Journal
of Hospital
Assay
Infection
of antiseptic
(1991)
agents in cell culture: affecting cytotoxicity
F. M. Tatnall, Department
17, 287-296
of Dermatology, Accepted
I. M. Leigh The Royal
for publication
conditions
and J. R. Gibson London
Hospital,
12 December
London
El
1BB
1990
Summary:
In-vivo studies suggest that chlorine-releasing antiseptic agents inhibit wound healing. Studies which have used cell culture systems to evaluate cytotoxicity have generated conflicting results for the toxicity of free-chlorine agents relative to other antiseptics. Here we examine the following three factors which may influence the toxicity of individual agents within a cell culture assay: (1) cell number; (2) duration of exposure; and (3) the nature of the antiseptic diluent. Three agents (sodium hypochlorite, chlorhexidine and hydrogen peroxide) were tested on transformed human keratinocytes (SVK 14 cells). It was found that increasing cell number, and using serum or medium as a diluent, reduced the toxicity of all agents but had the greatest effect on sodium hypochlorite. In contrast, increasing the duration of exposure increased the toxicity of all agents but had the greatest effect on hydrogen peroxide. These observations may explain the high toxicity of hydrogen peroxide and relatively low toxicity of sodium hypochlorite which have been observed in vitro and are the reverse of in-vivo findings. Culture systems in which high cell numbers are coupled with an agent diluted in serum or medium, and a long exposure time, seem likely to decrease the toxicity of chlorine-releasing agents relative to hydrogen peroxide. Keywords:
Antiseptics;
cytotoxicity;
cell
culture
Introduction
Increasing interest is being shown in the use of cell culture systems to evaluate the toxicity of antiseptics used on wounds. Whilst a number of studies have been published which show that all agents tested are toxic to the various cell types used in the assays, scant regard has been paid to the interpretation of these results and their relevance to in-vivo toxicity. Whilst methods of assessing the antibacterial activity of these agents have been standardized’, choice of experimental conditions for evaluating the cytotoxicity of antiseptics in cell culture have varied greatly. Most
Correspondence London El
to:
Dr
F. M.
Tat&l,
Department
of Dermatology,
The
Royal
London
Hospital,
1BB.
0,9556701/91/040287+
10 SO3.00/0
;rs 1991 The Hospml
287
Infectmn
Soarty
288
F. M. Tatnall et al.
studies have used fibroblasts, although recently cultured oral and cutaneous keratinocytes have also been used. 2,3Exposed cell numbers have varied from 5 x lo3 4 to 2 x 10’ ‘, length of exposure has varied from .5 min6 to 24 h4 and 4 days.3 The agents have been diluted in phosphate-buffered saline (PBS),’ saline,’ medium3v8 or medium plus foetal calf serum.* Most studies have examined cell killing and a small number have looked at the effect of antiseptic agents on cell growth.*v3 Techniques used have not allowed for an accurate method of comparison of agents, and relative toxicities are judged by the number of ten-fold dilutions necessary to eradicate toxicity. Using this method of analysis it has been demonstrated that agents containing free chlorine are less toxic than some commonly used antiseptics’,’ or more toxic than other agents.4 Previously we have used a cell culture system to compare the cytotoxic effect of three antiseptic agents (sodium hypochlorite, chlorhexidine and hydrogen peroxide) on transformed human keratinocytes, normal human keratinocytes and fibroblasts. 7 This study showed that the three cell types showed similar susceptibilities to the agents tested. These findings suggest that the transformed cell line, which has the advantage of ready availability and immortality, can replace fibroblasts and keratinocytes in studies the adverse effects of antiseptics in vitro. designed to investigate Comparison of the toxicity of individual antiseptics showed that sodium hypochlorite was the most toxic agent and chlorhexidine the least. These findings are in agreement with recent in-vivo findings.5,9,‘0 Since published studies present conflicting findings for the relative toxicities of agents tested, we have examined a series of conditions in a cell culture system which may alter the toxic effect of a range of antiseptic agents. Here we confine the antiseptic cytotoxicity assay to the SVK 14 cell type. Materials All chemicals
were
supplied
and methods
by the Sigma
Chemical
Company.
