3. Description of different cytotoxicity tests

The cellular damage caused by different chemical compounds can be various and thus, the methods to measure this effect are numerous. To select the proper test, we must know: number of treated cells, number of treatments, what kind of treatment the cells got, do we need these cells later, or the chosen method can terminate them? Also, do we want to know the kinetics of the post-treatment population's changes or simply what happens with the cells after a short incubation with the selected chemical? The price, the reliability, the user-friendliness of the kit is an important question too. The mechanism of cytotoxicity can be various so a single method only gives a simple view on a chosen material. Multiple tests and methods must be used before anyone can make a solid point about the biocompatibility of a chemical compound. Also, it must be noted, that a compound can interfere with the detection mechanism of a certain assay, resulting in a false positive or negative result; thus, multiple methods are needed to avoid such cases.

MTT assay is a cheap, popular way, to measure cell death [21]. The reduction of the tetrazolium structure in the MTT dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide leads to a colored formazan product—this is the basic chemical reaction in every tetrazolium based assay. The MTT dye has a positive charge, thus it is taken up by the cells and intracellular oxidoreductases catalyze the mentioned reaction. The oxidative state of the cell and the mitochondrial respiratory chain are essential in the conversion. Basically, the concentration of NADH limits the process and any chemical compound that modifies the oxidative potential of the cell can possibly decrease or increase the signal of the assay [22]. If this effect is not linked with direct cytotoxic activity, then, it may lead to a false positive or negative result. Various reports have already indicated that certain test compounds have radically different results in tetrazolium assay, than in other methods because of the possible antioxidant capabilities [23]. The reaction starts immediately, but 1–4 h should pass before measuring the absorbance of the test system the exact time must be setup according to the parameters of the current test system. A too short amount of time might result in low signal strength; a too long may mask the difference between different treatments/concentrations. Various organic solvents might be used to dissolve final product, the insoluble, purple formazan crystals, but we suppose an isopropanol:hydrochloride acid (25:1) solution, because it is safe and cheap to use. The acidification of the system is required, to reduce the amount of the original yellow dye, thus, give us a stronger main signal. The

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XTT, MTS, WST-1, and WST-8 assays are the improved versions of the old MTT. The final product of the reaction is soluble in water/cell culture medium; yet, the solubility of the original dye is greatly reduced, they must be used at 1–2 mg/ml concentrations instead of the 0.2–0.5 mg/ml of the original MTT dye [24]. The XTT and the WST-1/8 compounds have a negative charge, so they cannot penetrate the cell membrane; their reduction takes place in the extracellular space [22]. To enhance the effectiveness of transmembrane oxidoreductases, an

absorbance must be measured at 570 nm (Figures 1 and 2).

Figure 1. MTT dye reaction, the tetrazolium ring opens as a result of the reduction.

Figure 2. Simplified mechanisms of MTT and MTS assays.

#### 3.1. Assays

Cell viability assays are usually cheap, easy-to-perform methods, where after a given incubation with the selected chemical compound, the number of the surviving cells is measured by some method. They use no antibodies or radioactive chemicals. Usually, these assays are carried out on 96- or 384-well plates, making them ideal for screening experiments. It must be noted, that in some special cases, multiple measurements can be made, but these methods are not suited for long-term, kinetical or time-dependent killing studies. We must be aware of the fact, that most methods use some kind of "signal molecule" which—in normal cases—is directly proportional with the number of cells. If there is an uncontrolled factor that increases or decreases the strength of the signal, we can get false positive of false negative results. Such factor is usually increased uptake into the cell or increased activity of the specific enzyme, responsible for the creation of the signaling molecule. Usually not a single enzyme, but multiple proteins catalyze the reaction, so the overall metabolic state of the cell must be considered. It can be said that additional filters for background measuring can greatly increase the sensitivity. Nearly all eukaryotic cells can perform these biochemical reactions, but previous research can avoid incompatibility with certain cell lines. In the following table, the most well-known cell viability assays are listed. Prices are approximating, and they mean the price of 1000 tests of the kit, according to the manufacturer (Table 2).


Table 2. Most common cytotoxicity assay methods.

