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The effects of prednisolone, indomethacin, and Aloe vera gel on tissue culture cells
Section
of the
Federal dental services
The effects of prednisolone, indomethacin, and Aloe Vera gel on tissue culture cells 77’. James Brmhe.r, Major, DC, USA,” E. R. Zinanzerntm~~~, D.D.S., M.A.,“’ ad C. K. Colli~gs, M.A., D.D.S.,“” Dallas, Tents BROOKE
GENERAL
BAYLOR
UNIVERSITY
HOSPIT~4L, COLLEGF,
PORT
S.431 HOUSTON,
TEXAS,
BND
OF DENTISTRY
P
rednisolone and Aloe 2rera gel have been used to reduce the symptoms and promote healing of inflammatory lesions of the oral cavity. Indomethacin is a relatively new nonsteroid drug with anti-inflammatory, analgesic, and antipyretic activity.* Most of the reports evaluating these agents have been concerned with clinical and animal experiments, Since several investigators have been able to demonstrate stimulation or retardation of growth of cells in tissue culture by certain therapeutic agents,2-4 we decided to investigate the effect.s of prednisolone, indomethacin, and Atoe veTa gel on tissue culture cells. Prednisolone is a potent cortieosteroid with anti-inflammatory action and other metabolic effects. Corticosteroids have been used in the treatment of inflammatory lesions of the oral mucous membrane, acute pulpal inflammation, and hypersensitive dentine and to minimize undesirable postoperative sequela.e.5-g The reported systemic effects of corticosteroids have caused many investigators to discourage their use in dentistry. lo Others have stated that topically applied corticosteroids produced none of t,he undesirable side effects of systemic administration.ll Most of the literature available on indomcthacin concerns clinical trials in the treatment of rheumatic diseases. Little information has been published regarding the possible effects of indomethacin on wound healing or on any oral From a thesis submitted by the senior autkor to the faculty of Baylor University College of Dentistry in partial fulfillment of the requirements for the M. 8. D. degree. This research was supported in part by grants from the Medical Research Foundation of Texas and the Procter and Gamble Company. *Periodontics Service, Department of Dentistry, Brooke General Hospital. **Baylor University College of Dentistry.
122
Volume 27 Number 1
EfSects of corticosteroids
on tissue culture
cells
123
tissue changes associated with therapeutic doses. Aloe vera gel is said to enhance healing in certain conditions. It is an extract prepared from the leaf of the Aloe Vera, which belongs to the Liliaceae plant family. These plants are typical perennial xerophytes with spiny cuticularized leaves similar to a cactus. A viscous watery gel is present in thin-walled tubular cells which run the long axis through the leaves. As a folk remedy, Aloe vera has been commonly used for many years for the treatment of burns, skin diseases, eye afflictions, and stomach and intestinal disorders in many countries.lzl I3 The first reported use of Moe vera gel in modern medicine was published by Collins and Collins14 in 1935. Their report, as well as several others published thereafter,15-20 concerned the use of Aloe vera gel in the treatment of radiation injuries. CreweZ1 attributed the following properties to’ extracts of Aloe Vera: (1) the relief of pain, burning, and itching; (2) an antiseptic action; and (3) stimulation of rapid granulation tissue formation. In 1953 Lushbaugh and Halez2 reported that the use of fresh Aloe vera gel in the treatment of experimental radiodermatitis of rabbits hastened both the degenerative and the regenerative phases of the lesions so that complete healing occurred in half the time required for untreated control lesions. MATERIALS AND METHODS Prednisolone, indomethacin, and Aloe vera gel were the agents tested in this study. Solutions of prednisolone and indomethacin were prepared by dissolving the powdered drugs in cell-maintenance medium. The liquid AZoe vera gel was diluted with the maintenance medium. Preliminary studies indicated that a 5 x 1O-4 solution of indomethacin, a 1 x 1O-3 solution of prednisolone, and a 5 x 10-l solution of Aloe vera gel were completely to’xic to the Gey strain of HeLa cells. These concentrations were chosen as the strongest solutions of the drugs to be used in the experimental study. From these concentrations of the drugs, serial dilutions were made to obtain a total of four dilutions of each test agent to be used in the study. HeLa cells of the Gey strain and rabbit kidney fibroblasts were used in this study. The HeLa cellsz3 were grown in a medium consisting of 0.5 per cent lactalbumin hydrolysate, 15 per cent calf serum, and 84.5 per cent Hanks’ balanced salt solution and were maintained on the same medium except that the concentrations of the last two solutions were changed to 10 per cent and 89.5 per cent, respectively. R.abbit kidney fibroblasts were grown and maintained in medium consisting of 0.5 per cent la&albumin hydrolysate, 5 per cent calf serum, and 94.5 per cent Hanks’ balanced salt solution. One hundred units of penicillin and 100 pg of streptomycin per milliliter were included in the media for the two types of cells, along with 1 ml, of a 0.2 per cent phenol red solution per 100 ml. of medium. Using techniques described by Scherer, Syverton, and Gey,24 we inoculated approximately 250,000 actively growing HeLa cells in 3 ml. of growth medium into l-ounce glass prescription bottles. The rabbit kidney fibroblast cells were inoculated directly from the source culture procedure into similar bottles. The bottles containing the cells were closed and incubated horizontally at 37’ C. The
