IN VITRO Vol. 12, No. 5. 1976

T H E E F F E C T OF F E T A L B O V I N E S E R U M ON P O L Y E N E M A C R O L I D E ANTIBIOTIC CYTOTOXICITY AND A N T I F U N G A L ACTIVITY 1 D. P. BONNER, P. B. FISHER, ~ N. I. GOLDSTEIN, W. MECHLINSKI, V. BRYSON, AND C. P. SCHAFFNER

Waksman Institute of Microbiology, Rutgers University, New Brunswick, New Jersey 08903 SUMMARY

The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers to 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth of Saccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2. 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminating

Candida albieans, Saccharomyces cerevisiae, Aspergillus niger or Fusarium rnoniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems.

Key words: amphotericin B; amphotericin B methyl ester; fetal bovine serum; polyene macrolide antibiotic antifungal agents.

makes certain polyene macrolide antibiotics quite toxic to many cell lines normally employed in Amphotericin B (AB), a polyene macrolide tissue culture studies (12-17). However. a slight antifungal antibiotic, is used extensively in the preference for binding with the fungal sterols control of fungal and yeast contamination of tissue permits AB to be effectively used in the control of culture systems (1, 2). It is usually employed some of these microbial contaminants (3-5). as Fungizone ®, the commercially available desThe presence of sterols in the medium has been oxycholate complex of AB (E. R. Squibb & Sons) reported to have an antagonistic effect on the (3-5). The polyene macrolide antibiotics exert biological activity of the polyene macrolide antitheir effects by binding with sterols in the fungal biotics (18-21). Since fetal bovine serum (FBS), cell membrane, an interaction that may result which contains sterols, is a common constituent in permeability alterations and eventually may in many tissue culture media, its presence might lead to cell death (6-11). The ability to interact be expected to influence both the cytotoxic and also with membrane sterols in mammalian ceils antimicrobial activities of the added polyene macrolide antibiotics. An effect of serum on cyto1 Portions of this paper were presented at the 25th toxicity has been previously reported by Kinsky Annual Meeting of the Tissue Culture Association at (22). Using a hemoglobin release assay system. Miami, Florida, 1974. 2 Present address: Department of Microbiology and he demonstrated the antagonistic properties of Immunology, Albert Einstein College of Medicine, homologous sera on AB induced lysis of rat and human red blood cells. In the present study Bronx, N.Y. 10461 399 INTRODUCTION

400

BONNER ET AL.

we have investigated effects of the sterol-containing FBS in antagonizing cytotoxic activities of AB and its derivative, amphotericin B methyl ester (AME) (23-25). Also, the ability of AME to eliminate fungi from the KB cell line has been demonstrated. MATERIALS AND METHODS To assess the antagonism of FBS on AB or AME mediated cytotoxicity, studies were performed on a tumor-derived human cell line, KB (26) (originated from an epidermoid carcinoma of the mouth), grown in the presence of different concentrations of FBS and AB or AME. Fixed volumes of KB cells (ATCC CCL 17). 1 to 4 × 10 ~ cells, in 1 ml of medium, were inoculated into replicate Leighton tubes or 35 x 10 mm tissue culture plates on day 0. The medium consisted of Eagle's minimum essential medium supplemented with nonessential amino acids, 50 /xg per ml of gentamicin, and 2, 5, 10, 15 or 20% FBS from the same initial FBS lot (Microbiological Associates). A 10-/xl aliquot of varying concentrations of either AB or AME in dimethylsulfoxide (DMSO) was added on day 0 to the tubes or plates followed by incubation at 37°C in a CO2 incubator (5% CO2). AB was generously supplied by E. R. Squibb & Sons, Inc., while AME was synthesized in our laboratory aceording to procedures previously described (23). Control cultures received either 10 /~l of DMSO or no treatment. Viable cell counts were performed on day 1, 2 and 3 using the trypan blue dye exclusion technique and a hemacytometer (27). Our index of comparison among the differing % FBS media was designated as the relative TCDs0, that is, the concentration of AB or AME resulting in a 50% reduction in viable cell number after a 3-day test period (as compared to the day 3 controls) (14-17). All experiments, using differing % FBS concentrations, were performed a minimum of three times using duplicate samples in each trial test. For determining antagonism of antiyeast activity, Sabouraud's dextrose medium (4% dextrose, 1% neopeptone) with 5, 10 or 15% FBS was employed. Saeeharomyces cerevisiae (ATCC 9763) was the assay organism. A logarithmic phase culture of S. cerevisiae was diluted to 85% light transmission (at 560 nm) using a Coleman model 44 spectrophotometer, further diluted 1:100 in media containing the proper percentage