Cell culture Transformed human keratinocytes (SVK 14 cells)” were grown in RPM1 1640 medium supplemented with 10% foetal calf serum (FCS) in an atmosphere of 8% CO, at 37°C. Confluent flasks were trypsinized and seeded into multiwell plates. Cytotoxicity assay The following procedure was used for all standard experiments with subsequent modifications as noted below: 1 x 10’ SVK 14 cells were seeded into 24-well plates (Becton Dickinson UK Ltd.) and cultured in RPM1 1640 plus 10% FCS for 24 h. Three antiseptics were tested: hydrogen peroxide, sodium hypochlorite and chlorhexidine. The agents were prepared by serially diluting them (half or third dilutions) in PBS from the
Toxicity
of antiseptic
agents
in cell culture
289
concentrations in clinical use which are as follows: hydrogen peroxide, 0.88 M; sodium hypochlorite, 0.14 M; chlorhexidine, 0.00056 M. Each concentration was tested on four replicate wells. The cells were washed twice prior to the addition of 1 ml of the agent for 15 min. Control wells were exposed to PBS alone. The drug was then removed, the cells washed and RPM1 1640 plus 10% FCS replaced for a further 24 h. Percentage of viable cells for each test concentration was established by comparing values to the control concentrations. The standard conditions outlined above were compared to the following test conditions: (1) diluting the test agent in PBS containing 10% FCS; (2) diluting the test agent in RPM1 1640; (3) increasing the number of exposed cells to 5 x 10’; (4) extending the exposure time to 60 min. For the experiments which used a variation in cell number, 6-well plates were used for the control and test experiments. For each variable standard and test, experiments were run in parallel and each set of experiments was repeated three times. The number of viable cells remaining in each well was evaluated using a calorimetric assay based on the tetrazolium salt 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT).12 This compound is a yellow tetrazolium salt which is reduced by mitochondrial enzymes in viable cells to an insoluble blue formazan product. Previously we have shown that there is good correlation between this calorimetric assay and a counting method when evaluating numbers of viable cells remaining in an antiseptic cytotoxicity assay. l3 The medium was removed from each well and 1 ml of RPM1 1640 plus 200 ~1 MTT (5 mg ml-‘) was added to each well. The cells were incubated at 37°C in 8% CO, for 4 h. The unreacted dye was removed and 1 ml of isopropyl alcohol was added to each well to solubilize the intracellular blue formazan product. The optical density of the extract in isopropyl alcohol in each well was read in a spectrophotometer at 570 nm and expressed as a percentage of the values for the control wells, to generate a percentage viability for each concentration under test. The percentage viability, based on the MTT reading for each concentration was used to establish log-dose response curves. For each experiment the ED50 value was defined as the concentration allowing 50% survival of cells when compared to control wells and was determined by regression analysis. The significance of the difference between ED50 values for test conditions and standard conditions was determined using the Student paired t-test. Results
Mean ED50 values for experiments using test and standard conditions are summarized in Table I, and the ratio of the test to standard values is shown in Table II. An increase in ED50 concentration for a given test condition when compared to the standard value reflects decreased sensitivity of the
10% Foetal calf serum Medium Cell number Time
Variable
1.44f0.250 x lo-* l~lof0~04x 10-2 1.30f 0.08 x lo-* 0~18f0.014x 10-l
Test
9.3 f0.8 9.5 f0.9 7.7f0.8 8.450.5
x x x x
10-S 10-j 10-l 10-l
peroxide
Standard
Hydrogen
0.0030* 0.0340* 0~0005’ 0~0015*
P-value
1.6fO.l 2.8f0.13 4.6*0.8x 7.9fO.l
Test
x 10-j X 1O-3 lo-+ x 10-s
Standard
hypochlorite P-value
9.ozko.47 x 10 5 0~00007* 8.2f0.6 x 10ms 0.0007* 1.1 f0.1 x 10-4 0.018* 9.1 f0.5 x 10-1 0.041*
Sodium
7.5+1.4x 3.7zkO.l 3.7jzO.l 2.1 f0.2
Test
10 i x10-s x 10-S x 10-5
4.1 f0.12 x 10 5 3.6f0.4 x 10ms 2.8f0.06 x 10m5 3.9 f 0.07 x 10-S
Standard
Chlorhexidine
0.015* 0.5 0.25 0.002*
P-value
Table I. Mean ED50 concentrations f 1 SD (N = 3-4) for th ree agents comparing valuesfor standard experiments to those of test conditions. *Indicates significantly dsperent results at the 5% level (PC 0.05paired student t-test). Standard conditions were asfollows: 1 x 10s SVK cells were exposedfor 15 min to a test agent which was diluted in PBS, Standard conditions were compared to the following test conditions: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium (RPMI 1640); (3) increasing cell number to 5 X 10s; (4) increasing exposure time to 1 hour
Toxicity Table II. Ratio were asfollows: PBS. Standard PBS containing
of antiseptic
of mean ED50 values for 1 x 105 SVK 14 cells were conditions were compared 10% FCS; (2) diluting number to 5 x 105; (4)
agents
291
in cell culture
test and standard experiments. Standard conditions exposedfor 15 min to a test agent which was diluted in to the following test conditions: (1) diluting agent in agent in medium (RPMI 1640); (3) increasing cell increasing exposure time to 1 hour
Agent Variable
Hydrogen peroxide
Test/standard 10% Foetal Medium Cell number Time
Sodium hypochlorite
Test/standard calf
serum
Test/standard
I.55 I.15 1.72 0.21
Chlorhexidine Test/standard
17.8 33.5 4.2 0.89
I.8 I.0 I.0 0.54
cells, or increased survival, and therefore, decreased toxicity of the test agent. Representative dose-response curves are shown in Figures 1-3, comparing test and standard conditions for each agent. Eflect of foetal calf serum Comparison of mean ED.50 values showed that diluting
Log concentration
(M) sodium
hydrogen
peroxide
hypochorite
Figure 1. Dose response curves for the effect of sodium hypochlorite on SVK 14 cells showing one representative experiment. Points represent the means of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium; (3) increasing cell number from 1 x IO5 to 5 X 105; (4) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x IO5 cells were exposed for 15 min to the test agent which was diluted in PBS. -O10% FCS (test); -+FCS (standard); -Omedium (test); -0 medium (standard); -mcell no. (test); -0cell no. (standard) and time (standard); -A - time (test).