\\‘hcn microscopic esaminatiol~ 0 1: t tI(l IWt tlw ~!Ollti~illirlg tltc cells lyy(:nl(:(l complctc monolayer formation, the growth medium in the bottles was replaced wit,11 the serially diluted solutions of the three test agents. Two bottles of each cell type for each of four dilutions of the three test, agents for 24, 48-, and 72 hour test periods \vere prepared as ~~11 as controls. At the end of these time periods, the ~11s were trypsinized and a 0.5 per cent solution of trypan blue was added to the cell suspension. After 3 minutes, 12 aliquots of both experimental and control cells were counted in a hemacytometer. The total cell count and the, ratio of viable to nonviable cells were determined. Cell viability was determined by the ion-exclusion principle.“” RESULTS
Measurement of the hydrogen ion concentration of the HeLa and rabbit kidney cell media a,t the beginnin g of the experiment and at the end of each test period revealed no pattern to the change in pH. Some of the solutions increased in pH while others decreased, with no apparent relation to the concentration of the test agent, in the medium. The lack of a consistent pattern in the change in pH of the solutions throughout the test period indicated that the increase or decrease in numbers of cells in each bottle was apparently not directly rclatcd to t,he increase or decrease in hydrogen ion concentration in each bottle. Tables I, II, and III show the mean viable cell counts of the two cell lines following expo’sure to various dilutions of the test agents for the three time periods, and Table IV presents a comparison of these counts. For the three dilutions of indomethacin and prcdnisolone which could be compared at the same time periods, the HeLa cell count,s were significantly higher at certain time
I. The mean viable cell count per milliliter of medium of cells exposed to dilutions of indomethacin for certain time periods (~2,500) Table
i-
-7'irnQ "..""
(hours)
Control
cells
Concentration
5 x10-4
10-4
neI;a
24 48 72
120.1 + 12.4” 181.7 + 17.5t 140.5 k 11.2
46.1 I! 6.3 2.3 2 0.5t Toxict: Rabbit
19.4 + 2.0
72
40.3 f 4.0 45.0 + 4.6
4.2 + 0.9 1.3 + 0.49 Toxic
of indomethacin 10-S
I
10-S
cc1l.s
105.1 i: 14.2 70.9 I! 8.1 70.7 2 6.8 kidney
115.7 + 12.5 95.7 2 11.3 206.3 5 20.5
163.6 T 16.5 130.0 2 16.3 121.6 2 13.4
37.8 + 3.6 51.3 + 5.op 45.4 + 5.8
44.5 A 6.7 56.8 2 6.15 31.2 + 3.0
cells
23.4 + 2.4 33.7 2 3.8 36.82 4.0
*Mean viable cell count k the standard error. tThe difference between the low mean and the high mean is significant at the 0.05 level of probability. $No viable cells present. $The difference between the low mean anal the two highest means is significant at the 0.05 level of probability.
Efects of corticosteroids
0% tissue culture cells
125
intervals in the bottles exposed to prednisolone than in the bottles exposed to indomethacin. The rabbit kidney cell counts were significantly higher at certain time intervals for those bottles of cells exposed to indomethacin than in the bottles exposed to prednisolone, although fewer significant differences could be demonstrated among the counts for the rabbit kidney cells tha.n for the HeLa cells. The mean viable cell counts of the bottles of HeLa cells which had been exposed to a 10e3 dilution of Aloe vera gel were significantly higher at all three time periods than were the counts of the bottles of HeLa cells which had been exposed to the same concentration of prednisolone. The mean viable cells counts of the bottles of rabbit kidney cells which had been exposed to a 1O-3 dilution of Aloe Vera gel were significantly higher at the 48- and 72-hour time periods than the counts of the bottles of rabbit kidney cells which had been exposed to the same dilution of prednisolone.
II. The mean viable cell count per milliliter of medium of cells exposed to dilutions of prednisolone for certain time periods (~2,500) Table
Concentration
Time (hours)
cells
Control
10-s
10-4
H&a 24 48 72
195.6 2 18.8* 180.0 + 19.3 192.1% 8.5t
24 48 72
19.4 f. 2.0 40.3 f 4.0 45.0 ? 4.6
I
cells 149.3 + 15.7 172.6 2 17.9 118.2 + 13.3t
66.2 2 8.7 34.3 + 3.6 1.6 + 0.4t Babbit 15.2 + 1.5 18.4% 2.0 21.1+ 2.4
of prednisolone
kidney cells 26.0 2 2.8 41.5 + 4.2 32.0 2 3.4
10-J
10-S
138.4 5 13.5 164.8 + 18.4 181.9 ? 17.lt
147.5 f 14.3 230.9 + 23.4 192.2 f 18.6t
25.0 2 4.0 35.9 + 6.3 37.8 t 4.4
23.8 5 2.7 32.5 +_3.3 35.6 2 3.6
*Mean viable cell count k the standard error. tThe differences between the lowest mean and the three highest means and between the second lowest and the highest mean are significant at the 0.05 level of probability.
