In vitro cytotoxicity of antimicrobial agents to human keratinocytes

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  • ELSEVIER Journal of the European Academy of Dermatology and Venereology

    6 (1996) 159-166

    In vitro cytotoxicity of antimicrobial agents to human keratinocytes

    Florian Kautzky, Anton Hartinger, Lars D. Kiihler, Hermann-J. Vogt * a Department of Dermatology, Technische Uniuersitiit, Biedersteiner StraJ3e 29, 80802 Munich, Germany

    b Department of Microbiology, Klinikum Miinchen-Bogenhausen, Munich, Germany

    Abstract

    Backround The degree of bacterial contamination in the wound bed plays a key role in determining the level of take of keratinocyte grafts in burn patients. This study was done to evaluate the best suited antimicrobial agent for topical application in relation to antimicrobial activity and cytotoxic effect on graft cells.

    Objective The cytotoxicity of 7 antibacterial agents (gentamicin sulphate, ciprofloxacin hydrochloride, bacitracin, polymyxin B sulphate, diethanolamin salt of fusidic acid, teicoplanin, vancomycin hydrochlo- ride), one antifungal substance (amphotericin B sodium deoxycholate) and one antiseptic agent (povi- done iodine) on cultured human keratinocytes, HaCaT keratinocytes and fibroblasts was investigated.

    Material and methods The effects of these agents were tested using two separate assays - the neutral red assay and the Bradford protein assay. In order to assess toxicity of the agent, the obtained midpoint cytotoxicity (MC.50) was compared to the respective minimal inhibitory concentration (MIC) for receiving the cytotoxic potential in microbiologically effective doses.

    Results Antibiotics effective against Staphylococcus aureus with a small potential of toxicity are Bacitracin, Fusidic acid, Vancomycin and Teicoplanin. Gentamicin and Ciprofloxacin are effective against both Staphylococcus aureus and Pseudomonas aeruginosa with a low potential of toxicity.

    Conclusion The use of antimicrobial agents with a midpoint cytotoxicity (MC50) higher than the minimal inhibitory concentration (MIC) by at least a factor of 100 might reduce the risk of toxic injury to graft cells.

    Keywords: Antimicrobial agents; Keratinocytes; Cell culture; Cytotoxicity

    1. Introduction

    The immediate covering of severe and exten- * Corresponding author. Tel.: (089) 3849 3177; fax: (089) sive burns avoids dangerous complications and

    3849 3323. insures an increased survival rate of these pa-

    0926-9959/96/$15.00 0 1996 El sevier Science B.V. All rights reserved SSDIO926-9959(95)00153-O

  • 160 F. Kautzky et al. /J. Eur. Acad. Dermatol. Venereal. 6 (1996) 159-166

    tients [4,19]. The successful use of autologous cultured epidermis for permanent cover or allo- geneic cultured epidermis to induce reepitheliza- tion of skin lesions has been reported extensively in the literature [7,8,10,14,23].

    However. the great advantage of this method is limited by the fact that the level of transplant take is not satisfactory, varying from 5 to 95% Ill]. This was mainly attributed to the level of bacterial contamination of the wound bed, which seems to play a key role in a graft take [7,9].

    The present study was done to evaluate the best suited antimicrobial agent for topical appli- cation without cytotoxic effects on the graft cells. To quantify the cytotoxicity of the antimicrobial agents, their effects on basal keratinocytes, which play a fundamental role in the healing process, were studied and compared with the behaviour of HaCaT cells and 3T3 fibroblasts.

    2. Materials and methods

    2.1. Tissue culture conditions

    Primary cultures of human keratinocytes were derived from skin of patients undergoing reduc- tion mammoplasty. Small skin fragments were floated in PBS. After incubating EDTA (0.2%) the separated epidermis was trypsinized for 18 h at 4 c. Disaggregated epidermis cells were fil- tered, washed, and then cultured using the method of Rheinwald, Green et al. [181 on a feeder layer of mitomycin-treated 3T3 cells. Tis- sue culture was continued in a mixture of Dul- beccos modified Eagles medium containing Hams F12 medium at a ratio of 4:1, containing epidermal growth factor, cholera toxin, adenine, triiodothyronin, gentamicin and 10% fetal bovine serum.