MTT assay is a cheap, popular way, to measure cell death [21]. The reduction of the tetrazolium structure in the MTT dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide leads to a colored formazan product—this is the basic chemical reaction in every tetrazolium based assay. The MTT dye has a positive charge, thus it is taken up by the cells and intracellular oxidoreductases catalyze the mentioned reaction. The oxidative state of the cell and the mitochondrial respiratory chain are essential in the conversion. Basically, the concentration of NADH limits the process and any chemical compound that modifies the oxidative potential of the cell can possibly decrease or increase the signal of the assay [22]. If this effect is not linked with direct cytotoxic activity, then, it may lead to a false positive or negative result. Various reports have already indicated that certain test compounds have radically different results in tetrazolium assay, than in other methods because of the possible antioxidant capabilities [23]. The reaction starts immediately, but 1–4 h should pass before measuring the absorbance of the test system the exact time must be setup according to the parameters of the current test system. A too short amount of time might result in low signal strength; a too long may mask the difference between different treatments/concentrations. Various organic solvents might be used to dissolve final product, the insoluble, purple formazan crystals, but we suppose an isopropanol:hydrochloride acid (25:1) solution, because it is safe and cheap to use. The acidification of the system is required, to reduce the amount of the original yellow dye, thus, give us a stronger main signal. The absorbance must be measured at 570 nm (Figures 1 and 2).

XTT, MTS, WST-1, and WST-8 assays are the improved versions of the old MTT. The final product of the reaction is soluble in water/cell culture medium; yet, the solubility of the original dye is greatly reduced, they must be used at 1–2 mg/ml concentrations instead of the 0.2–0.5 mg/ml of the original MTT dye [24]. The XTT and the WST-1/8 compounds have a negative charge, so they cannot penetrate the cell membrane; their reduction takes place in the extracellular space [22]. To enhance the effectiveness of transmembrane oxidoreductases, an

Figure 1. MTT dye reaction, the tetrazolium ring opens as a result of the reduction.

Figure 2. Simplified mechanisms of MTT and MTS assays.

of treated cells, number of treatments, what kind of treatment the cells got, do we need these cells later, or the chosen method can terminate them? Also, do we want to know the kinetics of the post-treatment population's changes or simply what happens with the cells after a short incubation with the selected chemical? The price, the reliability, the user-friendliness of the kit is an important question too. The mechanism of cytotoxicity can be various so a single method only gives a simple view on a chosen material. Multiple tests and methods must be used before anyone can make a solid point about the biocompatibility of a chemical compound. Also, it must be noted, that a compound can interfere with the detection mechanism of a certain assay, resulting in a false positive or negative result; thus, multiple methods are needed to avoid such

Cell viability assays are usually cheap, easy-to-perform methods, where after a given incubation with the selected chemical compound, the number of the surviving cells is measured by some method. They use no antibodies or radioactive chemicals. Usually, these assays are carried out on 96- or 384-well plates, making them ideal for screening experiments. It must be noted, that in some special cases, multiple measurements can be made, but these methods are not suited for long-term, kinetical or time-dependent killing studies. We must be aware of the fact, that most methods use some kind of "signal molecule" which—in normal cases—is directly proportional with the number of cells. If there is an uncontrolled factor that increases or decreases the strength of the signal, we can get false positive of false negative results. Such factor is usually increased uptake into the cell or increased activity of the specific enzyme, responsible for the creation of the signaling molecule. Usually not a single enzyme, but multiple proteins catalyze the reaction, so the overall metabolic state of the cell must be considered. It can be said that additional filters for background measuring can greatly increase the sensitivity. Nearly all eukaryotic cells can perform these biochemical reactions, but previous research can avoid incompatibility with certain cell lines. In the following table, the most well-known cell viability assays are listed. Prices are approximating, and they mean the price

Name Mechanism How to detect Price (€) XTT Enzymatic activity Spectrophotometer 235 MTT Enzymatic activity Spectrophotometer 129 WST-1 Enzymatic activity Spectrophotometer 235 WST-8 Enzymatic activity Spectrophotometer 420 MTS Enzymatic activity Spectrophotometer 172 LDH Enzymatic activity Spectrophotometer 315 Resazurin Enzymatic activity Spectrofluorometer 110 Neutral Red Lysosomal uptake Spectrophotometer 339

of 1000 tests of the kit, according to the manufacturer (Table 2).