Ill. The mean viable cell count per milliliter
Table
dilutions Time (how-s) 24 48 72 24 48 72
of medium of cells exposed to of Aloe VW~ gel for certain time periods (~2,500) Concentration Control
cells
213.9 + 19.9” 232.3 2 21.7 192.3 !I 18.5 :;:;
; fj
45.0 f 4:6
*Mean viable cell tNo viable cells $The differences are significant at the
5x10-1 Toxict Toxic Toxic
1
10-I
of Aloe veTa gel 10-2
HeLa cells 111.6 ? 10.6 63.6 + 6.3 17.6 ? 1.7$
Rabbit Toxic Toxic Toxic
kidney cells 17.2 I! 0.7 20.9 + 2.9 44.2 ? 4.2
count 2 the standard error. present. between the two low means and between 0.05 level of probability.
10-s
219.2 z! 20.0 187.9 2 19.6 194.5 + 18.9$
225.8 + 21.0 241.8 2 22.8 237.8 t 21.9t
36.3 k 4.0 38.8 + 3.7 42.7 k 4.4
31.3 2 3.3 50.7 +_5.3 66.4 t 6.6
the lowest
and highest
means
126
&mh.et,
Xintmctwcltr
ttl md
O.S., O.M. & 0.1’. .Januarv . 9 1969
Co%li~~~/s
Table IV. Comparison of the moan ~iablc ccl1 counts pc’r milliliter of medium of two cell lines following csposnrc t,o similar dilutions of test ilgc?lts for csrt:iin t,ime periods (~2,500) Time (how-s)
Test agent and
concentration Indomethacin PrednisoIone
10-b 10-d
Indomethacin Prednisolone
10-s 10-S
115.7 + 12.5 138.4 5 13.5
95.7 f: 11.3t 164.8 + 18.4t
206.3 + 20.5 181.9 f. 17.1
Indomethacin Prednisolone
10-G lo-6
163.6 2 16.5 147.5 + 14.3
130.0 2 16.3t 230.9 + 23.4t
121.6 5 13.4t 192.2 ? 18.6t
225.8 t 20.9 t 66.2 2 8.7 t
241.8 5 22.8t 34.3 + 3.6t
237.8 ? 21.9t 1.6 +_0.4t
Aloe vera gel 10-S Prednisolone 10-3 Indomethacin Prednisolone
10-i 10-b
Rabbit 23.4 + 2.4 26.0 t 2.8
Indomethacin Prednisolone
10-5 10-s
Indomethacin Prednisolone
10-e 10-e
Aloe ve-ra gel 10-s Prednisolone 10-3
48
__-----72
I HeLa cells 105.1 2 14.2x t 149.3 2 15.7t a4
70.9 + 8.1 t 172.6 F 17.9+
70.7? 6.8t 118.2 5 13.3t
kidney cells 33.7 5 3.8 41.5 + 4.2
36.8 ?: 4.0 32.0 + 3.4
37.8 t 3.6t 25.0 k 4.0t
51.3 2 5.0 35.9 + 6.3
45.3 2 5.8 37.8 + 4.4
44.5 + 6.7t 23.8 k 2.7t
56.8 It 6.lt 32.5 -c 3.3t
31.2 t 3.0 35.6 t 3.6
31.3 f 3.3 15.2 + 1.5
50.7 k 5.3t 18.4 + 2.0t
66.4 It 6.6 t 21.1 z! 2.4t
*Mean viable cell count + the standard tThe difference between the vertically probability.
