In vitro assessment for cytotoxicity screening of new antimalarial candidates

Abstract In antimalarial research there are no standard procedures to determine the toxicity of a drug candidate. Among the alternatives available, in vitro cytotoxicity assays are the most widely used to predict toxic effects of future therapeutic products. They have the advantage over the in vivo assays, in that they offer the possibility to restrain the number of experimental variables. The objective of the present study was to compare in vitro cytotoxic methods by testing various compounds currently used to treat malaria against different cell lines. Neutral red (NR) uptake and methylthiazoletetrazolium (MTT) colorimetric in vitro assays were used to determine preliminary toxicity of commercially available antimalarial drugs against tumor and non-tumor cells lines. Toxicity through brine shrimp lethality bioassay and hemolytic activity were also evaluated. Significant differences were observed in the tests measured by NR uptake. The tumor cell lines TOV-21G and HepG2 and non-tumor WI-26VA4 cells showed relatively uniform toxicity results, with TOV-21G being the most sensitive cell tested, presenting the lowest concentration to cause death to 50% of viable cells (CC50) values. The results of this study support the use of TOV-21G, HepG2 and WI-26VA4 cells lines as the choice for cytotoxicity tests to evaluate potential bioactive compounds.

Effect of alveolar macrophages phagocytosis on prognosis in patients with acute respiratory distress syndrome caused by abdominal infection: a multicenter study / 中华危重病急救医学

Objective To investigate the effects of alveolar macrophage phagocytosis on prognosis in patients with acute respiratory distress syndrome (ARDS) caused by abdominal infection. Methods ARDS patients caused by severe intra-abdominal infection admitted to intensive care unit (ICU) of Tianjin Fourth Central Hospital, Tianjin Nankai Hospital, Tianjin First Central Hospital and Tianjin Fifth Central Hospital from June 2016 to March 2018 were enrolled. The gender, age, acute physiology and chronic health evaluationⅡ(APACHEⅡ) within 24 hours of admission, neutral red phagocytosis and alkaline phosphatase activity of macrophages in bronchoalveolar lavage fluid, the length of ICU stay, total hospitalization time, hospitalization expenses, and prognosis were recorded. According to the prognosis, the patients were divided into death group and survival group, and the parameters were compared between the two groups. Pearson test was used to analyze the correlation between neutral red phagocytosis function of macrophages and alkaline phosphatase activity and other indicators. The prognosis was analyzed by binary Logistic regression combined with neutral red phagocytosis and alkaline phosphatase activity in patients, and the predictive value of both subjects on prognosis was analyzed by the receiver operating characteristic (ROC) curve. Results Twenty patients were enrolled in the study, with 8 in the death group and 12 in the survival group. Compared with the survival group, the death group was older (years old: 58.50±14.86 vs. 46.67±13.40), APACHEⅡ score was higher (21.50±3.93 vs. 13.58±4.12), neutral red phagocytosis ability and alkaline phosphatase activity of alveolar macrophages were significantly decreased (A value:0.265±0.050 vs. 0.338±0.016; μmol/L: 12.06±1.24 vs. 17.96±3.90), and the length of ICU stay was significantly longer (days: 22.00±14.59 vs. 11.50±3.17), hospitalization cost was significantly increased (10 thousand Yuan:24.17±11.02 vs. 13.44±3.53), the total hospitalization time was shorter (days: 25.25±15.01 vs. 35.67±8.58), and the difference was statistically significant (all P < 0.05). There was no significant difference in gender between the survival group and the death group [male (case): 8 vs. 6, P > 0.05]. The neutral red phagocytosis ability of alveolar macrophages in ARDS patients caused by abdominal infection was negatively correlated with age, APACHEⅡ score and the length of ICU stay (r value was -0.328, -0.572, -0.809, respectively, all P < 0.05); alkaline phosphatase activity was negatively correlated with age, APACHEⅡ score, the length of ICU stay and hospitalization expenses (r value was -0.334, -0.583,-0.470, -0.517, respectively, all P < 0.05). Binary Logistic regression analysis showed that neutral red phagocytosis [odds ratio (OR) = 0.596, 95% confidence interval (95%CI) = 0.212-0.997] and alkaline phosphatase activity (OR = 0.573, 95%CI = 0.339-0.968) were the influencing factors of prognosis (both P < 0.05). ROC curve analysis showed that the AUC of neutral red phagocytosis ability for prognosis of ARDS patients caused by abdominal infection was 0.948, and the sensitivity and specificity were 91.7% and 87.5% when the off-cut value was 0.317. The AUC of alkaline phosphatase for the prognosis of ARDS patients caused by abdominal infection was 0.813; when the cut-off value was 19.72 μmol/L, the sensitivity was 75.0%, and the specificity was 87.5%. Conclusion The alveolar macrophage phagocytosis dysfunction in ARDS patients caused by severe abdominal infection was not only related to the severity of the disease, but also increased the medical burden of patients, and significantly affected the mortality of such patients.