Table 2. Most common cytotoxicity assay methods.

cases.

136 Cytotoxicity

3.1. Assays

intermediate electron acceptor (IEA in figure) is required, which gets reduced by NADH and intensifies the final signal of the assay. The specific IEA (such as phenazine ethyl sulfate) is usually part of the assay kits and its concentration must be determined according to the given test system. The MTS is at least partially reduced in the intracellular compartment, thus an IEA is not essentially needed [22]. Because, the final formazan product is soluble in water, the solubilization step is unnecessary; these assays are more flexible to combine with additional methods and easier to carry out (Figures 3 and 4).

Resazurin assay (or Alamar Blue) is based on the enzymatic conversion of the cell permeable blue resazurin to the pink resorufin [25]. Both chemicals are soluble in water and the assay provides higher sensitivity than the previous methods, because the detection is based on fluorescence, not spectrophotometry. Also, it must be noted that the reagent is toxic, so the appropriate reaction time must be based on the sensitivity of the given cell line so the toxicity of the reagent can be distinguished from the tested chemical. The fluorescence should be measured with 560 nm excitation/590 nm emission filters. Also, it can be combined with other techniques such as caspase activity assay because of the different detection method and the non-interference of the respective mechanisms [26] (Figure 5).

the LDH assay or multiple wash steps are needed before applying the assay reagent on the cells. Another problem is that if the cells are dead (or growth is inhibited), but the specific cytotoxic chemical does not distort the cell membrane, then the actual number of dead cells is underestimated. A solution of it can be the usage of control compounds which have a similar

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Neutral red assay is not based on a directly enzymatic biochemical reaction, but the dye is taken up by the cells and it stains the lysosomes of the cells [29]. A weak cationic compound, the neutral red is taken up by micropinocytosis or by non-ionic diffusion and is accumulated into the lysosomes [30]. After the cytotoxic treatment with the possibly cytotoxic chemical, the cells should be washed, then the staining solution must should be added to the test system. After an appropriate time of incubation, the dye must be removed and the cells are washed again. The incubation with a solubilization solution forces the cells to excrete the neutral red dye and thus, the concentration of it can be measured at 540 nm. As damaged cells can only take up and store neutral red at a decreased rate and dead cells are not stained at all, it is a sensitive assay, but, the cells are washed, disturbed multiple times which—if not carried out smoothly enough—can decrease the cell number, destroy the monolayer or other kind of

The simple cytotoxicity assays are limited in case of kinetical or time-dependent killing experiments. Simply, multiple measurements cannot be executed, because the test system is disturbed, some xenobiotics (tetrazolium dyes, other cofactors, etc.) are added to the cells or in case of some methods, the cells are solubilized. The reagents used in the assays can be directly

way-of-killing, than the tested chemical [28] (Figure 6).

Figure 5. Mechanism of the Resazurin assay.

collateral damage can happen to the cells (Figure 7).

Figure 6. Mechanism of LDH membrane leakage assay.

3.2. RT-CES

LDH assay is based on the activity of a cytoplasmic enzyme, the lactate dehydrogenase which reduces the NAD to NADH. NADH then reduces a tetrazolium dye (or other reagent) whose concentration can be measured spectrophotometrically [27]. The assay is mainly used for the detection of membrane leakage which correlates with the cell damage. The most common reagent is the 2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride (INT) which forms a red formazan product. The LDH is quite stable for the duration of the assay in the extracellular space and its amount is dependent on the cell size and oxidative activity, but similar among the cells of the same cell line. It is excellent when compared with other tetrazolium based methods because it can measure the damaged cells as well, not just the dead ones. Also in special cases, it can be used to detect the intracellular LDH concentration. A limitation of this method is that serum has a high LDH activity on its own, so serum-supplemented mediums are not ideal for

Figure 3. Chemical formulae of the new generation tetrazolium assays from left to right: MTS, XTT, and WST-1/8.

Figure 4. Simplified mechanism of the XTT and WTS-1/8 assay.

