced patients with NSCLC, but there was a trend for better HRQoL.

Dietary intake of melatonin from tropical fruit altered urinary excretion of 6-sulfatoxymelatonin in healthy volunteers.

This study assessed the melatonin content of six tropical fruits and examined whether human consumption could contribute to dietary melatonin as measured by 6-sulfatoxymelatonin (aMT6-s, a marker of circulating melatonin in the body). Melatonin was extracted using methanol and analyzed by high-performance liquid chromatography. In a clinical crossover study, 30 healthy volunteers consumed selected fruits one at a time, with a 1week wash-out period between fruits, until completing all six fruits. Most fruits had moderate melatonin content. Significant increases in urine aMT6-s concentrations were seen after the consumption of pineapple (266%, p = 0.004), banana (180%, p = 0.001), and orange (47%, p = 0.007). The need to analyze melatonin both in fruit and as in vivo uptake was demonstrated. Further study is warranted regarding the clinical effect of fruit consumption in people with age-related melatonin reduction problems such as sleeplessness and illnesses involving oxidative damage.

Glycemic and oxidative status of patients with type 2 diabetes mellitus following oral administration of alpha-lipoic acid: a randomized double-blinded placebo-controlled study.

Despite well-controlled blood glucose levels, diabetic complications still inevitably take place via several mechanisms including excessive generation of free radicals in patients who suffer from diabetes mellitus (DM). A randomized double-blind placebo-controlled clinical trial to investigate the effectiveness of oral supplementation of DL-alpha-lipoic acid (ALA) on glycemic and oxidative status in DM patients was conducted. Thirty eight outpatients with type 2 DM were recruited and randomly assigned to either placebo or treatment in various doses of ALA (300, 600, 900, and 1200 mg/day) for 6 months. Following the treatment, all subjects were evaluated for glucose status and oxidative biomarkers. Results showed that fasting blood glucose, HbA1c trended to decrease in a dose-dependent manner. Increase of urinary PGF2α-Isoprostanes (F2α-IsoP) was noted in placebo but not ALA-treated groups, indicating possible suppressing action of ALA on lipid peroxidation in DM subjects. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) levels, however, were similar in both placebo and ALA groups as well as urinary microalbumin and serum creatinine. Safety evaluation was monitored and treatment was found to be well tolerated despite some minor side effects. Results from this study reflected the benefits of ALA in glucose status with slight efficiency on oxidative stress-related deterioration in DM patients.

Bacillus anthracis endospores regulate ornithine decarboxylase and inducible nitric oxide synthase through ERK1/2 and p38 mitogen-activated protein kinases.

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide ((•)NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing L-ornithine at the expense of (•)NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.

Inducible nitric oxide synthase catalyzes ethanol oxidation to alpha-hydroxyethyl radical and acetaldehyde.

The physiologic function of nitric oxide synthases, independent of the isozyme, is well established, metabolizing L-arginine to L-citrulline and nitric oxide (NO). This enzyme can also transfer electrons to O2, affording superoxide (O2*-) and hydrogen peroxide (H2O2). We have demonstrated that NOS1, in the presence of L-arginine, can biotransform ethanol (EtOH) to alpha-hydroxyethyl radical (CH3*CHOH). We now report that a competent NOS2 with l-arginine can, like NOS1, oxidize EtOH to CH3*CHOH. Once this free radical is formed, it is metabolized to acetaldehyde as shown by LC-ESI-MS/MS and HPLC analysis. These observations suggest that NOS2 can behave similarly to cytochrome P-450 in the catalysis of acetaldehyde formation from ethanol via the generation of alpha-hydroxyethyl radical when L-arginine is present.

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron-dithiocarbamate complexes.

The free radical, nitric oxide ( radicalNO), is responsible for a myriad of physiological functions. The ability to verify and study radicalNO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of radicalNO at relevant cellular sites. With this in mind, several iron-chelates (Fe2+(L)2) have been developed, which have provided a useful tool for the study and identification of radicalNO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of radicalNO detection is dependent on the Fe2+(L)2 complex. The development of more efficient and stable Fe2+(L)2 chelates may help to better understand the role of radicalNO in vivo. In this paper, we present data comparing several proline derived iron-dithiocarbamate complexes with the more commonly used spin traps for radicalNO, Fe2+-di(N-methyl-D-glutamine-dithiocarbamate) (Fe2+(MGD)2) and Fe2+-di(N-(dithiocarboxy)sarcosine) (Fe2+(DTCS)2). We evaluate the apparent rate constant (kapp) for the reaction of radicalNO with these Fe2+(L)2complexes and the stability of the corresponding Fe2+(NO)(L)2 in presence of NOS I.