292
F. M. Tatnall
-4
-3 Log concentration
et al.
-2 (M) hydrogen
-I
0
peroxide
Figure 2. Dose response curves for the effect of hydrogen peroxide on SVK 14 cells showing one representative experiment. Points represent the mean of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium; (3) increasing cell number from 1 x lo5 to 5 x 105; (4) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x I O5 cells were exposed for 15 min to the test agent which was diluted in PBS. -A ~ Time (test); -Ocell no. (standard); -Wcell no. (test); -+10% FCS (standard); - A - time (standard); - 0 - medium (standard); - 0 - medium (test); -Cl - 10% FCS (test).
and chlorhexidine in PBS containing 10% FCS significantly reduced their toxicity. However, the effect of loo/o FCS on sodium hypochlorite was more dramatic, the ED50 concentrations in standard condition experiments being 17.8 times lower than test values (Table II), indicating that serum substantially reduces the toxicity of the chlorine-releasing agent. Effect of medium (RPMI 1640) Use of medium as a diluent had no significant effect on the toxicity of chlorhexidine and there was a small but significant decrease in the toxicity of hydrogen peroxide (Table I). Again, the greatest reduction in toxicity was seen with sodium hypochlorite, where the concentration allowing 50% cell survival for the standard conditions was 33.8 times lower than the test value (Table II). Efjcect of cell number In the standard and test experiments that examined the effect of cell number on toxicity, the assays were conducted in 30 mm dishes, rather than 24-well
Toxicity
of antiseptic
agents
Log concentration
chlorhexidine
in cell
293
culture
, (M)
Figure 3. Dose reponse curves for the effect of chlorhexidine on SVK 14 cells showing one representative experiment. Points represent the mean of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) increasing cell number from 1 X IO5 to 5 x 10S; (3) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x IO’ cells were exposed for 15 min to the test agent which was diluted in PBS. Data for diluting agent in medium are not shown, as responses for test and standard conditions were similar. - 0 Time (test); - W - time (standard); - 0 - cell no. (standard); - 0 - cell no. (test); ~ 0 10% FCS (standard); -+10% FCS (test).
plates, to accommodate the larger number of cells. It was notable that the ED50 values generated for the standard conditions for this series of experiments differed from those of the other experiments. Altering the size of culture dish increased the toxic effects of hydrogen peroxide and chlorhexidine and decreased the toxicity of sodium hypochlorite. Whilst chlorhexidine was as active on 5 x 10’ cells as 1 x 10’ cells, both hydrogen peroxide and sodium hypochlorite showed reduced toxicity in the presence of an increased cell number. This effect was greatest for sodium hypochlorite where there was four-fold increase in ED50 concentration compared to a 1.7-fold increase for hydrogen peroxide.
E#ect of increasing
exposure time
Increasing exposure time to 60 min increased the toxicity of all agents. The least effect was on sodium hypochlorite where a longer exposure produced an ED50 value which was close to that of the standard conditions: for chlorhexidine the test ED50 value was half of the value generated for the standard conditions. The greatest effect was on hydrogen peroxide, where
294
increasing exposure four-fold the control experiment.
F. M. Tatnall
et al.
decreased the ED50 concentration
to 20% of
Discussion
Difficulties arise in establishing appropriate in-vitro experimental conditions that mimic the clinical situation, since no data are available on the fate of topical antiseptics in wounds. Using in-vitro systems such as ours, important questions which need to be addressed are the length of exposure, the cell number used and whether or not the cells are tested in the presence of serum. Short periods of contact are generally used to test the antibacterial activity of these agents. Although some agents act rapidly, others exhibit persistence when tested on intact skin. l4 The best methods of testing these agents in vitro may necessitate varying periods of exposure for different agents. Although the antibacterial activity of antiseptics is routinely tested in the presence of 10% serum, this has not been routinely used in toxicity testing in cell culture. Serous exudate is always present in a wound, but repeated washings with an antiseptic agent may reduce the amount present. Foetal calf serum is used to culture most cell types and cannot be completely removed by routine washing procedures so that equally, a cell culture system is never completely serum free. Theoretical considerations may indicate that an agent should be tested in the presence or absence of serum and the exposure time. However, reported data suggest that practical aspects of performing cytotoxicity assays in cell culture may dictate the experimental conditions used and may, for instance, particularly influence the choice of cell number used in an attached cell assay. Within the limitations of an in-vitro cytotoxicity assay, we believe that the standard experimental conditions outlined in this study may reflect conditions in cutaneous wounds and, importantly, the assay appears to predict the in-vivo toxicities of the agents tested. The observations presented in this study may explain why the toxicity of individual agents varies between published studies. Increasing cell number and using medium or serum as a diluent decreases