Figure 5. Mechanism of the Resazurin assay.

intermediate electron acceptor (IEA in figure) is required, which gets reduced by NADH and intensifies the final signal of the assay. The specific IEA (such as phenazine ethyl sulfate) is usually part of the assay kits and its concentration must be determined according to the given test system. The MTS is at least partially reduced in the intracellular compartment, thus an IEA is not essentially needed [22]. Because, the final formazan product is soluble in water, the solubilization step is unnecessary; these assays are more flexible to combine with additional

Resazurin assay (or Alamar Blue) is based on the enzymatic conversion of the cell permeable blue resazurin to the pink resorufin [25]. Both chemicals are soluble in water and the assay provides higher sensitivity than the previous methods, because the detection is based on fluorescence, not spectrophotometry. Also, it must be noted that the reagent is toxic, so the appropriate reaction time must be based on the sensitivity of the given cell line so the toxicity of the reagent can be distinguished from the tested chemical. The fluorescence should be measured with 560 nm excitation/590 nm emission filters. Also, it can be combined with other techniques such as caspase activity assay because of the different detection method and the

LDH assay is based on the activity of a cytoplasmic enzyme, the lactate dehydrogenase which reduces the NAD to NADH. NADH then reduces a tetrazolium dye (or other reagent) whose concentration can be measured spectrophotometrically [27]. The assay is mainly used for the detection of membrane leakage which correlates with the cell damage. The most common reagent is the 2-p-iodophenyl-3-p-nitrophenyl tetrazolium chloride (INT) which forms a red formazan product. The LDH is quite stable for the duration of the assay in the extracellular space and its amount is dependent on the cell size and oxidative activity, but similar among the cells of the same cell line. It is excellent when compared with other tetrazolium based methods because it can measure the damaged cells as well, not just the dead ones. Also in special cases, it can be used to detect the intracellular LDH concentration. A limitation of this method is that serum has a high LDH activity on its own, so serum-supplemented mediums are not ideal for

Figure 3. Chemical formulae of the new generation tetrazolium assays from left to right: MTS, XTT, and WST-1/8.

methods and easier to carry out (Figures 3 and 4).

138 Cytotoxicity

non-interference of the respective mechanisms [26] (Figure 5).

Figure 4. Simplified mechanism of the XTT and WTS-1/8 assay.

the LDH assay or multiple wash steps are needed before applying the assay reagent on the cells. Another problem is that if the cells are dead (or growth is inhibited), but the specific cytotoxic chemical does not distort the cell membrane, then the actual number of dead cells is underestimated. A solution of it can be the usage of control compounds which have a similar way-of-killing, than the tested chemical [28] (Figure 6).

Neutral red assay is not based on a directly enzymatic biochemical reaction, but the dye is taken up by the cells and it stains the lysosomes of the cells [29]. A weak cationic compound, the neutral red is taken up by micropinocytosis or by non-ionic diffusion and is accumulated into the lysosomes [30]. After the cytotoxic treatment with the possibly cytotoxic chemical, the cells should be washed, then the staining solution must should be added to the test system. After an appropriate time of incubation, the dye must be removed and the cells are washed again. The incubation with a solubilization solution forces the cells to excrete the neutral red dye and thus, the concentration of it can be measured at 540 nm. As damaged cells can only take up and store neutral red at a decreased rate and dead cells are not stained at all, it is a sensitive assay, but, the cells are washed, disturbed multiple times which—if not carried out smoothly enough—can decrease the cell number, destroy the monolayer or other kind of collateral damage can happen to the cells (Figure 7).

#### 3.2. RT-CES

The simple cytotoxicity assays are limited in case of kinetical or time-dependent killing experiments. Simply, multiple measurements cannot be executed, because the test system is disturbed, some xenobiotics (tetrazolium dyes, other cofactors, etc.) are added to the cells or in case of some methods, the cells are solubilized. The reagents used in the assays can be directly

Figure 6. Mechanism of LDH membrane leakage assay.

cytotoxic or at least they modify the cells biochemical equilibrium and activity in a way that the results are no longer relevant for the latter in vivo experiments. This means that after a certain treatment with the probably cytotoxic compound, we can only make an end-point measurement because the test system is irreversibly changed after the addition of the given signaling molecule(s). Also, if the tested chemical can absorb light or is capable of fluorescence, it can interfere with the detection system. Because of these limitations the need for a noninvasive, yet precise method resulted in the invention of real-time cell electronic sensor.