of FBS, and dispensed in 2-ml quantities. Various concentrations of AB or AME in 10 gl of DMSO were added and after 24 hr incubation at 28°C, tube turbidities were read at 560 nm. Increasing turbidity indicated growth of the yeast and antagonism of the antibiotic by FBS. The antibiotics were evaluated in the different media as to the amount required for a 50% growth inhibition of the test organism when compared to the proper control tubes receiving no antibiotic. The minimum fungicidal concentrations (MFC's) for AB and AME in media containing differing percentages of FBS were determined by plating each tube at each dilution onto Sabouraud's plates. When two out of the three plates from each dilution showed less than three colonies, this was considered the MFC (survival less than 0.1%). The ability of AB and AME to eliminate various yeasts and fungi from KB cells grown in the presence of 5% or 10% FBS was also tested. KB cells were grown as previously described in media containing 5% or 10%FBS. Yeasts or filamentous fungi (Candida albicans, Saccharomyces cerevisiae, Aspergillus niger or Fusarium moniliforme) were added to flasks of KB cells in the proper concentrations to yield approximately 1000 CFU per ml. The "contaminated" cultures were then dispensed in 1-ml aliquots (2 × 105 KB cells) into 35 × 10 mm tissue culture plates (Falcon Plastics). AB (not Fungizone ®) and AME were then added in 10/xl of DMSO. Plates were incubated at 37°C in 5% CO2 for 48 hr. Triplicate samples (0.1 ml per sample) were then removed and plated on Sabouraud's dextrose agar and incubated at 28°C for up to 2 weeks for observation of colonies. In addition, control cultures and the artificially contaminated KB cells treated with the polyene antibiotics were observed using phase optics for a 7-day period to detect presence of the fungi and to monitor KB cell growth. RESULTS The results of FBS antagonism of AB and AME cytotoxicity are presented in Table 1. In the 3-day test, AB and AME were approximately 60-fold less toxic toward KB cells grown in 20% as opposed to 2% FBS. AB at 6 p.g per ml or AME at 20/xg per ml yielded a 50% reduction in viable cell number in 20% FBS, whereas a similar reduction was evident at 0.09/zg per

401

POLYENE ANTIBIOTICS AND SERUM ml of AB or 0.3 /xg per ml of AME when the cells were grown in 2% FBS. AME was less toxic than AB toward KB cells grown in 2, 5, 10, 15 or 20% FBS. The antagonism of the antiyeast activity of AB and AME by FBS is presented in Table 2. The concentration of antibiotic necessary to inhibit growth by 50% was used as the comparative index. As the concentration of FBS in the medium increased so did the amount of AB or AME required for effective inhibition of S. cerevisiae growth. At all three FBS concentrations (5%, 10% or 15%), the level of AB or AME required to inhibit the growth of S. cerevisiae was below the TCD~0 level for KB cells. The differences in absolute inhibitory concentrations of the two antibiotics are probably more a matter of relative purity of the two preparations involved rather than inherent potency, since previous reports (25, 28) have confirmed for many fungi the relative equivalent, in vitro antimicrobial activity of parent compound and derivative. In our studies AB was approximately 95% pure while AME was approximately 80% pure, as determined by spectrophotometric analysis. Higher levels of AB and AME were required to induce a fungicidal effect (Table 3) than to inhibit the growth of S. eerevisiae by 50% (Table 2). The MFC's were dependent on FBS concert-