the cytotoxicity of the free-chlorine-containing agent to a greater degree than for chlorhexidine and hydrogen peroxide and it seems likely that the combination of medium and serum used to routinely culture cells would decrease the toxicity of the chlorine-releasing agents further. This effect is likely to be related to the increase in organic material present. Lengthening the exposure time increases the cytotoxic effect of hydrogen peroxide but has little effect on the toxicity of sodium hypochlorite. Cell culture systems that contain a high cell number, serum or medium and use long exposure times increase the toxicity of hydrogen peroxide relative to the chlorine-releasing agents. These observations may explain reports of high toxicity of hydrogen peroxide and relatively low toxicity of sodium hypochlorite based on in-vitro observations and are the reverse of in-vivo findings, where hydrogen
Toxicity
of antiseptic
agents
in cell
culture
295
peroxide, unlike chlorine-releasing agents, has no adverse effects on the 5,9*‘5,16 Lineaweaver et ~1.~ examined the effect of a range of healing process. antiseptic agents on fibroblasts and found that the toxicity of sodium hypochlorite could be eradicated at a loo-fold dilution from the ‘in-use’ concentration, whereas the toxicity of hydrogen peroxide was abolished at a lOOO-fold dilution from the ‘in-use’ concentration. The findings for hydrogen peroxide are in line with our own results and it seems likely that the differences in toxicity of sodium hypochlorite are related to the use of a higher cell number (2 X lo8 cells) used by Lineaweaver et aZ.’ Leaper & Brennan’ found that the toxicity of chloramine T and hydrogen peroxide could be eradicated at a loo-fold dilution and a 10 OOO-fold dilution respectively from their ‘in-use’ concentrations. In that case the high toxicity of hydrogen peroxide may have been related to the length of exposure (24 h) and the relatively low toxicity of the chlorine-releasing agent to the use of medium as a diluent. In one study cell culture was used to establish a safe concentration at which antiseptics can be administered to wounds, i.e. a concentration which does not harm cutaneous cells but kills bacteria.’ Such a concentration could be established for povidone-iodine and sodium hypochlorite, agents whose toxicity is influenced by the amounts of organic material present. Although equal numbers of bacteria and cells (fibroblasts) were used in this study, these may not represent equal organic loads, bacteria being smaller than fibroblasts. Hence, at a given concentration, hypochlorites may be rendered less toxic to fibroblasts than to bacteria. Such differences might otherwise be attributed to different modes of action on bacteria and mammalian cells; however, at high concentrations it is more likely that most antiseptic agents kill by non-specific means, without species differences.14 Our findings suggest that the results of in-vitro toxicity testing should be interpreted cautiously and within the context of the experimental conditions.
References 1. Bruch MK. Methods of testing antiseptics: antimicrobials used topically in humans. In: Block SS, Ed. Disinfection, Sterilization and Preservation. Philadelphia: Lea and Febiger 1983; 946-963. 2. Shakespeare V, Shakespeare PG, Evans BT. Effect of proprietary oral rinses containing chlorhexidine, hexetidine and benzydamine on the proliferation of human buccal epithelial cells in culture. Arch Oral Biol 1988; 33: 881-885. 3. Cooper ML, Boyce ST, ,Hansborough JF et al. Cytotoxicity to cultured human keratinocytes of topical antimicrobial agents. J Surg Res 1990; 48: 190-195. 4. Deas J, Billings P, Brennan S et al. The toxicity of commonly used antiseptics on fibroblasts in tissue culture. Phlebology 1986; 1: 205-209. 5. Lineaweaver W, Howard R, Saucy D et al. Topical antimicrobial toxicity. Arch Surg 1985; 120: 257-270. 6. Blenkharn JI. The differential cytotoxicity of antiseptic agents. J Pharmncol 1987; 39: 477479. 7. Tatnall FM, Leigh I, Gibson JR. A comparative study of antiseptic toxicity on basal
296
F. M. Tatnall
et al.
keratinocytes, transformed human keratinocytes and fibroblasts. Skin Pharmacol 1990; 3: 157-163. 8. Leaper DG, Brennan S. Let’s have a rethink about the use of antiseptics for venous ulcers. In: Negus D, Jantet G, Eds. Phlebology ‘85. London: John Libbey & Co Ltd 1986; 580-583. 9. Brennan SS, Leaper DJ. The effect of antiseptics on the healing wound: a study using the rabbit ear chamber. Br J Surg 1985; 72: 780-782. SS, Foster ME, Leaper DJ. Antiseptic toxicity in wounds healing by secondary 10. Brennan intention. J Hosp Infect 1986; 8: 263-267. 11. Taylor-Papadimitriou J, Purkis P, Lane EB et al. Effects of SV 40 transformation on the cytoskeleton and behavioural properties of human keratinocytes. CeZE Differ 1982; 11: 169-179. 12. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63. 13. Tatnall FM, Gibson JR, Leigh IM. Evaluation of the cytotoxicity of antiseptics using a calorimetric assay (abstract). Skin Pharmacol 1988; 1: 62. J, Gray KC. Chlorhexidine. In: Block SS, Ed. Disinfection, Sterilization and 14. Gardner Preseroation. Philadelphia: Lea and Febiger 1983; 251-270. 15. Gruber RP, Vistnes, L, Pardoe R. The effect of commonly used antiseptics on wound healing. Plast and Reconstr Surg 1975; 55: 472-476. 16. Leyden JJ, Bartelt NM. Comparison of topical antibiotic ointments, a wound protectant and antiseptics for the treatment of blister wounds contaminated with Staphylococcus aureus. J Fam Prac 1987; 24: 601-604.