This technique is based on the impedance changes of the cell populations [31]. The cells are seeded into special E-plates, which are available in multiple size (4-, 8-, 16-, 96-wells, etc.). These special devices have a positive and a negative electrode in every well, and a low voltage alternate current flows through the well. As the cells grow, they have a higher impedance (resistance in AC circuits); and as they die, the impedance value lowers. This effect has literally no impact on the cells, so it is a non-invasive technique. The length of the experiment is theoretically unlimited, as there is no end-point of AC current flow. For this reason, the cell growth can be measured during multiple treatments of the cells, and not just the cytotoxic or non-toxic effects, but the possible recovery of the cells can be studied as well. It is important, that first, a part of the cell medium and the solution of the screened chemical must be placed into the wells of the E-plate, thus the connected software can detect it as a background, with zero impedance, so chemicals with ionic charges do not interfere with the measured signal. The cells should be added to the wells after the background detection in a high-density suspension. Also, the whole experiment can be stopped at any point, to remove the test solution or to add a new compound to test system (Figure 8).

requires an experienced crew to execute perfectly. Also, the total protein only works, if the cells grew in the presence of the cytotoxic chemical, otherwise, the dead cells cannot be distinguished from the viable ones. However, there are several studies indicating that the sulforhodamin B results correlate well with the MTT results [34]. A slight advantage of this method is that because of the multiple wash steps, the tested chemicals can hardly interact with the dye, unlike other enzyme-based methods. The optimization with the specific cell line

Figure 8. A typical RT-CES diagram showing a time and dose dependent cytotoxicity of 2,6-dichloro-(1,4)-benzoquinone

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Calcein-AM/Hoechst 33342 and propidium iodide are dyes that stain viable and dead cells [35]. In appropriate concentration, Calcein-AM, a lipophilic derivative of calcein is capable pass through cell membranes and stains the cell, as intracellular enzymes cleave the lipophilic carbon chain from the dye [36]. Hoechst 33342 binds the A-T rich regions of the DNA. Propidium iodide stains the nucleus of the cell, but cannot penetrate the cell membrane, thus it only binds to the dead cells. As the two reagents can be detected at different wavelengths, multiple emission and excitation filters are needed. Also, every cell line has a different binding rate and the ideal concentration must be found through testing, as the cytosol of the cells can be stained by each dye and instead of spectrophotometric detection, manual counting is

is also much easier because the lack of dependence on metabolic activity (Figure 9).

needed with a microscope with the specific filters/lamps (Figure 10).

[32].

Figure 9. Sulforhodamin B.

However, this system is ideal for cell viability studies, it has some disadvantages as well. The devices and the E-plates have a high price and a limited number of slots to use. The E-plates can be used multiple times after a specific cleaning protocol, but the sensitive, microelectronic sensing arrays are easily damaged by washing and organic solvents. This means, that it is not suited for high performance screening experiments, because multiple assays can be done during the same amount of time.

#### 3.3. Other methods

Sulforhodamin B is a dye which stains the total protein amount of the cells [33]. The reagent is an aminoxanthene dye which binds stoichiometrically with the amino-acids under acidic pH. First, the cells must be fixed with trichloroacetic acid, then washed and dried and the wells respective optical density measured for background detection. The sulforhodamin B must be added after this, and it should stain the cells for 20–30 min. After a wash step, the stained cells must be solubilized and the absorbance measured at 565 nm. The protocol is quite long and

Figure 7. Neutral red dye.

Figure 8. A typical RT-CES diagram showing a time and dose dependent cytotoxicity of 2,6-dichloro-(1,4)-benzoquinone [32].

requires an experienced crew to execute perfectly. Also, the total protein only works, if the cells grew in the presence of the cytotoxic chemical, otherwise, the dead cells cannot be distinguished from the viable ones. However, there are several studies indicating that the sulforhodamin B results correlate well with the MTT results [34]. A slight advantage of this method is that because of the multiple wash steps, the tested chemicals can hardly interact with the dye, unlike other enzyme-based methods. The optimization with the specific cell line is also much easier because the lack of dependence on metabolic activity (Figure 9).