A comparative study of neuronal and inducible nitric oxide synthases: generation of nitric oxide, superoxide, and hydrogen peroxide.

Nitric oxide synthases (NOS) independent of the isozyme, produce nitric oxide (.NO), superoxide (O2.-), and hydrogen peroxide (H2O2). Since .NO has been implicated in many physiological processes, the importance of O2.- and H2O2 in regulating cell signaling by .NO cannot be overlooked. Before addressing these questions, we investigated the production of .NO, O2.-, and H2O2 by purified NOS. NOS 1 and NOS 2 were chosen, as the flux of .NO from each isozyme supports differential biological activity. We found that the initial rate and sustained production of .NO was considerably greater for NOS 2 as compared to NOS 1. In the absence of L-arginine, however, NOS 1 generation of O2.- and H2O2 was found to be substantially greater than that measured for NOS 2. Differences between NOS 1 and NOS 2 production of .NO, O2.-, and H2O2 may define the specific physiologic function of each isozyme.

Contamination of aflatoxins in herbal medicinal products in Thailand.

Twenty-eight herbal medicinal products from Thailand were investigated for aflatoxin (AF) contaminations by employing a specific HPLC assay for the determination of AFB1, B2, G1 and G2. The samples were extracted with 80% (v/v) methanol in water before further cleaned up with an immunoaffinity column and followed by the detection of AFs by using an electrochemically post-column derivatization with iodine and fluorescence detector. The extraction procedure was optimized in order to obtain the best recovery. The method was successfully carried out with all the herbal products diversified as to compositions and dosage forms. The results revealed that five (18%) of herbal samples were contaminated with detectable amount of the total AFs ranging from 1.7 to 14.3 ng/g. The association between particular herbal/plant and the AF contaminated could not be determined due to the low frequency of positive samples. The contaminated products were those in tablet (4) and capsule (1) dosage forms. It was possible that the original fungal infection of these products may have been derived from either the crude herbal or other ingredients making these preparations, such as starch. In conclusion, none of the AF contaminated level found was above the current legislative level permissible in Thailand (20 ng/g). A word of caution, however, exporting some high AF-contaminated herbal products to countries where more stringent permissable level of aflatoxins exist could result in trade Barriers.

The effect of divalent cations on neuronal nitric oxide synthase activity.

Neuronal nitric oxide synthase (NOS I) is a Ca(2+)/calmodulin-binding enzyme that generates nitric oxide (NO*) and L-citrulline from the oxidation of L-arginine, and superoxide (O(2)*(-)) from the one-electron reduction of oxygen (O(2)). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca(2+), which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca(2+) binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I-mediated catalysis. As in the case of Ca(2+), we demonstrate that Ni(2+), Ba(2+), and Mn(2+) can activate NOS I to metabolize L-arginine to L-citrulline and NO*, and afford O(2)*(-) in the absence of L-arginine. In contrast, Cd(2+) did not activate NOS I to produce either NO* or O(2)*(-), and the combination of Ca(2+) and either Cd(2+), Ni(2+), or Mn(2+) inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO*, O(2)*(-) and products derived from these free radicals.

Hemin: a possible cause of oxidative stress in blood circulation of beta-thalassemia/hemoglobin E disease.

A correlation between endogenous hemin and pro-oxidant activity was revealed in serum of beta-thalassemia/hemoglobin E disease (beta-thal/Hb E), which is the most common prevalent type of thalassemia in Thailand. The technique of low temperature electron spin resonance spectroscopy was used for characterization and quantification of high spin ferric heme, which had been identified as hemin (iron (III)-protoporphyrin IX). Hemin was present at levels ranging from 50 to 280 microM in serum of beta-thal/Hb E but not detectable in serum of non-thalassemia. Pro-oxidant activity in serum of beta-thal/Hb E was demonstrated by luminol-mediated chemiluminescence, a sensitive method for screening of free radical generation in vitro. In the presence of H2O2, the chemiluminescence intensity (CL) was about 20 fold enhanced in serum of beta-thal/Hb E, indicating its extensive pro-oxidant activity. The CL showed a good correlation with serum heroin, r = 0.778 (p < 0.001), while the correlations with total serum iron and serum ferritin were 0.260 (p = 0.259) and 0.519 (p = 0.004), respectively. Our finding suggested that serum hemin readily catalyzed free radical reactions and it may contribute a major pro-oxidant in blood circulation of beta-thal/Hb E.

The generation of free radicals by nitric oxide synthase.

