Measurement of Drug-Induced Cell Damage Using Lactate Dehydrogenase - Coursework Example

Measurement of Drug-Induced Cell Damage Using Lactate Dehydrogenase as a Biomarker The bioactivity of LDH as a measure of cytotoxi of H40 tumor cells in culture medium was done by incubation with doxorubicin, an anticancer drug. Quantification of the LDH release which is linearly proportional to the amount of color produced due to conversion of lactate to pyruvate in presence of tetrazolium dye was measured spectrophotometrically. The results showed dose dependent cytotoxicity for cells in complete medium and assay medium used for comparison. Although the complete medium had more serum, and consequently higher LDH than those in AM, the AM showed higher cytotoxicity of up to 39.4%. Together these results suggest that LDH can be used to quantitatively measure doxorubicin-induced damage H40 cells. Introduction Development and validation of biomarkers as tools for diagnostic and prognostic use has been on the increase in the recent past. Diamantino et al (2000) observes that these tools are important because they can detect the toxic effects of the drugs before they can alter the physiological functions of the hosts. Some of the biomarkers developed include enzymes such as lactate dehydrogenase (LDH), an important glycolytic enzyme found in all tissues with high concentrations in the heart and liver. It is made of two subunits M (muscle) and H (heart) which makes five different isoenymes and is mainly found in the heart and erythrocytes. This enzyme has been widely used in clinical chemistry and toxicology to diagnose cell, tissue and organ damage (Kaplan and Pesce, 1996). LDH is released into the culture medium following the loss of cell membrane integrity and the amount of LDH released into the assay medium is proportional to the amount of cellular damage. The aim of this study was to determine the validity of the use of LDH as a measure of tissue damage induced by Doxorubicin, an anticancer drug. Materials and Methods An already prepared 96-well flat bottomed microtitre plate containing 2.0x104 cells/ml in 200µl of normal complete medium incubated at 37°C 5% CO2 overnight and washed in 200µl of HBSS was provided. The HBSS was replaced with 200µl of complete medium (CM) or assay medium (AM) which is complete medium containing 1% fetal bovine serum (FBS), or 100µl of either AM or CM as shown in the plate matrix in Table 1. Serial drug dilutions from the 1mM doxorubicin stock solution in 1.5ml microfuge tubes were made by diluting the drug in either 1% FCS containing AM or CM to give a final volume of 1.2ml and 100µl of the drug solvent added to the wells as shown in Table 1 and incubated at 37°C for 3 days. The background or spontaneous NADH oxidation was measured by substituting 100µl aliquots of distilled water or triton X-100 lysis solution instead of media or lysate samples into the reaction mixture. The lysed samples were used as positive control samples and those with distilled water as negative controls. The microplate was removed from the incubator and 100µl of Triton X-100 solution added to each well in the high control medium and the microplate further incubated at 37°C for 30 minutes. The microplate was removed from the incubator and 100µl of the medium removed carefully without disturbing the pellet and transferred to the corresponding wells in the reaction microplate. Immediately before use, 110µl of the assay catalyst solution was added to the assay dye tube and mixed by rotating the tube gently to prevent introduction of air bubbles. The microplate was then incubated in the dark for 30 minutes at room temperature after which it was read on the microplate reader at 450nm and the plates disposed. The mean values for each experimental point was calculated and the mean background control subtracted. The cytotoxicity value calculated according to the formula below and the curves of the concentration verses percentage toxicity for each time points plotted. × 100 Table 1: Cell plate matrix 1% FCS AM 10% FCS CM 1 2 3 4 5 6 7 8-12 A X Background control - 200µl AM or CM (no cells) X B X Low control – 200µl AM or CM (cells) X C X High control – 100µl AM or CM + 100µl 1% Triton X-100 in medium X D X 300µM doxorubicin – 100µl AM or CM + 100µl drug x2 X E X 100µM doxorubicin – 100µl AM or CM + 100µl drug x2 X F X 30µM doxorubicin – 100µl AM or CM + 100µl drug x2 X G X 10µM doxorubicin – 100µl AM or CM + 100µl drug x2 X H X 3µM doxorubicin – 100µl AM or CM + 100µl drug x2 X Results The absorbance recorded during the study as shown in Table 2 below and the percentage toxicity in Table 3. The results show that the toxicity of Doxorubicin was dose dependent with the highest toxicity (39.4%) recorded at a concentration of 100µM in the assay medium. The toxicity in the complete medium was also dose dependent but lower than those recorded in cells incubated in the assay medium as shown in Figure 1. However, there was an unexpected decline in toxicity in cells incubated with assay medium at 300µl. Table 2: Absorbance of test and controls at 450nm AM AM AM CM CM CM Background Control 0.151 0.146 0.146 0.418 0.413 0.421 Low Control 0.417 0.447 0.399 0.707 0.709 0.703 High Control 1.130 1.721 1.564 1.803 1.585 2.090 300uM Doxorubicin 1.043 0.985 1.017 1.601 1.585 1.615 100uM Doxorubicin 0.901 0.883 0.904 1.167 1.173 1.142 30uM Doxorubicin 0.691 0.671 0.672 0.807 0.863 0.832 10uM Doxorubicin 0.495 0.385 0.553 0.756 0.708 0.777 3uM Doxorubicin 0.585 0.586 0.590 