Calcein-AM/Hoechst 33342 and propidium iodide are dyes that stain viable and dead cells [35]. In appropriate concentration, Calcein-AM, a lipophilic derivative of calcein is capable pass through cell membranes and stains the cell, as intracellular enzymes cleave the lipophilic carbon chain from the dye [36]. Hoechst 33342 binds the A-T rich regions of the DNA. Propidium iodide stains the nucleus of the cell, but cannot penetrate the cell membrane, thus it only binds to the dead cells. As the two reagents can be detected at different wavelengths, multiple emission and excitation filters are needed. Also, every cell line has a different binding rate and the ideal concentration must be found through testing, as the cytosol of the cells can be stained by each dye and instead of spectrophotometric detection, manual counting is needed with a microscope with the specific filters/lamps (Figure 10).

Figure 9. Sulforhodamin B.

cytotoxic or at least they modify the cells biochemical equilibrium and activity in a way that the results are no longer relevant for the latter in vivo experiments. This means that after a certain treatment with the probably cytotoxic compound, we can only make an end-point measurement because the test system is irreversibly changed after the addition of the given signaling molecule(s). Also, if the tested chemical can absorb light or is capable of fluorescence, it can interfere with the detection system. Because of these limitations the need for a noninvasive, yet precise method resulted in the invention of real-time cell electronic sensor.

This technique is based on the impedance changes of the cell populations [31]. The cells are seeded into special E-plates, which are available in multiple size (4-, 8-, 16-, 96-wells, etc.). These special devices have a positive and a negative electrode in every well, and a low voltage alternate current flows through the well. As the cells grow, they have a higher impedance (resistance in AC circuits); and as they die, the impedance value lowers. This effect has literally no impact on the cells, so it is a non-invasive technique. The length of the experiment is theoretically unlimited, as there is no end-point of AC current flow. For this reason, the cell growth can be measured during multiple treatments of the cells, and not just the cytotoxic or non-toxic effects, but the possible recovery of the cells can be studied as well. It is important, that first, a part of the cell medium and the solution of the screened chemical must be placed into the wells of the E-plate, thus the connected software can detect it as a background, with zero impedance, so chemicals with ionic charges do not interfere with the measured signal. The cells should be added to the wells after the background detection in a high-density suspension. Also, the whole experiment can be stopped at any point, to remove the test

However, this system is ideal for cell viability studies, it has some disadvantages as well. The devices and the E-plates have a high price and a limited number of slots to use. The E-plates can be used multiple times after a specific cleaning protocol, but the sensitive, microelectronic sensing arrays are easily damaged by washing and organic solvents. This means, that it is not suited for high performance screening experiments, because multiple assays can be done

Sulforhodamin B is a dye which stains the total protein amount of the cells [33]. The reagent is an aminoxanthene dye which binds stoichiometrically with the amino-acids under acidic pH. First, the cells must be fixed with trichloroacetic acid, then washed and dried and the wells respective optical density measured for background detection. The sulforhodamin B must be added after this, and it should stain the cells for 20–30 min. After a wash step, the stained cells must be solubilized and the absorbance measured at 565 nm. The protocol is quite long and

solution or to add a new compound to test system (Figure 8).

during the same amount of time.

3.3. Other methods

140 Cytotoxicity

Figure 7. Neutral red dye.

viability or cytotoxicity assays can be performed. Cytotoxicity assays based on membrane integrity changes are positive-readout assay which are most typically indicated for shorterterm exposure models (48 h or less). These assays may not properly determine the absolute degree of early or late stage cytotoxicity since the kinetics of biomarker emergence or degradation. Viability assays measure the level of biomarker activity inversely correlated with cytotoxicity, and therefore may be used at any endpoint during a compound/cell incubation period. Each biomarker of viability and cytotoxicity has advantages and disadvantages. Moreover in early drug discovery, in vitro evaluations of new drug candidates is often met with skepticism since their reliability and in vivo correlatability. It can be concluded that however there is some validity to this argument, it is important to put in vitro toxicity data into

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Ildikó Bácskay\*, Dániel Nemes, Ferenc Fenyvesi, Judit Váradi, Gábor Vasvári, Pálma Fehér,

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Debrecen,

[1] Horvath S. Cytotoxicity of drugs and diverse chemical agents to cell cultures. Toxicology.