Nitric oxide synthase (NOS) is an example of a family of heme-containing monooxygenases that, under the restricted control of a specific substrate, can generate free radicals. While the generation of nitric oxide (NO*) depends solely on the binding of L-arginine, NOS produces superoxide (O(2)*(-)) and hydrogen peroxide (H(2)O(2)) when the concentration of the substrate is low. Not surprisingly, effort has been put forth to understand the pathway by which NOS generates NO*, O(2)*(-) and H(2)O(2), including the role of substrate binding in determining the pathways by which free radicals are generated. By binding within the distal heme pocket near the sixth coordination position of the NOS heme iron, L-arginine alters the coordination properties of the heme iron that promotes formation of the perferryl complex NOS-[Fe(5+)=O](3+). This reactive iron intermediate has been shown to abstract a hydrogen atom from a carbon alpha to a heteroatom and generate carbon-centered free radicals. The ability of NOS to produce free radicals during enzymic cycling demonstrates that NOS-[Fe(5+)=O](3+) behaves like an analogous iron-oxo complex of cytochrome P-450 during aliphatic hydroxylation. The present review discusses the mechanism(s) by which NOS generates secondary free radicals that may initiate pathological events, along with the cell signaling properties of NO*, O(2)*(-) and H(2)O(2).

The role of tetrahydrobiopterin in the regulation of neuronal nitric-oxide synthase-generated superoxide.

Tetrahydrobiopterin (H(4)B) is a critical element in the nitric-oxide synthase (NOS) metabolism of l-arginine to l-citrulline and NO(.). It has been hypothesized that in the absence of or under nonsaturating levels of L-arginine where O(2) reduction is the primary outcome of NOS activation, H(4)B promotes the generation of H(2)O(2) at the expense of O(2)(-.). The experiments were designed to test this hypothesis. To test this theory, two different enzyme preparations, H(4)B-bound NOS I and H(4)B-free NOS I, were used. Initial rates of NADPH turnover and O(2) utilization were found to be considerably greater in the H(4)B-bound NOS I preparation than in the H(4)B-free NOS I preparation. In contrast, the initial generation of O(2)(-.) from the H(4)B-free NOS I preparation was found to be substantially greater than that measured using the H(4)B-bound NOS I preparation. Finally, by spin trapping nearly all of the NOS I produced O(2)(-.), we found that the initial rate of H(2)O(2) production by H(4)B-bound NOS I was considerably greater than that for H(4)B-free NOS I.

The activation of neuronal nitric-oxide synthase by various divalent cations.

Nitric-oxide synthase (NOS; EC 1.14.13.39) catalyzes the oxidation of L-arginine to nitric oxide (NO(.)) and L-citrulline via the intermediate N(omega)-hydroxy-L-arginine. Of the three distinct isoforms of NOS that have been characterized, the constitutive neuronal NOS (NOS I) generates NO(.) associated with long-term potentiation (LTP) and early brain development. All of the NOS isoforms contain an N-terminal oxidase and a C-terminal reductase domain connected by a Ca(2+)/calmodulin binding region. To activate NOS I, Ca(2+) has to bind to calmodulin, allowing electron transport through both domains. Calcium ions are tightly regulated in cells. However, a number of other metal ions that bind and activate calmodulin may also activate NOS I. One such metal ion may be Pb(2+), which is associated with neurobehavioral and psychological alterations, including the inhibition of LTP. The effect of various divalent cations on NOS I activity was tested, and the results presented herein demonstrate that Pb(2+) and Sr(2+) can activate NOS I to a level similar to that found for Ca(2+). Finally, there is a synergy between Pb(2+) and Ca(2+) resulting in maximal activation of NOS I using minimal concentrations of both metal ions.

Perferryl complex of nitric oxide synthase: role in secondary free radical formation.

Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO*) and superoxide (O(2)*-)during enzymatic cycling, the ratio of each free radical is dependent upon the concentration of L-arginine. Using spin trapping and electron paramagnetic resonance (EPR) spectroscopy, we recently reported that NOS I can oxidize ethanol (EtOH) to alpha-hydroxyethyl radical (CH(3)*CHOH). We speculated that the perferryl complex of NOS, (NOS-[Fe(5+)[double bond]O](3+)) was responsible for the generation of CH(3)*CHOH. Using potassium monopersulfate (KHSO(5)) to oxidize the heme of NOS I to NOS-[Fe(5+)[double bond]O](3+), we were able to demonstrate that this perferryl complex can oxidize L-arginine to L-citrulline and NO*. Even in the absence of L-arginine, EtOH was oxidized to CH(3)*CHOH by NOS-[Fe(5+)[double bond]O](3+). Sodium cyanide (NaCN), a heme blocker, inhibited the formation of CH(3)*CHOH by NOS.