of Hospital
Assay
Infection
of antiseptic
(1991)
agents in cell culture: affecting cytotoxicity
F. M. Tatnall, Department
17, 287-296
of Dermatology, Accepted
I. M. Leigh The Royal
for publication
conditions
and J. R. Gibson London
Hospital,
12 December
London
El
1BB
1990
Summary:
In-vivo studies suggest that chlorine-releasing antiseptic agents inhibit wound healing. Studies which have used cell culture systems to evaluate cytotoxicity have generated conflicting results for the toxicity of free-chlorine agents relative to other antiseptics. Here we examine the following three factors which may influence the toxicity of individual agents within a cell culture assay: (1) cell number; (2) duration of exposure; and (3) the nature of the antiseptic diluent. Three agents (sodium hypochlorite, chlorhexidine and hydrogen peroxide) were tested on transformed human keratinocytes (SVK 14 cells). It was found that increasing cell number, and using serum or medium as a diluent, reduced the toxicity of all agents but had the greatest effect on sodium hypochlorite. In contrast, increasing the duration of exposure increased the toxicity of all agents but had the greatest effect on hydrogen peroxide. These observations may explain the high toxicity of hydrogen peroxide and relatively low toxicity of sodium hypochlorite which have been observed in vitro and are the reverse of in-vivo findings. Culture systems in which high cell numbers are coupled with an agent diluted in serum or medium, and a long exposure time, seem likely to decrease the toxicity of chlorine-releasing agents relative to hydrogen peroxide. Keywords:
Antiseptics;
cytotoxicity;
cell
culture
Introduction
Increasing interest is being shown in the use of cell culture systems to evaluate the toxicity of antiseptics used on wounds. Whilst a number of studies have been published which show that all agents tested are toxic to the various cell types used in the assays, scant regard has been paid to the interpretation of these results and their relevance to in-vivo toxicity. Whilst methods of assessing the antibacterial activity of these agents have been standardized’, choice of experimental conditions for evaluating the cytotoxicity of antiseptics in cell culture have varied greatly. Most
Correspondence London El
to:
Dr
F. M.
Tat&l,
Department
of Dermatology,
The
Royal
London
Hospital,
1BB.
0,9556701/91/040287+
10 SO3.00/0
;rs 1991 The Hospml
287
Infectmn
Soarty
288
F. M. Tatnall et al.
studies have used fibroblasts, although recently cultured oral and cutaneous keratinocytes have also been used. 2,3Exposed cell numbers have varied from 5 x lo3 4 to 2 x 10’ ‘, length of exposure has varied from .5 min6 to 24 h4 and 4 days.3 The agents have been diluted in phosphate-buffered saline (PBS),’ saline,’ medium3v8 or medium plus foetal calf serum.* Most studies have examined cell killing and a small number have looked at the effect of antiseptic agents on cell growth.*v3 Techniques used have not allowed for an accurate method of comparison of agents, and relative toxicities are judged by the number of ten-fold dilutions necessary to eradicate toxicity. Using this method of analysis it has been demonstrated that agents containing free chlorine are less toxic than some commonly used antiseptics’,’ or more toxic than other agents.4 Previously we have used a cell culture system to compare the cytotoxic effect of three antiseptic agents (sodium hypochlorite, chlorhexidine and hydrogen peroxide) on transformed human keratinocytes, normal human keratinocytes and fibroblasts. 7 This study showed that the three cell types showed similar susceptibilities to the agents tested. These findings suggest that the transformed cell line, which has the advantage of ready availability and immortality, can replace fibroblasts and keratinocytes in studies the adverse effects of antiseptics in vitro. designed to investigate Comparison of the toxicity of individual antiseptics showed that sodium hypochlorite was the most toxic agent and chlorhexidine the least. These findings are in agreement with recent in-vivo findings.5,9,‘0 Since published studies present conflicting findings for the relative toxicities of agents tested, we have examined a series of conditions in a cell culture system which may alter the toxic effect of a range of antiseptic agents. Here we confine the antiseptic cytotoxicity assay to the SVK 14 cell type. Materials All chemicals
were
supplied
and methods
by the Sigma
Chemical
Company.