error. paired
figures
is significant
at the 0.05 level
of
DISCUSSION
Most studies reported on the effects of anti-inflammatory agents on animals and human subjects pertain to the effects upon the animal as a whole, rather than effects at the cellular level. 1n vitro investigations of the effects of antiinflammatory agents and Aloe vera gel have not been reported. The use of an established strain of epithelial cells (HeLa) and a primary culture of fibroblastic cells offered the opportunity to observe the reactions of two different types’ of cells to the test agents. Because of the extreme sensitivity of tissue culture cells to changes in their environment, the drugs tested were diluted in medium used for growing and maintaining the cells before being added to the actively growing cells. When evaluating the effects of drugs on tissue culture cells, serial dilutions of the drugs should be used if effects other than complete toxicity are to be observed. When the cells were counted in the hemacytometer, the total viable and nonviable cells were counted separately. In computing the mean cell count for each dilution at each time period, the total number of viable cells only was considered. The nonviable cell count was not considered reliable, for as the cells degenerate and become nonviable the majority of them become detached from the surface and float in the medium which is poured off during the staining procedure. The mean viable cell counts from the bottles of HeLa cells were consistently much
Volume Number
27 1
Efects of corticosteroids
on tissue culture cells
127
higher than those from bottles of rabbit kidney cells, except for bottles in which toxicity resulted from exposure to the more concentrated solutions of the test agents. The differences in the numbers of the two types of cells in the bottles made direct comparison of the results difficult. Attempts were made to inoculate the same number of cells into each bottle during the experimental procedure. The reasons for the larger cell counts from the bottles of HeLa cells are not definitely known.26 It is interesting to note the apparently opposite effects of prednisolone and indomethacin on the two cell lines. From the data presented in Table IV, it may be seen that the HeLa cells exposed to prednisolone apparently grew more rapidly than did the HeLa cells exposed to indomethacin, while the rabbit kidney cells exposed to prednisolone apparently grew slower than did the rabbit kidney cells exposed to indomethacin. The data presented in Table IV suggest that prednisolone was more toxic to rabbit kidney cells and less toxic to HeLa cells than indomethacin and that AZoe Vera gel was less toxic to both cell types than prednisolone. This study was concerned with evaluating the toxicity of three test agents on tissue culture cells. The value of drugs in clinical treatment cannot be estimated until adequate in vivo studies have been conducted. SUMMARY Prednisolone, indomethacin, and Aloe ‘Uenctgel were tested for their effects on the Gey strain of HeLa cells and rabbit kidney fibroblasts. Actively growing monolayer cultures of HeLa cells and rabbit kidney fibroblasts were exposed to various concentrations of the three test agents. Toxicity of the test agents was estimated by counting the number of viable cells remaining at the end of three different time intervals. Results suggest that prednisolone was more toxic to rabbit kidney cells and less toxic to HeLa cells than indomethacin and that Aloe vera gel was less toxic to both types of cells than prednisolone. REFERENCES
1. Norcross, B. M.: Treatment of Connective Tissue Diseases With a New Non-steroidal Compound (Indomethacin). Arthritis & Rheumat. 6: 290, 1963. 2. Shafer, W.‘ G.: Effect of’Dilantin Sodium on Growth of Human Fibroblast-like Cell Cultures, Proc. Sot. Exper. Biol. & Med. 104: 198-201, 1960. 3. Kreth. K. K., Zimmermann. E. R., and Callings, C. K.: Effect of Periodontal Dressings ‘on Tissue Culture Cells, J. Periodont. 37: 48-53; i966. 4. De la Rosa, A. M.: Cytotoxicity of Mouthwashes in Tissue Culture Systems, Thesis submitted to Baylor University, Dallas, Texas, 1966. 5. Fisher, E. T.: Effects of Cortisone in Treatment of Gingivitis, Locally and Systemically Administered. With and Without Other Forms of Treatment, Northwestern Univ. Bull. 52: 13-20, 195’6. for the 6. Fry, A. E., Watkins! R. F., and Phatak, N. M.: T,opical Use of Corticosteroids Relief of Pain Sensitivity of Dentine and Pulp, ORAL SURG., ORAL MED. & ORAL PATH. 13: 594-597, 1960. 7. Bowers, G. M., and Elliott, J. R.: Topical Use of Prednisolone in Periodontics, J. Periodont. 35: 486488, 1964. and Corticosteroids in the Reduction ,of Post8. Stewart, G. G.: The Antihistamines operative Sequelae Following Endodontic Therapy, ORAL SURG., ORAL MED. & ORAL PATH. 9: 216-220, 1956. 9. Saad, L. J., and Swenson, H. M.: Corticosteroid and Periodontal Packs, J. Periodont. 36: 407-412, 1965. 10. Rendershot, L. C.: The Use of Adrenocorticosteroid Hormones in Dentistry, D. Clin. North America, pp. 503-512, July, 1963.
