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Cell-based assay methods are being widely used in drug discovery and development. After treatment of cells with drugs cytotoxicity is a common parameter to be measured in preclinical level. There are four cytotoxicity assays named as LDH assay, protein assay, neutral red assay and MTT assay of which LDH assay is discussed here. The LDH leakage assay is a colorimetric assay based on the measurement of lactate dehydrogenase activity in the extracellular medium. When the plasma membrane is damaged due to cell death LDH is released in to culture medium thus proportionating to the lysed cells. LDH activity was determined as NADH oxidation by LDH catalyzed conversion of lactate to pyruvate and INT reduction to form red formazan over a defined time interval. Reaction products were then assayed using a microplate reader (96 wells) and measured spectrophotometrically at 490nm. This method was successfully applied to determine cell death activity on H460 cells. This LDH release assay combines the advantages of reliability and simple evaluation characteristic of radioisotope release assays with the convenience of speed and avoidance of radioactivity.
Cell-based assay methods are now characteristically integrated into high throughput screening activities for drug discovery and development. Cytotoxicity is considered as common biological parameters which are measured after experimental treatment because it can be easily measured possessing the dose dependant paradigm. Drugs always produce physiological and therapeutic actions at lower concentration and toxic effects including necrosis or apoptosis at higher concentrations. Necrosis involves mitochondrial swelling and increased permeability of plasma membrane whereas, apoptosis deals with damage and breakdown of cells in to membrane bound apoptosis bodies. Mammalian cells after interacting with toxins undergoes structural and morphological changes which will lead to loss of membrane integrity. http://www.funakoshi.co.jp/data/datasheet/CAY/10008882.pdf
There are four cyototoxicity assays named as LDH assay, protein assay, neutral red assay and MTT assay. Protein assay is the method of indirect measurement of cell viability as it measures the protein content of viable cells which are left after washing of the treated plates. The neutral red assay is also a method to measure cell viability. It is used as an indicator of cytotoxicity in cultures of primary hepatocytes and other cell lines.
The MTT assay is another cell viability assay which is often used to determine cytotoxicity. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) is a water soluble tetrazolium salt, which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes and therefore it accumulates in healthy cells. The MTT assay was tested for its validity in various cell lines. (Fotakis and Timbrell, 2006), and the cytotoxicity assay which is carried here is LDH leakage assay.
LDH leakage assay:
The LDH leakage assay is the most common assay which is based on the measurement
of lactate dehydrogenase activity in the extracellular medium. Lactate dehydrogenase (LDH) is a stable soluble cytoplasmic enzyme present in all eukaryotic cells. LDH is released into culture medium upon cell death due to damage of plasma membrane. The increase of the LDH activity in culture supernatant is directly proportional to the number of lysed cells. http://www.sciencellonline.com/site/productsheets/8078.pdf
The LDH activity is determined by an enzymatic colorimetric test. The reaction takes place in following steps. The first step is the reduction of NAD+ to NADH/H+ by the LDH catalyzed conversion of lactate to pyruvate. In a second step, the catalyst (diaphorase) transfers H/H+ from NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)- 3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT), which is reduced to a red formazan. This irreparable damage to cellular architecture allows free movement of previously excluded molecules into the cell and their enzymatic contents to leak into the culture medium.(Weyermann et al.,2005) Therefore, measurement of intracellular enzyme marker activity in the extra-cellular environment is the basis of this cytotoxicity assays. http://www.clontech.com/images/pt/PT3947-1.pdf
This assay use the microtiterplates (96-well format). This miniaturization allows many samples to be analyzed rapidly and simultaneously. The amount of the highly colored and soluble formazan can be measured at 490 nm spectrophotometrically.
Figure 1: In the first step, released Lactate dehydrogenase (LDH) reduces NAD+ to NADH+H+ by oxidation of lactate to pyruvate. In the secondenzymatic reaction 2H are transferred from NADH+H+ to the yellow tetrazolium salt INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyl tetrazolium chloride) by a catalyst.
Materials and methods:
96-well microplate with H460 tumor cell are added which has been processed previously are needed. 3-96-well microplate (empty) for assay reactions are taken. The tube solutions used are as follows, 50ml tube containing assay medium, 1.5ml tube containing drug solvent master stock,1.5ml tube containing 2% Triton X-100/medium solution,1-1.5ml tube containing 160 μl of assay catalyst (store at 4 0C),3-30ml tube containing 2.25ml of assay dye solution (store at 4 0C) and class II cabinet 37 0C incubator, Gilson micropipettes and sterile pipette tips, Microplate reader are also needed.
Preparations for working solution:
The catalyst for the LDH release assay is prepared by dissolving the lyophilizate in 1ml of double distilled water for 10 min and then mixing it thoroughly. It is then stored for 4 weeks at +2 to +80C and then at room temperature for 2 days. The reaction mixture for LDH release assay is prepared by mixing the catalyst solution prepared above with dye solution just before the start of the experiment.
Inorder to calculate the percentage toxicity the controls are used in each experimental setup. The function of the three controls to be used are given below.
Background control: determines the LDH activity contained in the assay medium.
The low control: determines the LDH activity released from the untreated normal cells and this is equal to the spontaneous LDH release.
High control: determines the maximium releasable LDH activity in the cells and is equal to the maximum LDH release.
When preparing the controls the lysis reagents are added to the samples at the correct time to estimate the maximum releasable LDH because, the control cells grow during the period of exposure to the cytotoxic compounds and LDH release is underestimated to avoid this the lysis reagent are added to the high control at the end of the exposure period. The preparation of the control is given as under.