Cell culture Transformed human keratinocytes (SVK 14 cells)” were grown in RPM1 1640 medium supplemented with 10% foetal calf serum (FCS) in an atmosphere of 8% CO, at 37°C. Confluent flasks were trypsinized and seeded into multiwell plates. Cytotoxicity assay The following procedure was used for all standard experiments with subsequent modifications as noted below: 1 x 10’ SVK 14 cells were seeded into 24-well plates (Becton Dickinson UK Ltd.) and cultured in RPM1 1640 plus 10% FCS for 24 h. Three antiseptics were tested: hydrogen peroxide, sodium hypochlorite and chlorhexidine. The agents were prepared by serially diluting them (half or third dilutions) in PBS from the
Toxicity
of antiseptic
agents
in cell culture
289
concentrations in clinical use which are as follows: hydrogen peroxide, 0.88 M; sodium hypochlorite, 0.14 M; chlorhexidine, 0.00056 M. Each concentration was tested on four replicate wells. The cells were washed twice prior to the addition of 1 ml of the agent for 15 min. Control wells were exposed to PBS alone. The drug was then removed, the cells washed and RPM1 1640 plus 10% FCS replaced for a further 24 h. Percentage of viable cells for each test concentration was established by comparing values to the control concentrations. The standard conditions outlined above were compared to the following test conditions: (1) diluting the test agent in PBS containing 10% FCS; (2) diluting the test agent in RPM1 1640; (3) increasing the number of exposed cells to 5 x 10’; (4) extending the exposure time to 60 min. For the experiments which used a variation in cell number, 6-well plates were used for the control and test experiments. For each variable standard and test, experiments were run in parallel and each set of experiments was repeated three times. The number of viable cells remaining in each well was evaluated using a calorimetric assay based on the tetrazolium salt 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT).12 This compound is a yellow tetrazolium salt which is reduced by mitochondrial enzymes in viable cells to an insoluble blue formazan product. Previously we have shown that there is good correlation between this calorimetric assay and a counting method when evaluating numbers of viable cells remaining in an antiseptic cytotoxicity assay. l3 The medium was removed from each well and 1 ml of RPM1 1640 plus 200 ~1 MTT (5 mg ml-‘) was added to each well. The cells were incubated at 37°C in 8% CO, for 4 h. The unreacted dye was removed and 1 ml of isopropyl alcohol was added to each well to solubilize the intracellular blue formazan product. The optical density of the extract in isopropyl alcohol in each well was read in a spectrophotometer at 570 nm and expressed as a percentage of the values for the control wells, to generate a percentage viability for each concentration under test. The percentage viability, based on the MTT reading for each concentration was used to establish log-dose response curves. For each experiment the ED50 value was defined as the concentration allowing 50% survival of cells when compared to control wells and was determined by regression analysis. The significance of the difference between ED50 values for test conditions and standard conditions was determined using the Student paired t-test. Results
Mean ED50 values for experiments using test and standard conditions are summarized in Table I, and the ratio of the test to standard values is shown in Table II. An increase in ED50 concentration for a given test condition when compared to the standard value reflects decreased sensitivity of the
10% Foetal calf serum Medium Cell number Time
Variable
1.44f0.250 x lo-* l~lof0~04x 10-2 1.30f 0.08 x lo-* 0~18f0.014x 10-l
Test
9.3 f0.8 9.5 f0.9 7.7f0.8 8.450.5
x x x x
10-S 10-j 10-l 10-l
peroxide
Standard
Hydrogen
0.0030* 0.0340* 0~0005’ 0~0015*
P-value
1.6fO.l 2.8f0.13 4.6*0.8x 7.9fO.l
Test
x 10-j X 1O-3 lo-+ x 10-s
Standard
hypochlorite P-value
9.ozko.47 x 10 5 0~00007* 8.2f0.6 x 10ms 0.0007* 1.1 f0.1 x 10-4 0.018* 9.1 f0.5 x 10-1 0.041*
Sodium
7.5+1.4x 3.7zkO.l 3.7jzO.l 2.1 f0.2
Test
10 i x10-s x 10-S x 10-5
4.1 f0.12 x 10 5 3.6f0.4 x 10ms 2.8f0.06 x 10m5 3.9 f 0.07 x 10-S
Standard
Chlorhexidine
0.015* 0.5 0.25 0.002*
P-value
Table I. Mean ED50 concentrations f 1 SD (N = 3-4) for th ree agents comparing valuesfor standard experiments to those of test conditions. *Indicates significantly dsperent results at the 5% level (PC 0.05paired student t-test). Standard conditions were asfollows: 1 x 10s SVK cells were exposedfor 15 min to a test agent which was diluted in PBS, Standard conditions were compared to the following test conditions: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium (RPMI 1640); (3) increasing cell number to 5 X 10s; (4) increasing exposure time to 1 hour
Toxicity Table II. Ratio were asfollows: PBS. Standard PBS containing
of antiseptic
of mean ED50 values for 1 x 105 SVK 14 cells were conditions were compared 10% FCS; (2) diluting number to 5 x 105; (4)
agents
291
in cell culture
test and standard experiments. Standard conditions exposedfor 15 min to a test agent which was diluted in to the following test conditions: (1) diluting agent in agent in medium (RPMI 1640); (3) increasing cell increasing exposure time to 1 hour
Agent Variable
Hydrogen peroxide
Test/standard 10% Foetal Medium Cell number Time
Sodium hypochlorite
Test/standard calf
serum
Test/standard
I.55 I.15 1.72 0.21
Chlorhexidine Test/standard
17.8 33.5 4.2 0.89
I.8 I.0 I.0 0.54
cells, or increased survival, and therefore, decreased toxicity of the test agent. Representative dose-response curves are shown in Figures 1-3, comparing test and standard conditions for each agent. Eflect of foetal calf serum Comparison of mean ED.50 values showed that diluting
Log concentration
(M) sodium
hydrogen
peroxide
hypochorite
Figure 1. Dose response curves for the effect of sodium hypochlorite on SVK 14 cells showing one representative experiment. Points represent the means of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium; (3) increasing cell number from 1 x IO5 to 5 X 105; (4) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x IO5 cells were exposed for 15 min to the test agent which was diluted in PBS. -O10% FCS (test); -+FCS (standard); -Omedium (test); -0 medium (standard); -mcell no. (test); -0cell no. (standard) and time (standard); -A - time (test).