V. H.: The Gorticosteroids in the 1lanagement of Diseases of the Skin, Ann. 11. Witten, Xew York Acad. Se. 82: 9X3-992, 1959. 12. Bovik, E. G.: Aloe wera: Panacea or Oltl \Vives’ Tales? Texas 1). 1. 84: 13-16, 1966. Exploitation of Aloe Leaf Pulp, Econ. Bot. 15: 13. Morton, F.: Folk Uses and Commercial 311-319, 1961. 14. Collins. C. E.. and Collins. (1.: Roenteen 1)ermatitis Treated n’ith Fresh Whole Leaf of Bloc 2;&a, Am. J. Roentgenol. 33: X)6:97, 1935. 15. TVright, C. S.: Aloe rcrn in the Treatment of Roentgen Ulcers and Telangiectasis, .J. A: M. A. 106: 136:+136l, 19:{6. S6. Lovermarl, A. B.: J,eaf of ;lloc l‘c,‘e in Treatment of Roentgen Ray Ulcers; Report of Two Additional Cases, Arch. 1)ernmt. & Syph. 36: 838-843, 1937. 17. Fine, A., and Brown, S.: (lultivation ant1 (Xnical Application of ,410~ 2’6ro Leaf, Radiology 31: 735-736, 1938. 18. Mandeville, F. B.: AZoc I’C~(C in the Treatment of Radiation Ulcers of Mucous Membranes, Radiology 32: 598-599, 1939. 19. Rowe, T. I>.: Effect of Fresh Alec wcro Gel in Treatment of Third-Degree Roentgen Reaction on White Rats; Preliminary Report, J. ilm. Pharm. A. (Scient. Ed.) 29: 348350, 1940. 20. Rowe, T. B., Lovell, B. I<., and Parks, 1,. M.: Further Observations on the Use of Aloe we~a Leaf in the Treatment of Third-Degree X-ray Reactions, J. Am. Pharm. A. (Scient. Ed.) 30: 266-269, 1941. 21. Crewe, J. E.: The External Use of Aloes, Minnesota Med. 20: 670-673, 1937. 22. Lushbauah. C. C.. and Hale. B. R.: Exuerimental Acute Radiodermatitis Following Beta Irradiation. ‘V. Histopathological Stndy of the Mode of Action ‘of Therapy With Aloe wera, Cancer 6: 690-698, 1953. 23. Gey, G. O., Coffman, \V. I)., and Kubicek, M. T.: Tissue (:ulture Studies of the Proliferative Capacit,y of Cervical Carcinoma and Normal Epithelium, Cancer I&es. 12: 264-265, 1952. J. T., and Gey, G. 0.: Stutlies on Propagation In Vitro of 24. Scherer, W. F., Syverton, Poliomyelitis Viruses, ,T. Exper. Med. 97: 695-710, 1953. of Cell Viability, Proc. See. Exper. 25. Hanks, J. H., and Wallace, J. H.: Determination Biol. & Med. 98: 188-192, 1958. W. J.: The Efl’ect of Anti-inflammatory Agents on Tissue Culture Cells. 26. Brasher, Thesis submitted to Baylor University, Dallas, Texas, 1968.
of the
Federal dental services
The effects of prednisolone, indomethacin, and Aloe Vera gel on tissue culture cells 77’. James Brmhe.r, Major, DC, USA,” E. R. Zinanzerntm~~~, D.D.S., M.A.,“’ ad C. K. Colli~gs, M.A., D.D.S.,“” Dallas, Tents BROOKE
GENERAL
BAYLOR
UNIVERSITY
HOSPIT~4L, COLLEGF,
PORT
S.431 HOUSTON,
TEXAS,
BND
OF DENTISTRY
P
rednisolone and Aloe 2rera gel have been used to reduce the symptoms and promote healing of inflammatory lesions of the oral cavity. Indomethacin is a relatively new nonsteroid drug with anti-inflammatory, analgesic, and antipyretic activity.* Most of the reports evaluating these agents have been concerned with clinical and animal experiments, Since several investigators have been able to demonstrate stimulation or retardation of growth of cells in tissue culture by certain therapeutic agents,2-4 we decided to investigate the effect.s of prednisolone, indomethacin, and Atoe veTa gel on tissue culture cells. Prednisolone is a potent cortieosteroid with anti-inflammatory action and other metabolic effects. Corticosteroids have been used in the treatment of inflammatory lesions of the oral mucous membrane, acute pulpal inflammation, and hypersensitive dentine and to minimize undesirable postoperative sequela.e.5-g The reported systemic effects of corticosteroids have caused many investigators to discourage their use in dentistry. lo Others have stated that topically applied corticosteroids produced none of t,he undesirable side effects of systemic administration.ll Most of the literature available on indomcthacin concerns clinical trials in the treatment of rheumatic diseases. Little information has been published regarding the possible effects of indomethacin on wound healing or on any oral From a thesis submitted by the senior autkor to the faculty of Baylor University College of Dentistry in partial fulfillment of the requirements for the M. 8. D. degree. This research was supported in part by grants from the Medical Research Foundation of Texas and the Procter and Gamble Company. *Periodontics Service, Department of Dentistry, Brooke General Hospital. **Baylor University College of Dentistry.