    For cytotoxicity assays the human ker- atinocytes were grown according to the technique of Eisinger et al. [9] without a feeder layer. Hu- man keratinocytes were suspended in serum free medium (Gibco) containing epidermal growthfac- tor and an extract of hypophyse (Gibco). without gentamicin and seeded into 96 well tissue culture plates from subconfluent primary cultures at a

    higher density (4 x lo4 cells per well for neutral red assay and 6 X lo3 per well for the Bradford protein assay). HaCaT cells were grown also in serum free medium (Gibco) containing epidermal growthfactor and an extract of hypophyse (Gibco). 3T3 cells were cultured in Dulbeccos modified Eagles medium containing 10% newborn bovine serum. Like human keratinocytes both the 3T3 and HaCaT cells were placed into 96 well tissue culture plates (Greiner) for the toxicity assays (3T3 cells: 1 X lo4 per well for neutral red assay and 2 X lo3 per well for the Bradford protein assay; HaCaT cells: 1.8 x lo4 per well for neutral red assay and 4 x lo3 per well for the Bradford protein assay).

    3. Cytotoxicity assays

    The cytotoxicity of 7 antibacterial agents, gen- tamicin sulphate (Sigma), ciprofloxacin hydro- chloride (Bayer), bacitracin (Sigma), polymyxin B sulphate (Sigma), diethanolamin salt of fusidic acid (Thomae), teicoplanin (Merel), vancomycin hydrochloride (Lilly), one antifungal substance (amphotericin B sodium deoxycholate (Sigma) and one antiseptic agent (povidone iodine, Sigma) on cultured human keratinocytes, HaCaT ker- atinocytes and fibroblasts was investigated using neutral red assay and Bradford protein assay.

    3.1. Neutral red assay

    Only cells with intact cell membrane take up neutral red into their lysosomes. The quantity of intralysosomal dye therefore is proportional to the function of cell membranes and to the num- ber of vital cells. When cells reached subconflu- ency in 96 well tissue culture plates, the medium was replaced by diluted test agents. Both medium without any antimicrobial agent and medium con- taining sodium dodecyl sulphate (50 mg/ml) were placed in quadruplicates to obtain a value of reference for maximum vitality and toxicity, re- spectively. After a 24 h incubation with various concentrations of the test agents, the cells were removed and medium containing 0.05 mg/ml neutral red was added to each well for 3 h at 37

  • F. Kautzky et al. /J. Eur. Acad. Dermatol. Venereal. 6 (1996) 159-166 161

    Table 1 MIC against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans in 10m3 mg/ml

    MIC for MIC for MIC for

    Gentamicin sulfate Ciprofloxacin hydrochloride Bacitracin Polymyxin B sulfate Fucidic acid Vancomycin hydrochloride Teicoplanin

    Amphotericin B sodium deoxycholate Povidone iodine

    Staphylococcus aureus

    1.00 0.50 12.5

    0.06 1.60 3.10

    1000

    Pseudomonas aeruginosa

    8.00 0.50 - 5.00 -

    - 1000

    Candida albicans

    - - - -

    - -

    3.70 1000

    c. The cells were then washed, fixed and neutral red was extracted as discribed before [2,3]. The intensity of neutral red was measured spectopho- tometrically at a wavelength of 540 nm.

    The quantity of intralysosomal dye is propor- tional to the function of cell membranes and to the number of vital cells.

    3.2. Bradford protein assay

    The binding of the dye coomassie brilliant blue G 250 causes a shift in the maximum absorption of the dye from 465 to 595 which can be quanti- fied spectophotometrically. After 24 h the medium was removed from the wells and re-

    Table 2 The midpoint cytotoxicity data (MC50) of antimicrobial agents to human keratinocytes, 3T3-fibroblasts and HaCaT-cells measured by Bradford protein assay and neutral red assay in mg/ml

    Bradford protein assay

    Gentamicin sulfate Ciprofloxacin hydrochloride Bacitracin Polymyxin B sulfate Fucidic acid Vancomycin hydrochloride Teicoplanin Amphotericin B

    sodium deoxycholate Povidone iodine

    3T3-fibroblasts

    5.00 0.26 2.80 0.29 0.05

    20.80 9.20

    0.11 0.52

    Neutral red assay

    HaCaT- cells Keratinocytes

    8.00 1.90 0.22 0.56 9.10 12.2 0.38 0.56 0.13 0.12

    18.1 10.7 16.7 1.00

    0.19 0.02 0.31 0.26

    Gentamicin sulfate Ciprofloxacin hydrochloride Bacitracin Polymyxin B sulfate Fucidic acid Vancomycin hydrochloride Teicoplanin Amphotericin B

    sodium deoxycholate Povidone iodine

    3T3-fibroblasts HaCaT-cells Keratinocytes

    2.60 5.00 1.000 0.25 0.76 0.710

    10.0 9.60 10.20 0.39 0.37 0.240 0.46 0.19 0.190

    10.10 5.60 13.60 3.00 4.80 1.500

    0.10 0.04 0.055 1.10 1.40 0.58

  • 162 F. Kautzky et al. /J. Eur. Acad. Dermatol. Venereal. 6 (1996) 159-166

    placed by adequate fresh medium (control wells), or by medium containing test agents in various concentrations. The test medium was decanted after 48 h and cells were washed twice with PBS and then incubated with 0.1 N NaOH for 15 min at 37 Co [20]. The diluted dye solution was added to each well and the absorption was measured at 405 nm and 630 nm. To determine a defined equivalent of protein quantity, serial dilutions of bovine serum albumin were tested in the same fashion in the 96 well plates.