292
F. M. Tatnall
-4
-3 Log concentration
et al.
-2 (M) hydrogen
-I
0
peroxide
Figure 2. Dose response curves for the effect of hydrogen peroxide on SVK 14 cells showing one representative experiment. Points represent the mean of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) diluting agent in medium; (3) increasing cell number from 1 x lo5 to 5 x 105; (4) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x I O5 cells were exposed for 15 min to the test agent which was diluted in PBS. -A ~ Time (test); -Ocell no. (standard); -Wcell no. (test); -+10% FCS (standard); - A - time (standard); - 0 - medium (standard); - 0 - medium (test); -Cl - 10% FCS (test).
and chlorhexidine in PBS containing 10% FCS significantly reduced their toxicity. However, the effect of loo/o FCS on sodium hypochlorite was more dramatic, the ED50 concentrations in standard condition experiments being 17.8 times lower than test values (Table II), indicating that serum substantially reduces the toxicity of the chlorine-releasing agent. Effect of medium (RPMI 1640) Use of medium as a diluent had no significant effect on the toxicity of chlorhexidine and there was a small but significant decrease in the toxicity of hydrogen peroxide (Table I). Again, the greatest reduction in toxicity was seen with sodium hypochlorite, where the concentration allowing 50% cell survival for the standard conditions was 33.8 times lower than the test value (Table II). Efjcect of cell number In the standard and test experiments that examined the effect of cell number on toxicity, the assays were conducted in 30 mm dishes, rather than 24-well
Toxicity
of antiseptic
agents
Log concentration
chlorhexidine
in cell
293
culture
, (M)
Figure 3. Dose reponse curves for the effect of chlorhexidine on SVK 14 cells showing one representative experiment. Points represent the mean of three replicates. Test conditions were as follows: (1) diluting agent in PBS containing 10% FCS; (2) increasing cell number from 1 X IO5 to 5 x 10S; (3) increasing exposure time from 15 min to 1 h. For each experiment the test conditions were compared to the following standard conditions: 1 x IO’ cells were exposed for 15 min to the test agent which was diluted in PBS. Data for diluting agent in medium are not shown, as responses for test and standard conditions were similar. - 0 Time (test); - W - time (standard); - 0 - cell no. (standard); - 0 - cell no. (test); ~ 0 10% FCS (standard); -+10% FCS (test).
plates, to accommodate the larger number of cells. It was notable that the ED50 values generated for the standard conditions for this series of experiments differed from those of the other experiments. Altering the size of culture dish increased the toxic effects of hydrogen peroxide and chlorhexidine and decreased the toxicity of sodium hypochlorite. Whilst chlorhexidine was as active on 5 x 10’ cells as 1 x 10’ cells, both hydrogen peroxide and sodium hypochlorite showed reduced toxicity in the presence of an increased cell number. This effect was greatest for sodium hypochlorite where there was four-fold increase in ED50 concentration compared to a 1.7-fold increase for hydrogen peroxide.
E#ect of increasing
exposure time
Increasing exposure time to 60 min increased the toxicity of all agents. The least effect was on sodium hypochlorite where a longer exposure produced an ED50 value which was close to that of the standard conditions: for chlorhexidine the test ED50 value was half of the value generated for the standard conditions. The greatest effect was on hydrogen peroxide, where
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increasing exposure four-fold the control experiment.
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et al.
decreased the ED50 concentration
to 20% of
Discussion
Difficulties arise in establishing appropriate in-vitro experimental conditions that mimic the clinical situation, since no data are available on the fate of topical antiseptics in wounds. Using in-vitro systems such as ours, important questions which need to be addressed are the length of exposure, the cell number used and whether or not the cells are tested in the presence of serum. Short periods of contact are generally used to test the antibacterial activity of these agents. Although some agents act rapidly, others exhibit persistence when tested on intact skin. l4 The best methods of testing these agents in vitro may necessitate varying periods of exposure for different agents. Although the antibacterial activity of antiseptics is routinely tested in the presence of 10% serum, this has not been routinely used in toxicity testing in cell culture. Serous exudate is always present in a wound, but repeated washings with an antiseptic agent may reduce the amount present. Foetal calf serum is used to culture most cell types and cannot be completely removed by routine washing procedures so that equally, a cell culture system is never completely serum free. Theoretical considerations may indicate that an agent should be tested in the presence or absence of serum and the exposure time. However, reported data suggest that practical aspects of performing cytotoxicity assays in cell culture may dictate the experimental conditions used and may, for instance, particularly influence the choice of cell number used in an attached cell assay. Within the limitations of an in-vitro cytotoxicity assay, we believe that the standard experimental conditions outlined in this study may reflect conditions in cutaneous wounds and, importantly, the assay appears to predict the in-vivo toxicities of the agents tested. The observations presented in this study may explain why the toxicity of individual agents