122
Volume 27 Number 1
EfSects of corticosteroids
on tissue culture
cells
123
tissue changes associated with therapeutic doses. Aloe vera gel is said to enhance healing in certain conditions. It is an extract prepared from the leaf of the Aloe Vera, which belongs to the Liliaceae plant family. These plants are typical perennial xerophytes with spiny cuticularized leaves similar to a cactus. A viscous watery gel is present in thin-walled tubular cells which run the long axis through the leaves. As a folk remedy, Aloe vera has been commonly used for many years for the treatment of burns, skin diseases, eye afflictions, and stomach and intestinal disorders in many countries.lzl I3 The first reported use of Moe vera gel in modern medicine was published by Collins and Collins14 in 1935. Their report, as well as several others published thereafter,15-20 concerned the use of Aloe vera gel in the treatment of radiation injuries. CreweZ1 attributed the following properties to’ extracts of Aloe Vera: (1) the relief of pain, burning, and itching; (2) an antiseptic action; and (3) stimulation of rapid granulation tissue formation. In 1953 Lushbaugh and Halez2 reported that the use of fresh Aloe vera gel in the treatment of experimental radiodermatitis of rabbits hastened both the degenerative and the regenerative phases of the lesions so that complete healing occurred in half the time required for untreated control lesions. MATERIALS AND METHODS Prednisolone, indomethacin, and Aloe vera gel were the agents tested in this study. Solutions of prednisolone and indomethacin were prepared by dissolving the powdered drugs in cell-maintenance medium. The liquid AZoe vera gel was diluted with the maintenance medium. Preliminary studies indicated that a 5 x 1O-4 solution of indomethacin, a 1 x 1O-3 solution of prednisolone, and a 5 x 10-l solution of Aloe vera gel were completely to’xic to the Gey strain of HeLa cells. These concentrations were chosen as the strongest solutions of the drugs to be used in the experimental study. From these concentrations of the drugs, serial dilutions were made to obtain a total of four dilutions of each test agent to be used in the study. HeLa cells of the Gey strain and rabbit kidney fibroblasts were used in this study. The HeLa cellsz3 were grown in a medium consisting of 0.5 per cent lactalbumin hydrolysate, 15 per cent calf serum, and 84.5 per cent Hanks’ balanced salt solution and were maintained on the same medium except that the concentrations of the last two solutions were changed to 10 per cent and 89.5 per cent, respectively. R.abbit kidney fibroblasts were grown and maintained in medium consisting of 0.5 per cent la&albumin hydrolysate, 5 per cent calf serum, and 94.5 per cent Hanks’ balanced salt solution. One hundred units of penicillin and 100 pg of streptomycin per milliliter were included in the media for the two types of cells, along with 1 ml, of a 0.2 per cent phenol red solution per 100 ml. of medium. Using techniques described by Scherer, Syverton, and Gey,24 we inoculated approximately 250,000 actively growing HeLa cells in 3 ml. of growth medium into l-ounce glass prescription bottles. The rabbit kidney fibroblast cells were inoculated directly from the source culture procedure into similar bottles. The bottles containing the cells were closed and incubated horizontally at 37’ C. The
\\‘hcn microscopic esaminatiol~ 0 1: t tI(l IWt tlw ~!Ollti~illirlg tltc cells lyy(:nl(:(l complctc monolayer formation, the growth medium in the bottles was replaced wit,11 the serially diluted solutions of the three test agents. Two bottles of each cell type for each of four dilutions of the three test, agents for 24, 48-, and 72 hour test periods \vere prepared as ~~11 as controls. At the end of these time periods, the ~11s were trypsinized and a 0.5 per cent solution of trypan blue was added to the cell suspension. After 3 minutes, 12 aliquots of both experimental and control cells were counted in a hemacytometer. The total cell count and the, ratio of viable to nonviable cells were determined. Cell viability was determined by the ion-exclusion principle.“” RESULTS
Measurement of the hydrogen ion concentration of the HeLa and rabbit kidney cell media a,t the beginnin g of the experiment and at the end of each test period revealed no pattern to the change in pH. Some of the solutions increased in pH while others decreased, with no apparent relation to the concentration of the test agent, in the medium. The lack of a consistent pattern in the change in pH of the solutions throughout the test period indicated that the increase or decrease in numbers of cells in each bottle was apparently not directly rclatcd to t,he increase or decrease in hydrogen ion concentration in each bottle. Tables I, II, and III show the mean viable cell counts of the two cell lines following expo’sure to various dilutions of the test agents for the three time periods, and Table IV presents a comparison of these counts. For the three dilutions of indomethacin and prcdnisolone which could be compared at the same time periods, the HeLa cell count,s were significantly higher at certain time
I. The mean viable cell count per milliliter of medium of cells exposed to dilutions of indomethacin for certain time periods (~2,500) Table
i-
-7'irnQ "..""