TABLE 1 TOXICITY OF AMPROTERICIN B (AB) AND AMPHOTERICIN B METHYL ESTER (AME) TOWARD KB CELLS GROWN IN MEDIA CONTAINING DIFFERENT CONCENTRATIONS OF FETAL BOVINE SERUM Fetal Bovine Serum Concentration (%)

TCDsoa AB qzg per ml)

TCD~0 AME Igg per ml)

TABLE2 EFFECT OF FETAL BOVINE SERUM CONCENTRATION ON THE ABILITY OF AB AND A M E TO INHIBIT THE GROWTH OF SACCHAROMYCES CEREVISIAEa Fetal Bovine Serum Concentration (%)

50% Inhibitionb AB (/xg per ml)

50% Inhibition AME (/xg per ml)

5

0.078

0.095

10

0.091

0.132

15

0.101

0.160

a Test was performed in triplicate in Sabouraud's medium (4% dextrose, 1% neopeptone). Organism was diluted to 85% transmission Cat 560 nm) on Coleman model 44 spectrophotometer, then diluted 1:100 in media containing proper % of FBS and dispensed in 2 ml aliquots. Antibiotics were added in 10/xl of DMSO. Cultures were then incubated for 24 hr at 28°C, read at 560 nm and computed as % growth of control. b Refers to concentration of polyene macrolide antibiotic resulting in a 50% reduction in growth of S. eerevisiae in comparison with DMSO or untreated controls.

tration, with higher levels of AB and AME required to kill the test organism in 10% or 15% FBS than in 5% FBS (Table 3). AB was toxic toward KB cells grown in 5%, 10% or 15% FBS at levels required for fungicidal activity, whereas AME did not exhibit toxicity (less than 5% reduction in viable cell number after 72 hr) at the MFC's for the three different FBS concentrations. AB was most toxic toward KB cells at the MFC for S. cerevisiae in media containing 5% (30% reduction in viability at 72 hr) or 10% FBS (40% reduction in viability at 72 hr). A relationship between the type of "contam-

TCDs0 Ratk AME/AB

2

0.09

0.3

3.33

5

0.8

3.0

3.75

10

1.0

7.0

7.0

15

3.0

10.0

3.30

20

6.0

20.0

3.33

a TCDs0, concentration of polyene macrolide antibiotic which results in approximately a 50% reduction in 72-hr KB viable cell number in comparison with DMSO (1.0%) or untreated controls. The experiments were performed in duplicate (a minimum of three times) as described in Materials and Methods section.

TABLE 3 MINIMUM FUNGICIDAL CONCENTRATION (MFC) OF AB OR A M E AGAINST S. CEREVISIAE IN THE PRESENCE OF FETAL BOVINE SERUM a Fetal Bovine Serum Concentration (%)

MFC AB (ttg per ml)

MFC AME (ttg per ml)

5

0.4

0.4

10

0.8

0.6

15

0.8

0.8

a MFC determined by plating out each tube at each dilution in Sabouraud's plates. When two out of the three plates from each dilution showed less than three colonies this was considered the MFC.

402

BONNER ET AL.