varies between published studies. Increasing cell number and using medium or serum as a diluent decreases the cytotoxicity of the free-chlorine-containing agent to a greater degree than for chlorhexidine and hydrogen peroxide and it seems likely that the combination of medium and serum used to routinely culture cells would decrease the toxicity of the chlorine-releasing agents further. This effect is likely to be related to the increase in organic material present. Lengthening the exposure time increases the cytotoxic effect of hydrogen peroxide but has little effect on the toxicity of sodium hypochlorite. Cell culture systems that contain a high cell number, serum or medium and use long exposure times increase the toxicity of hydrogen peroxide relative to the chlorine-releasing agents. These observations may explain reports of high toxicity of hydrogen peroxide and relatively low toxicity of sodium hypochlorite based on in-vitro observations and are the reverse of in-vivo findings, where hydrogen
Toxicity
of antiseptic
agents
in cell
culture
295
peroxide, unlike chlorine-releasing agents, has no adverse effects on the 5,9*‘5,16 Lineaweaver et ~1.~ examined the effect of a range of healing process. antiseptic agents on fibroblasts and found that the toxicity of sodium hypochlorite could be eradicated at a loo-fold dilution from the ‘in-use’ concentration, whereas the toxicity of hydrogen peroxide was abolished at a lOOO-fold dilution from the ‘in-use’ concentration. The findings for hydrogen peroxide are in line with our own results and it seems likely that the differences in toxicity of sodium hypochlorite are related to the use of a higher cell number (2 X lo8 cells) used by Lineaweaver et aZ.’ Leaper & Brennan’ found that the toxicity of chloramine T and hydrogen peroxide could be eradicated at a loo-fold dilution and a 10 OOO-fold dilution respectively from their ‘in-use’ concentrations. In that case the high toxicity of hydrogen peroxide may have been related to the length of exposure (24 h) and the relatively low toxicity of the chlorine-releasing agent to the use of medium as a diluent. In one study cell culture was used to establish a safe concentration at which antiseptics can be administered to wounds, i.e. a concentration which does not harm cutaneous cells but kills bacteria.’ Such a concentration could be established for povidone-iodine and sodium hypochlorite, agents whose toxicity is influenced by the amounts of organic material present. Although equal numbers of bacteria and cells (fibroblasts) were used in this study, these may not represent equal organic loads, bacteria being smaller than fibroblasts. Hence, at a given concentration, hypochlorites may be rendered less toxic to fibroblasts than to bacteria. Such differences might otherwise be attributed to different modes of action on bacteria and mammalian cells; however, at high concentrations it is more likely that most antiseptic agents kill by non-specific means, without species differences.14 Our findings suggest that the results of in-vitro toxicity testing should be interpreted cautiously and within the context of the experimental conditions.
References 1. Bruch MK. Methods of testing antiseptics: antimicrobials used topically in humans. In: Block SS, Ed. Disinfection, Sterilization and Preservation. Philadelphia: Lea and Febiger 1983; 946-963. 2. Shakespeare V, Shakespeare PG, Evans BT. Effect of proprietary oral rinses containing chlorhexidine, hexetidine and benzydamine on the proliferation of human buccal epithelial cells in culture. Arch Oral Biol 1988; 33: 881-885. 3. Cooper ML, Boyce ST, ,Hansborough JF et al. Cytotoxicity to cultured human keratinocytes of topical antimicrobial agents. J Surg Res 1990; 48: 190-195. 4. Deas J, Billings P, Brennan S et al. The toxicity of commonly used antiseptics on fibroblasts in tissue culture. Phlebology 1986; 1: 205-209. 5. Lineaweaver W, Howard R, Saucy D et al. Topical antimicrobial toxicity. Arch Surg 1985; 120: 257-270. 6. Blenkharn JI. The differential cytotoxicity of antiseptic agents. J Pharmncol 1987; 39: 477479. 7. Tatnall FM, Leigh I, Gibson JR. A comparative study of antiseptic toxicity on basal
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keratinocytes, transformed human keratinocytes and fibroblasts. Skin Pharmacol 1990; 3: 157-163. 8. Leaper DG, Brennan S. Let’s have a rethink about the use of antiseptics for venous ulcers. In: Negus D, Jantet G, Eds. Phlebology ‘85. London: John Libbey & Co Ltd 1986; 580-583. 9. Brennan SS, Leaper DJ. The effect of antiseptics on the healing wound: a study using the rabbit ear chamber. Br J Surg 1985; 72: 780-782. SS, Foster ME, Leaper DJ. Antiseptic toxicity in wounds healing by secondary 10. Brennan intention. J Hosp Infect 1986; 8: 263-267. 11. Taylor-Papadimitriou J, Purkis P, Lane EB et al. Effects of SV 40 transformation on the cytoskeleton and behavioural properties of human keratinocytes. CeZE Differ 1982; 11: 169-179. 12. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63. 13. Tatnall FM, Gibson JR, Leigh IM. Evaluation of the cytotoxicity of antiseptics using a calorimetric assay (abstract). Skin Pharmacol 1988; 1: 62. J, Gray KC. Chlorhexidine. In: Block SS, Ed. Disinfection, Sterilization and 14. Gardner Preseroation. Philadelphia: Lea and Febiger 1983; 251-270. 15. Gruber RP, Vistnes, L, Pardoe R. The effect of commonly used antiseptics on wound healing. Plast and Reconstr Surg 1975; 55: 472-476. 16. Leyden JJ, Bartelt NM. Comparison of topical antibiotic ointments, a wound protectant and antiseptics for the treatment of blister wounds contaminated with Staphylococcus aureus. J Fam Prac 1987; 24: 601-604.