(hours)
Control
cells
Concentration
5 x10-4
10-4
neI;a
24 48 72
120.1 + 12.4” 181.7 + 17.5t 140.5 k 11.2
46.1 I! 6.3 2.3 2 0.5t Toxict: Rabbit
19.4 + 2.0
72
40.3 f 4.0 45.0 + 4.6
4.2 + 0.9 1.3 + 0.49 Toxic
of indomethacin 10-S
I
10-S
cc1l.s
105.1 i: 14.2 70.9 I! 8.1 70.7 2 6.8 kidney
115.7 + 12.5 95.7 2 11.3 206.3 5 20.5
163.6 T 16.5 130.0 2 16.3 121.6 2 13.4
37.8 + 3.6 51.3 + 5.op 45.4 + 5.8
44.5 A 6.7 56.8 2 6.15 31.2 + 3.0
cells
23.4 + 2.4 33.7 2 3.8 36.82 4.0
*Mean viable cell count k the standard error. tThe difference between the low mean and the high mean is significant at the 0.05 level of probability. $No viable cells present. $The difference between the low mean anal the two highest means is significant at the 0.05 level of probability.
Efects of corticosteroids
0% tissue culture cells
125
intervals in the bottles exposed to prednisolone than in the bottles exposed to indomethacin. The rabbit kidney cell counts were significantly higher at certain time intervals for those bottles of cells exposed to indomethacin than in the bottles exposed to prednisolone, although fewer significant differences could be demonstrated among the counts for the rabbit kidney cells tha.n for the HeLa cells. The mean viable cell counts of the bottles of HeLa cells which had been exposed to a 10e3 dilution of Aloe vera gel were significantly higher at all three time periods than were the counts of the bottles of HeLa cells which had been exposed to the same concentration of prednisolone. The mean viable cells counts of the bottles of rabbit kidney cells which had been exposed to a 1O-3 dilution of Aloe Vera gel were significantly higher at the 48- and 72-hour time periods than the counts of the bottles of rabbit kidney cells which had been exposed to the same dilution of prednisolone.
II. The mean viable cell count per milliliter of medium of cells exposed to dilutions of prednisolone for certain time periods (~2,500) Table
Concentration
Time (hours)
cells
Control
10-s
10-4
H&a 24 48 72
195.6 2 18.8* 180.0 + 19.3 192.1% 8.5t
24 48 72
19.4 f. 2.0 40.3 f 4.0 45.0 ? 4.6
I
cells 149.3 + 15.7 172.6 2 17.9 118.2 + 13.3t
66.2 2 8.7 34.3 + 3.6 1.6 + 0.4t Babbit 15.2 + 1.5 18.4% 2.0 21.1+ 2.4
of prednisolone
kidney cells 26.0 2 2.8 41.5 + 4.2 32.0 2 3.4
10-J
10-S
138.4 5 13.5 164.8 + 18.4 181.9 ? 17.lt
147.5 f 14.3 230.9 + 23.4 192.2 f 18.6t
25.0 2 4.0 35.9 + 6.3 37.8 t 4.4
23.8 5 2.7 32.5 +_3.3 35.6 2 3.6
*Mean viable cell count k the standard error. tThe differences between the lowest mean and the three highest means and between the second lowest and the highest mean are significant at the 0.05 level of probability.
Ill. The mean viable cell count per milliliter
Table
dilutions Time (how-s) 24 48 72 24 48 72
of medium of cells exposed to of Aloe VW~ gel for certain time periods (~2,500) Concentration Control
cells
213.9 + 19.9” 232.3 2 21.7 192.3 !I 18.5 :;:;
; fj
45.0 f 4:6
*Mean viable cell tNo viable cells $The differences are significant at the
5x10-1 Toxict Toxic Toxic
1
10-I
of Aloe veTa gel 10-2
HeLa cells 111.6 ? 10.6 63.6 + 6.3 17.6 ? 1.7$
Rabbit Toxic Toxic Toxic
kidney cells 17.2 I! 0.7 20.9 + 2.9 44.2 ? 4.2
count 2 the standard error. present. between the two low means and between 0.05 level of probability.