Interaction of phenylethanoid glycosides and calf thymus DNA / 中国中药杂志

In physiological condition, the interaction of acteoside and forsythoside B with calf thymus DNA using neutral red (NR) as a fluorescence probe were investigated by fluorescence, UV-visible spectrophotometry, viscosity, DNA melting techniques, and molecular docking. It is observed that acteoside and forsythoside B can react with DNA. The major mode of recognition between drug and DNA is groove binding by hydrogen bonds, and the interaction of acteoside with DNA is stronger than that of forsythoside B.

Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847) from the Narta Lagoon (Albania)

ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora (Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.

Neutral red staining and trypsin treatment to study viability and culture yield of Dermatophytes.

Dermatophytosis is common fungal infection of human being. To diagnose dermatophytic infections microscopic examination should be followed by culture which is essential step. Many times fungus may fail to grow on culture even after direct microscopy is positive due to non-Viability of fungus and it has been revealed that trypsinization enhances the rate of isolation of fungus on culture. Therefore, the study was undertaken with an aim to look for the viability and yield of dermatophytes on neutral red staining and trypsinization respectively.

Caracterização do extrato de aroeira (Myracrodruon urundeuva) e seu efeito sobre a viabilidade de fibroblastos gengivais humanos / Characterization of extract of mastic (M. urundeuva) and its effect on the viability of human gingival fibroblasts

A aroeira (Myracrodruon urundeuva) é uma árvore cujas folhas vem sendo estudadas com fins terapêuticos na medicina e odontologia por apresentar potencial antiinflamatório e antimicrobiano, além de favorecer o processo de reparo e cicatrização. O objetivo do trabalho foi caracterizar quimicamente o extrato de aroeira e avaliar seu efeito sobre fibroblastos gengivais humanos. Foi realizado a caracterização química da aroeira por meio do processo de triagem cromatográfica que consistiu na coleta das folhas sadias, maceração em metanol (MeOH) 80%, partição entre os solventes hexano, acetato de etila (AcOEt) e n-butanol (n-(BuOH)) Em seguida, realizaram-se análises dos compostos químicos em cromatografia liquida de alta eficiência acoplado a detector de arranjo de fotodiodo HPLC-PAD, análises por espectrometria de massas MS e cromatografia liquida acoplada a espectrometria de massas (LC-MS). Posteriormente foi realizada a análise de citotoxicidade do extrato hidroalcoólico de aroeira em fibroblastos gengivais humanos (linhagem FGH). Para a realização dos experimentos foram plaqueadas 2x103 células/poço em placas de 96 poços. O meio de cultivo foi substituído por meio de cultura de Eagle modificado por Dullbecco complementado por 10% soro fetal bovino (SFB) em diferentes concentrações do extrato hidroalcoólico de aroeira (extrato bruto; 1:10; 1:100; 1:1000; 1:10000 e controle). As análises de viabilidade foram feitas nos tempos experimentais de 24, 48, 72 e 96 horas, por meio dos testes de redução do MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazólio), captação do vermelho neutro e coloração pelo cristal violeta. A estatística foi realizada em análise de variância de 2 critérios seguido de uma análise de teste Tukey (p<0,05). Os compostos encontrados no extrato foram os derivados de ácido gálico, galotaninos, além de flavonoides. Para a redução do MTT os resultados mostraram uma ligeira oscilação da absorbância em todos os grupos...

Aroeira (M. urundeuva) is a tree whose leaves have been studied with therapeutic purpose in dentistry and medicine, due it antimicrobial and antinflamatory potencial protect, besides the repair and cicatrization processes. The aim of the study was characterize the aroeiras extract and evaluate effect over human gingival fibroblast. The aroeiras characterization was realized by means of chromatography screenuy, process that consists in healthy leaves maceration in 80% MeOH, partition in the BuOH, AcOEt and hexanes solvents. Next, the chemicals compounds were analysed in High Performance Liquide Chromatography-Photo Diode Array (HPLC-PAD), Liquide Chromatography Mass Spectrometry (LCMS) and Mass Spectrometry (MSs). Subsequently was realized the citotoxicitys analysis from the aroeira hidroalcooholic extract in human gingival fibroblast (FGH lineage). For the experiments realization was platted 2x103 cels/well in 96 wells plate. The cultives mean was substituted for Eagles means culture changed for complemented Dullbeco for 10% bovine fetal serum in aroeiras hidroalcooholic extracts concentrations different (brute extract, 1:10, 1:100, 1:1000, 1:10000). The viability analysis was carried out in experimental times 24, 48, 72 and 96 hours, by means of MTT reductions test (brometo3-(4,5-dimetiltiazol-2-yl)-2,5-difeniltetrazolio), neutral red captation and violet crystal). The statistics were performed for analysis of variance in 2 criteria continue with a Tukey test analysis (p<0.05). The founded compounds in the extract was the galic acid, gallotanine linked to glucose, besides flavonoids. For the MTTs reduction the results display an absorbance oscillation lightness in all the groups in experimental periods. Distinction for the control group and 1:100, 1:1000 and 1:10000 dilution, that present, by and large, for all the periods, the biggest valuable, while the brute extract group and 1:10 dilution present valuable lower to the others groups (p<0.05)...