0.799 0.665 0.706 Table 3: Concentration of Doxorubicin and the corresponding percentage toxicity in Assay and Complete medium Concentration of Doxorubicin Toxicity (%) Assay Medium Complete Medium 3µM 28.06 11.9 10µM 33.97 15.44 30µM 34.16 14.62 100µM 39.38 22.96 300µM 33.28 23.74 Figure 1: Comparison of toxicity (%) of cells with increasing concentrations of Doxorubicin in 1% FCS assay medium or 10% FCS complete medium Dicussion Doxorubicin is a widely used anticancer drug whose toxicity to target cells is mediated by induction of free radicals (Gewirtz, 1999) and exacerbated by iron from hemoglobin breakdown. The results showed that its toxicity was dose dependent. This is in agreement with other studies which have shown that Doxorubicin accumulates in the cells and inhibits cell proliferation for more than 48 hours before demonstrating cytotoxicity (Wolf and Baynes 2005). This explains why the cells were incubated for 3 days after introducing the drug into the cultured cells. Serial dilutions of the drug were done to eliminate possible solubility concerns which would interfere with the observations by giving a false positive or negative test results. The LDH released from the damaged cells reacted with the solution containing lactate, NAD+, diaphorase and a dye in the assay medium. Conversion of lactate to pyruvate leads to production of NADH/H+ and diaphorase transfers H/H+ from NADH/H+ to the dye (tetrazolium salt INT) which is reduced to a fluorescent product (formazan) absorbing at the range of 490-520nm (Wolterbeeek and Van deer Meer, 2005). The intensity of color formed depends on the number of lysed cells. The sensitivity of this method is however limited since the phenol red indicator present in the culture medium absorbs at 490nm The other limitation of using LDH assay is that the assay medium contains serum which has considerable LDH activity (Sepp et al 1996) thus limiting its sensitivity. Serum concentrations of 10% are necessary for growth and viability of cells in culture medium. Attempts to reduce background LDH activity by minimizing the amount of serum in the media by using 1% FBS, mainly used for maintenance of culture cells without affecting cell survival or promoting growth, resulted in high toxicity of the cells. This result was in agreement with observations by other authors for culture cells (Batchelor and Zhou, 2004). Other enzyme based cytotoxicity assays with greater sensitivity have been developed which use enzymes of the glycolytic pathways such as adenylate kinase (AK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes necessary for ATP generation. These assays use luminesce and thus produce high signal-to-noise ratio in short time periods from relatively few numbers of dead cells. However, AK and GAPDH have relatively poor activity half-lives outside the cellular compartment thus limiting their usage (Niles et al 2008). Trypan Blue Exclusion Assay (TBA) is another technique used to determine cell cytotoxicity. this method treats cells with trypsin which is subsequently stained with trypan blue which is a diazo dye taken up by dead cells only which can be expressed as a percentage of the total cells. Microculture Tetrazolium Assay (MTA) is a metabolic assay which provides indirect information on the number of viable cell population in a culture relative to the controls using tetrazolium salts which is reduced into insoluble purple formazan and quantified spectrophotometically. The limitation of this technique is that certain human cell lines are inefficient at processing tetrazolium salt reagents lengthens the assay due to their insolubility and also risk of exposure to harzadous levels of the salt (Scudiero et al 1988). Techniques such as Clonogenic Assay or Colony Forming Efficiency (CFE) methods have also been utilized in determination of cytotoxicity. This method allows for the assessment of decreased or increased survival and proliferation over extended periods of time of several weeks (Hillegass et al 2010). Conclusion Several cytotoxicity assays have been developed for in vitro testing and the method used depends on the sensitivity required, practical use for many samples, time and also the cost of the assay. None of the assays used for cytotoxicity currently are perfect. Cytotoxicity using LDH remains one of the most utilized methods as it gives good results in presence of appropriate controls. Therefore from the results we suggest that LDH is a reliable marker for measurement of Doxorubicin induced cytotoxicity. Reference Batchelor RH and Zhou M, (2004) Use of cellular glucose-6-phosphate dehydrogenase for cell quantitation: applications in cytotoxicity and apoptosis assays. Analytical Biochemistry 329: 35-42 Diamantino TC, Almeida E, Soares A and Gulhermino L (2001). Lactata dehydrogenase activity as an effect criterion in toxicity tests with Daphnia magna straus. Chemosphere 45: 553-560 Gewirtz, DA(1999). 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Improved protocol for calorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity. Journal of Immunological Methods 196: 175- 180 Wolf MB and Baynes JW (2006) The anti-cancer drug, doxorubicin, causes oxidant stress-induced endothelial dysfunction. Biochimica et Biophysica Acta 1760: 267 – 271 Wolterbeek HT and van deer Meer JGM (2005) Optimization, application, and interpretation of lactate dehydrogenase measurements in microwell determination of cell number and toxicity. Assay and Drug Development Technology 3(6) 675-682 Read More