inating" organism and the MFC of AB and AME was demonstrated. C. albicans, S. cerevisiae and A. niger were eliminated from KB cultures grown in 5% or 10% FBS when treated with 0.625 /xg per ml of AB or 1.25 txg per ml of AME, whereas 5 p~g per ml of AB or 10 /.~g per ml of AME was required to kill F. moniliforme in KB cultures. The apparent discrepancies in the MFC's of AB and AME are not highly significant since in this assay, a twofold serial dilution assay, there is an inherent 50% error. For significance, more than a one-tube difference in antibiotic activity is necessary. DtscusstoN Ideally, tissue culture systems should be maintained without antibiotics. However, in the majority of cases it has been necessary to incorporate specific antibiotics in tissue culture medium to both prevent and eliminate bacterial, fungal and mycoplasmal contamination (1, 2). The most widely used antifungal antibiotic has been amphotericin B, usually administered as Fungizone ®. However, recent studies have indicated that Fungizone ® can be toxic (12-17) and cause permeability alterations (12, 17, 29-34) in tissue culture cells. These findings suggest that this polyene macrolide antibiotic should be used with caution and only when absolutely necessary, e.g. in the primary isolation of cells from high potential contamination sources, or in the elimination of contamination. In determining which type of polyene macrolide antibiotic is to be used in preventing or treating cell cultures contaminated with fungi or yeast, two important factors must be considered: (a) the maximum level of polyene macrolide antibiotic which will not alter the properties or kill the infected cell culture; and (b) the concentration of polyene macrolide required to inhibit or kill the contaminant. Recently, Mechlinski and Schaffner (23) synthesized a water soluble derivative of AB, amphotericin B methyl ester. AME has been found to: (a) exhibit similar antifungal activity as its parent AB (25, 28)~ (b) be less toxic than AB or Fungizone ® when administered intravenously in mice (25, 35) and dogs (35); (c) be less toxic than AB or Fungizone ® toward mammalian cells in culture (14-17); (d) exhibit good stability (14, 17, 36); and (e) induce less membrane damage than either AB or Fungizone ® as indicated by 51Cr release after 1 hr treatment of mammalian cells with similar or higher AME

levels (17). In the present study, AME was found to be less toxic than AB in media containing different concentrations of fetal bovine serum. As FBS concentration increased, the level of AB and AME required to induce a 50% reduction in 72-hr viability of KB cells also increased. This ability of sterols as present in FBS to antagonize the cytotoxicity of polyene antibiotics must be considered in determining the permissible level of polyene macrolides used in tissue culture systems containing different concentrations of FBS. Since different cell types will vary in their sensitivity to AB, AME and Fungizone ® in media containing 10% FBS (14-17) and the same cell type (KB) will also vary in sensitivity to AB and AME in media containing 2% to 20% FBS, the proper permissible level of polyene macrolide antibiotic will cleariy depend on: (a) the type of cell being used; (b) the number of initial cells being exposed to the polyene macrolide (12, 14); (c) the type of polyene macrolide antibiotic (14-17); and (d) the composition of the media, in particular the concentration of serum employed (18, 37). Other studies have indicated differences in the innate sensitivity (relative sensitivity under standard culture conditions) of various species of yeast and fungi to both AB and AME (28). In the present investigation, FBS was found to antagonize the in vitro antifungal effects of AB and AME. Higher levels of both parent and derivative antibiotic were required to inhibit the growth of S. eerevisiae by 50% and to kill S. cerevisiae (MFC) when employing 15% as opposed to 5% or 10% FBS in the growth medium. The levels of AB required to kill S. cerevisiae in 5% or 10% FBS were found to exhibit toxicity toward KB cells, whereas the MFC's of AME in similar concentrations of FBS did not induce toxicity. In addition, the level of AB required to eliminate C. atbicans, S. cerevisiae orA. niger from KB cultures was found to be toxic toward KB cells, whereas the MFC's of AME against these organisms were not as toxic toward KB cells. In the case o f F . moniliforme, the MFC of AB (5 /.~g per ml) resulted in total KB cell death in both 5% or 10% FBS, whereas AME at 10 gg per ml resulted in survival of KB cultures and elimination of this contaminant. These results indicate that AME is superior to AB for the treatment of C. albicans, S. cerevisiae, A. niger or F. moniliforme contamination of KB cell culture, since it exhibits reduced toxicity.

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This investigation was supported in part by contract N I H 69-2161, N I H grant no. AI-02095 and N I H training grant no. GM 507 from the National Institute of General Medical Sciences.

ERRATUM Erratum: A. L. Boynton, J. F. Whitfield and R. J. Isaacs. The Different Roles of S e r u m and Calcium in the Control of Proliferation of BALB/c 3T3 Mouse Cells. In Vitro 12: 120-123, 1976. The graphical presentation of figures 1 and 2 should be exchanged.