Determination of the Concentration of New Antithrombotic Medicine Dermatan Sulfate in Blood or Urine by Spectrophotometry / 中国药房

OBJECTIVE:To determine the concentration of Dermatan Sulfate,a new antithrombotic medicine in blood or urine by spectrophotometry.METHODS:In Britton Robinson buffer solution(pH=5.8),color fading reaction occurred when Dermatan sulfate combined with neutral red dye to form ionic associate.The reduction of absorbance of the system was positively correlated to dermatan sulfate concentration,with the maximum absorption wavelength at 526 nm.RESULTS:The linear range of dermatan sulfate was 0.18～4.0?g?mL~(-1)(r=0.999 1) with a detection limit of 0.054?g?m~(-1).The average recovery was 100.3%(RSD=1.2%),with RSD of dermatan sulfate at low,medium,and high concentrations at 2.9%, 1.5%,and 1.1%,respectively.The coexisting substances did no interference on the determination results.CONCLUSION: The method is simple,accurate,and sensitive with good methodological selectivity and it achieved satisfactory results in the determination of dermatan sulfate in blood or urine.

Simultaneous Stain with Neutral Red and Tetrazolium Salt for Experimental Cerebral Infarction Morphometry

Serial changes in the size of infracted area induced by MCA occlusion(MCAO) were compared with Neutral Red(NR) and 2, 3, 5-Triphenyl tetrazolium chloride(TTC) stains. The differences in size of the infracted area as shown by the 2 stains and its significance were also evaluated. The experimental animals were divided into 7 troups, with each group consisting of rats;these groups were stained at 2, 4, 6, 8, 12, 24 and 48 hours after MCAO. After MCAO, NR was infused into the femoral vein, after which the brain was removed, the fraontal pole of the brain cut into 1.5mm sections, and each section photographed. Then, the NR-stained sections were immersed in TTC solution for 45 minutes and photographed. Results showed that the infracted area progressively increased according to time duration after MCAO(one-way ANOVA, p<0.01). Between 4 and 6 hour groups, the difference of the infracted area was greater than at any other timed groups, this being statistically significant(unpaired t-test, p<0.05). After 6 hours, the infracted area with NR stain became relatively stable. In contrast, however, the infracted area with TTC stain did not stabilize, but continued to increase up to 24 hours. Overall, the infracted area with NR stain was greater than with TTC stain in all the timed groups(paired t-test, p<0.05). As time progressed, the differences tended to decrease 48 hours post occlusion. In our study, serial changes of the ischemic penumbra area were evaluated by staining the ischemic area simultaneously with Neutral red and TTC stain. The results suggest that the ischemic penumbra area may still persist even after 48 hours post-MCAO.

MTT and Neutral red Biologic Assay for Human Bladder Cancer Cell Line(TCC-SuP) and Androgen Stimulated Cell Line of Mouse (SC-115): In Vitro Chemosensitivity Test / 대한비뇨기과학회지

To overcome the limitations of clonogenic assay (low plating efficiencies, clumping artifacts, long assay duration), we performed chemosensitivity test using MTT and neutral red biologic assay on SC-115 (androgen stimulated mouse tumor) and TCC-SuP (human bladder tumor) cell lines. The anticancer agents tested were adriamycin, cisplatin, methotrexate and combination of these drugs. And the following results were obtained ; 1) The growth of two cell lines were inhibited dose-dependently by application of adriamycin, cisplatin and combination of drugs. But methotrexate was not effective on TCC-SuP cell line. 2) The neutral red biologic assay is more convenient and rapid method than MTT assay but staining ability is superior in MTT assay. 3) The MTT and neutral red biologic assay offer advantages in speed, simplicity, cost & safety for the chemosensitivity test and for determining the new regimen.

The effects of different opiate peptides and ACTH on the phagocytosis of mouse macrophages / 中国免疫学杂志

Peritoneal macrophages were obtained from normal LACA mice and the phagocy-tosis of neutral red was measured after 24 hr.incubation with different opiate peptidesand ACTH.?-endorphin(10~-(10)-10~_6M)and dynorphin A(10~_(10)-1(10~_6M)were found tostimulate the phagocytosis and these increases in phagocytosis could be reversed byopiate receptor blocker,naltrexone(10~_5M).[D-Ala~2,D-Leu~5]-enkephalin(10~_(12)-10~_6M),however,had no effect on the phagocytosis.ACTH(10~_(10)-10~_6M)was fo-und to have suppressive effect on the phagocytosis.These results suggest that diffe-rent opiate peptides may cause different effects on phagocytosis of macrophages andthe increase in phagocytosis induced by ?-endorphin and dynorphin appears to be me-diated by opiate receptors on peritoneal macrophages.