    3.3. Calculation of cytotoxic potential

    In general 4 wells were used for each test agent concentration with 8 wells as controls. The results of each assay were expressed as mean and standard deviation of mean (SD) of 4 wells. Each experiment was repeated, with different isolates of human keratinocytes and twice with 3T3 and HaCaT cells.

    The degree of cytotoxicity of agents tested are represented by midpoint cytotoxicity (MC50) which correlates with the individual concentra- tion of a test agent that reduces the neutral red uptake by 50% compared with untreated sam- ples. Similar to the neutral red assay, the MC50 of the Bradford protein assay is the concentration

    of a test agent that reduces the quantity of cell protein by 50%. MC50 values are compared with the minimal inhibitory concentrations (MIC) of important microorganisms [1,11,16] (Table 1) us- ing the formula Qi = MCSO/MIC.

    4. Results

    The effects of antimicrobial agents on primary human keratinocytes, HaCaT cells and 3T3 fi- broblasts tested by neutral red assay and Brad- ford protein assay are listed in Table 2. Detailed data on neutral red assay of Ciprofloxacin are exemplarily shown in Fig. 1. Our first interest was to evaluate the cytotoxicity of antimicrobial agents on cells, which is demonstrated in Table 2 by cytotoxic midpoint concentrations (MC50). How- ever, the main factor for clinical use is the indi- vidual cytotoxic potential of agents tested in re- spect to their effect against the most problematic microorganisms. The comparison of cytotoxic midpoint concentrations (MC501 and minimal in- hibitory concentrations (MI0 [1,11,161 (Table 3) for Staphylococcus aureus (Fig. 21, Pseudomonas aeruginosa (Fig. 3) and Candida a&cans (Fig. 4) demonstrates their cytotoxic potential.

    MC50 of Gentamicin, Ciprofloxacin, Baci- tracin, Fusidic acid, Vancomycin and Teicoplanin

    Table 3 Relation of MC50 values and MIC expressed by the formula Qi = MCSO/MIC

    STS-Fibroblasts HaCaT-cells Keratinocytes

    MC50/MIC of Staphylococcus aureus Gentamicin sulfate 2600-5000 5000-8000 1000-1900 Ciprofloxacin hydrochloride 500-520 440-1520 1120-1420 Bacitracin 224-800 728-768 976-816 Fusidic acid 833-7667 2167-3167 200-3167 Vancomycin hydrochloride 6313-13000 3500-11313 6688-8500 Teicoplanin 968-2968 1548-5387 323-484 Povidone iodine 0.52-1.10 0.31-1.4 0.26-0.58

    MCSO/MIC of Pseudomonas aeruginosa Gentamicin sulfate 325-625 625-1000 125-238 Ciprofloxacin hydrochloride 500-520 440-1520 1120-1420 Polymyxin B sulfate 58-78 74-76 48-112 Povidone iodine 0.52-1.1 0.31-1.4 0.26-0.58

    MCSO/MIC of Candida albicans Amphotericin B sodium deoxycholate 27-30 11-51 15-70 Povidone iodine 0.52-1.1 0.31-1.4 0.26-0.58

  • F. Kautzky et al. /J. Eur. Acad. Dermatol. Venereol. 6 (1996) 159-166 163

    Extinction at 540 nm Cytotoxic potential:

    1o-3 10. 10 lo1 10 mg/ml

    Fig. 1. Example: detailed test results for testing cytotoxicity of Vancomycin hydrochloride by neutral red assay.

    Cytotoxic potential:

    Bacitracin

    Ciprofloxacin

    Fucidic Acid

    Gcntamicin

    Teicopianin

    Vancomycin

    Povidone iodine

    high middle small

    Fig. 2. The potential for toxicity of antimicrobial agents on Staphylococcus aureus are represented by the quotient of MC50 divided by the MIC (Qi). A Qi higher then 100 indicates a low potential for toxicity on HaCaT-cells and keratinocytes.