Contents of the well
Cell cell-free culture medium
Test substance or effector cells diluted in culture medium
LDH standard solution
1.0-10 5 cells/ml are added in 200μl of normal complete medium to a flat-bottomed 96-well plate according to the matrix in the table. The plate is then incubated overnight at 37 0C, 5% CO2. The plates are then washed in 200μl HBSS. Then HBSS is replaced with 200μl of assay medium (fetal calf serum).after this the serial dilutions of the drug is made. The drug is diluted in 1% FCS containing assay medium using 34mM drug solvent to make a final volume of 10ml.Then drug at 2X the on plate concentration is made. 100μl of the Triton X-100 solution are added to each of 9 wells in rows D-G as indicated in the microplate matrix table. The microplate is incubated at 37 0C at desired time point the microplate is removed from the incubator carefully and without disturbing the cell pellet 100μl of medium is removed from the selected wells and then it is transferred to the corresponding wells in the reaction microplate. Just immediately before use the 50μl of assay catalyst solution is added to one of the dye solution tubes and mixed gently to avoid air bubbles. The microplate is incubated at room temperature in dark for 30 minutes. The microplate is read on the microplate reader at 490nm.opnce it is read the contents of the pate are disposed in waste container and working place is kept tidy. The mean values for each experimental point are calculated and the mean background control are subtracted and thus the percentage cytotoxicity is calculated.
60 time point
120 time point
240 time point
Back control 200μl AM(No Cells)
Low control - 200μl AM(Cells)
High control - 100μl AM + 100μl 2% Triton X-100 medium
5.1μM Drug solvent + 100μl AM + 100μl drug solvent conc #1(5.1µM)
5.1μM Drug solvent + 100μl AM + 100μl drug solvent conc #2(17µM)
5.1μM Drug solvent + 100μl AM + 100μl drug solvent conc #3(51µM)
Table 1: The spectrometric readings of the spectrometric analysis of the plate at respective time points.
Concentration Vs percentage Cytotoxicity:
Figure2: Percentage cytotoxicity value corresponding to the drug concentration and different exposure time points (60', 120', 240'minutes) were plotted
The cytotoxicity of the H460 tumor cells were determined using LDH release assay. It showed that the cytotoxicity is proportional to the concentration of drug solvent and the time of exposure. The spectrometric readings obtained at the end of each time point using plate reader has been tabulated as shown in the table 1. The results obtained during the LDH assay were tabulated and mean values were calculated. The calculations were shown in the supplement documents. Finally, the endpoint has been depicted in graph showing the percentage toxicity against the drug concentration. From the figure2 it is clear that there is no considerable cytotoxicity at the low concentration of the drug solvent at the start i.e at the first hour of the assay. Although there is some cytotoxicity observed which is not significant when compared to the maximum time point of exposure. At the fourth hour, the exposure of the H460 cells to the drug solvent concentration of 51µM produced a considerable toxicity. The individual plot of the percentage cytotoxicity at various time points such as 60,120 and 240 were provided as separate graphs in the supplement documents.
The human toxicity and the general screening of the drug substances have been detected by in vitro cytotoxicity assays. The ultimate result of these cytotoxicty assays were accounted to be different based on the difference in the drug substance screened and methods employed. The results obtained from the LDH release cytotoxicity assay indicate that there are differences in the percentage cytotoxicity caused by the drug solvent. We can assume from the result that drug solvent causing cell membrane leakage depends on the drug solvent concentration and the time of exposure. Despite of obtaining little or zero cytotoxicity at the concentrations of 5.1µM and 17µM at the first and second hour time point there is little cytotoxicity at the 240'time point for the 17µM concentration.
The obtained result shows that the maximum cytotoxicity is well documented at the high concentration of (51µM) drug and at the maximum exposure time of 4 th hour. Therefore, rupture of the cell membrane can occurs when cells were exposed to higher concentrations for a prolonged period of time.
Although, the LDH release assay is more consistent and fast method to determine cytotoxicity it has its own negative aspect. There are considerable factors which needed to be highlighted that may interfere in the quantification of the cytotoxicity based on the LDH release from the cell membrane. The difference may be due to inhibition of LDH release by other proteases which are potent inhibitor of LDH release. This can also be result of either modulation in the LDH activity by the process other than cytotoxicity or due to insufficient half life activity of the LDH in the culture cells (H. Schafer et al 1997).
Furthermore, the not all putative target cells release sufficient amount of LDH to quantify the cell death. For instance in cell mediated cytotoxic systems the vast extensively present effector cells can release LDH together with other granules. It can interfere in the final reading of the assay due to the high background it produces. (Fotakis and Timbrell, 2006) .
From the results obtained in LDH assay it is slightly evident that this assay is suitable only to determine the toxicity due to membrane damage on the flip side it is not useful to estimate the variation in the cells due to membrane damage. Obviously, it is due to varying mechanism in the cell death. In other words, the cytotoxic agents can cause cell death in different ways.
To conclude, the trypan blue assay is appropriate method for verifying the number of cells with/without membrane damage. However, it is unsuitable to reveal cytotoxicity in cases of cells without membrane integrity losses. The MTT assay in addition to estimation of extent of cellular redox perturbation and mitochondrial dysfunction,it is flexible for assessing relative cell number estimations within a range of unchanged cellular reduction. Unfortunately, it is not suitable for the evaluation of cytotoxicity in cells with unchanged reduction fluctuation by a high level of basal cellular reduction capacity and/or oxidant defenses. In case of ambiguous extent of cytotoxicity caused by new chemicals it is always advisable to use two different methods of assay rather than implying on one.