[2] Diaz D, O'Brien PJ. Defining the sequence of events in cerivastatin toxicity using a high content multi-parametric cytotoxicity assay. European Pharmaceutical Review. 2006;11:38-45

[3] Bolt M, Card J, Racz W, et al. Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity. The Journal of Pharmacology and Experimental Therapeutics. 2001;298:1280-1289

[4] Buxser S, Vroegop S. Calculating the probability of detection for inhibitors in enzymatic or binding reactions in high throughput screening. Analytical Biochemistry. 2005;340:1-

[5] Ujhelyi J, Ujhelyi Z, Szalai A, Laszlo JF, Cayasso M, Vecsernyes M, Porszasz R. Analgesic and anti-inflammatory effectiveness of sitagliptin and vildagliptin in mice. Regulatory

[6] Xia M, Huang R, Witt K, et al. Compound cytotoxicity profiling using quantitative highthroughput screening. Environmental Health Perspectives. 2008;116:284-291. DOI: 10.1289/

Peptides. 2014;194-195:23-29. DOI: 10.1016/j.regpep.2014.09.006

consideration during pharmaceutical development.

\*Address all correspondence to: bacskay.ildiko@pharm.unideb.hu

Miklós Vecsernyés and Zoltán Ujhelyi

13. DOI: 10.1016/j.ab.2005.01.034

Author details

Debrecen, Hungary

1980;16:59-66

ehp.10727

References

Figure 10. Chemical formula of the Calcein-AM (upper image), Hoechst 33342 (left image) and the propidium iodide (right image).

#### 3.4. Comparison between in vitro cytotoxicity data and in vivo data

As the whole, medical science and industry is based on the modification, repair of damaged or badly functioning cells and tissues in human or animal body, the correlation between in vitro and in vivo data is crucial. Do these artificial test systems, cell lines truly replicate how a real tissue would react to a certain treatment or compound? The answer is based on the application of multiple in vitro methods and the careful planning of the in vivo experiments. A good example of the practice is the study of Yu et al. [37]. Xantii fructus is a traditional Chinese herbal drug and clinical reports indicated its renal toxicity. The study was based on MTT and LDH assays of the main components of the herbal drug on a renal cell line, as well as acute and chronic toxicity experiments in rats. While the main component of the drug, the atractyloside potassium salt showed no cytotoxicity on the cell lines, the water extract of the fruit had an inhibitory effect in case of high concentrations on the MTT assay, but no membrane damage on the LDH assay. These results indicate that the secondary components of the water extract have cytotoxic capabilities and the exact mechanism of killing might involve the suppressed metabolic activity of the cells, but not the damage of the cell membrane. The acute in vivo toxicity showed that only high concentrations could terminate the rats and cause abnormalities in the organs and the chronic toxicity showed only minor changes in the highest concentration group. Overall, this complex study created a much more accurate, scientific point of view about the toxicity of Xanthii fructus, what chemicals are responsible for its toxicity, what are the exact dosages, and what are the side effects that are caused by the herbal drug. It could only be made by the co-application of various in vivo and in vitro methods.

#### 4. Conclusion

The importance of cytotoxicity assays in early drug development is unquestionable although it must be concluded that no assay technology for detecting cytotoxicity in vitro is perfect. Strong arguments can be made for and against using cell viability or cytotoxicity assays as a reliable model of human medication. Depending upon the objectives of the investigation, either viability or cytotoxicity assays can be performed. Cytotoxicity assays based on membrane integrity changes are positive-readout assay which are most typically indicated for shorterterm exposure models (48 h or less). These assays may not properly determine the absolute degree of early or late stage cytotoxicity since the kinetics of biomarker emergence or degradation. Viability assays measure the level of biomarker activity inversely correlated with cytotoxicity, and therefore may be used at any endpoint during a compound/cell incubation period. Each biomarker of viability and cytotoxicity has advantages and disadvantages. Moreover in early drug discovery, in vitro evaluations of new drug candidates is often met with skepticism since their reliability and in vivo correlatability. It can be concluded that however there is some validity to this argument, it is important to put in vitro toxicity data into consideration during pharmaceutical development.