    Ciprofloxacin

    Gentamicin

    Polymyxin B

    Povidone

    Cytotoxic potential:

    medium 1OW

    7

    10 100 1000

    1

    Fig. 3. The potential for toxicity of antimicrobial agents on Pseudomonas aeruginosa are represented by the quotient of MC50 divided by the MIC (Qi).

    high middle SlIl2.11

    Amphotericin B

    P&done iodine

    Fig. 4. The potential for toxicity of antimicrobial agents on Candida albicans are represented by the quotient of MC50 divided by the MIC (Qi).

    is more than 100 times higher than their MIC against Staphylococcus aureus. In addition gen- tamicin also has an MC50 more than 100 times higher than the MIC for Pseudomonas aerugi- nosa. Therefore this agent has a low cytotoxic potential in regard to these microorganisms. All other agents tested show smaller differences be- tween the toxic concentrations (MC501 and mini- mal inhibitory concentrations (MIC), which means a higher toxic potential.

    5. Discussion

    Only a few studies so far have investigated the in vitro toxicity of topical antimicrobial agents on cultured human skin cells [5,22], which show a high cytotoxicity especially for antiseptic agents [6,12,211.

    This study confirms parts of earlier results and was done because of the clinical relevance not only in burn patients but also in the treatment of leg ulcers with grafts.

    In this study the effects of 9 antimicrobial agents were investigated using two separate as- says, the neutral red assay and the Bradford protein assay, in 3 different cell types. It is neces- sary to compare the antimicrobially effectiveness and cytotoxic concentrations of agents tested for determining their toxic potential in clinical use. The most difficult to control microorganisms in severe and extensive burns are Staphylococcus aureus, Pseudomonas aeruginosa and Candida al- bicans [13]. The known minimum inhibitory con-

  • 164 F. Kautzky et,al. /J. Eur. Acad. Derrnatol. Venereol. 6 (1996) 159-166

    centrations (MIC) of the agents tested against these microorganims are listed in Table 1 [1,11,16] and compared with the MC50 data of the agents tested (Table 3).

    All the MC50, except the MC50 of povidone iodine, are significantly higher then the MICs. The relation of cytotoxic concentrations (MC.50) and MIC as expressed by the quotient Qi, repre- sents the potential of cytotoxicity for effective microbiological treatment with the agents (Table 3). The risk of cytotoxic effect of the agents in an antimicrobial therapy must to be as small as possible. In nearly every biological system natural variations exist. Due to these fluctuations a low potential for toxicity in vivo can be supposed with a high possibility only for agents with an MC50 being 100 times higher than the respective MIC (Qi > 100). Thus a Qi smaller than 10 represents a high, a Qi between 10 and 100 a medium and a

    Qi higher than 100 a low potential for toxicity (Figs. 2, 3, 4).

    The data obtained are directly transferable to cell culture systems. The most important reason for the loss of cell culture grafts are microbiologi- cal infections. Thus, the prophylactic use of an- tibiotics can help to prevent this problem. To avoid cytotoxic effects, the concentration of the agent should be 100 times smaller than MC50 (Figs. 2, 3, 4). We recommend the use of agents with a small cytotoxic potential to reduce the risk of toxic injury of cells. In vivo many factors (i.e. exudation of different body fluids) influence drugs and their effects, such as the cytotoxic effect of antimicrobial agents. Therefore, the concentra- tion for topical application should be significantly higher than the concentrations used in cell cul- tures already examined [15,17].

    Figs. 5 and 6 show the results of keratinocyte

    Fig. 5. Excellent take of keratinocyte graft on a mesh graft donor site with an optimal aseptic wound bed.

  • F. Kautzky et al. /J. Eur. Acad. Dermatol. Venereal. 6 (1996) 159-166 165

    Fig. 6. Unsuccessful keratinocyte graft on a bacterially contaminated wound bed.

    grafts on an ideal clean wound and an infected wound, respectively. The success of using topical antibiotics in a keratinocyte graft on a contami- nated wound is indicated by recent clinical expiri- ences. However this clinical correlation needs to be confirmed by further clinical investigations.

    6. Conclusion

    The use of effective and cytotoxically harmless antimicrobial agents appears to improve the suc- cess of keratinocyte grafts in severely burned patients when adequate early cover is possible as well as in patients with chronic leg ulcers.

    Povidone iodine (the only antiseptic agent tested), as well as amphotericin (the only antimy- cotic tested), show high potential toxicity in the therapy of all sensitive reference microorganisms. Polymyxin has a medium potential for toxicity in

    regard to Pseudomonas aeruginosa. Antibiotics effective against Staphylococcus aureus with a low potential for toxicity are Bacitracin, Fusidic acid, Vancomycin as well as Teicoplanin. Gentamicin and Ciprofloxacin can be used for Staphylococcus aureus and Pseudomonas aeruginosa with a low potential for toxicity.

    Acknowledgement

    We thank Dr. W. Dahm for critical reading of the manuscript.

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