10-s
219.2 z! 20.0 187.9 2 19.6 194.5 + 18.9$
225.8 + 21.0 241.8 2 22.8 237.8 t 21.9t
36.3 k 4.0 38.8 + 3.7 42.7 k 4.4
31.3 2 3.3 50.7 +_5.3 66.4 t 6.6
the lowest
and highest
means
126
&mh.et,
Xintmctwcltr
ttl md
O.S., O.M. & 0.1’. .Januarv . 9 1969
Co%li~~~/s
Table IV. Comparison of the moan ~iablc ccl1 counts pc’r milliliter of medium of two cell lines following csposnrc t,o similar dilutions of test ilgc?lts for csrt:iin t,ime periods (~2,500) Time (how-s)
Test agent and
concentration Indomethacin PrednisoIone
10-b 10-d
Indomethacin Prednisolone
10-s 10-S
115.7 + 12.5 138.4 5 13.5
95.7 f: 11.3t 164.8 + 18.4t
206.3 + 20.5 181.9 f. 17.1
Indomethacin Prednisolone
10-G lo-6
163.6 2 16.5 147.5 + 14.3
130.0 2 16.3t 230.9 + 23.4t
121.6 5 13.4t 192.2 ? 18.6t
225.8 t 20.9 t 66.2 2 8.7 t
241.8 5 22.8t 34.3 + 3.6t
237.8 ? 21.9t 1.6 +_0.4t
Aloe vera gel 10-S Prednisolone 10-3 Indomethacin Prednisolone
10-i 10-b
Rabbit 23.4 + 2.4 26.0 t 2.8
Indomethacin Prednisolone
10-5 10-s
Indomethacin Prednisolone
10-e 10-e
Aloe ve-ra gel 10-s Prednisolone 10-3
48
__-----72
I HeLa cells 105.1 2 14.2x t 149.3 2 15.7t a4
70.9 + 8.1 t 172.6 F 17.9+
70.7? 6.8t 118.2 5 13.3t
kidney cells 33.7 5 3.8 41.5 + 4.2
36.8 ?: 4.0 32.0 + 3.4
37.8 t 3.6t 25.0 k 4.0t
51.3 2 5.0 35.9 + 6.3
45.3 2 5.8 37.8 + 4.4
44.5 + 6.7t 23.8 k 2.7t
56.8 It 6.lt 32.5 -c 3.3t
31.2 t 3.0 35.6 t 3.6
31.3 f 3.3 15.2 + 1.5
50.7 k 5.3t 18.4 + 2.0t
66.4 It 6.6 t 21.1 z! 2.4t
*Mean viable cell count + the standard tThe difference between the vertically probability.
error. paired
figures
is significant
at the 0.05 level
of
DISCUSSION
Most studies reported on the effects of anti-inflammatory agents on animals and human subjects pertain to the effects upon the animal as a whole, rather than effects at the cellular level. 1n vitro investigations of the effects of antiinflammatory agents and Aloe vera gel have not been reported. The use of an established strain of epithelial cells (HeLa) and a primary culture of fibroblastic cells offered the opportunity to observe the reactions of two different types’ of cells to the test agents. Because of the extreme sensitivity of tissue culture cells to changes in their environment, the drugs tested were diluted in medium used for growing and maintaining the cells before being added to the actively growing cells. When evaluating the effects of drugs on tissue culture cells, serial dilutions of the drugs should be used if effects other than complete toxicity are to be observed. When the cells were counted in the hemacytometer, the total viable and nonviable cells were counted separately. In computing the mean cell count for each dilution at each time period, the total number of viable cells only was considered. The nonviable cell count was not considered reliable, for as the cells degenerate and become nonviable the majority of them become detached from the surface and float in the medium which is poured off during the staining procedure. The mean viable cell counts from the bottles of HeLa cells were consistently much
Volume Number
27 1
Efects of corticosteroids
on tissue culture cells
127
higher than those from bottles of rabbit kidney cells, except for bottles in which toxicity resulted from exposure to the more concentrated solutions of the test agents. The differences in the numbers of the two types of cells in the bottles made direct comparison of the results difficult. Attempts were made to inoculate the same number of cells into each bottle during the experimental procedure. The reasons for the larger cell counts from the bottles of HeLa cells are not definitely known.26 It is interesting to note the apparently opposite effects of prednisolone and indomethacin on the two cell lines. From the data presented in Table IV, it may be seen that the HeLa cells exposed to prednisolone apparently grew more rapidly than did the HeLa cells exposed to indomethacin, while the rabbit kidney cells exposed to prednisolone apparently grew slower than did the rabbit kidney cells exposed to indomethacin. The data presented in Table IV suggest that prednisolone was more toxic to rabbit kidney cells and less toxic to HeLa cells than indomethacin and that AZoe Vera gel was less toxic to both cell types than prednisolone. This study was concerned with evaluating the toxicity of three test agents on tissue culture cells. The value of drugs in clinical treatment cannot be estimated until adequate in vivo studies have been conducted. SUMMARY Prednisolone, indomethacin, and Aloe ‘Uenctgel were tested for their effects on the Gey strain of HeLa cells and rabbit kidney fibroblasts. Actively growing monolayer cultures of HeLa cells and rabbit kidney fibroblasts were exposed to various concentrations of the three test agents. Toxicity of the test agents was estimated by counting the number of viable cells remaining at the end of three different time intervals. Results suggest that prednisolone was more toxic to rabbit kidney cells and less toxic to HeLa cells than indomethacin and that Aloe vera gel was less toxic to both types of cells than prednisolone. REFERENCES
1. Norcross, B. M.: Treatment of Connective Tissue Diseases With a New Non-steroidal Compound (Indomethacin). Arthritis & Rheumat. 6: 290, 1963. 2. Shafer, W.‘ G.: Effect of’Dilantin Sodium on Growth of Human Fibroblast-like Cell Cultures, Proc. Sot. Exper. Biol. & Med. 104: 198-201, 1960. 3. Kreth. K. K., Zimmermann. E. R., and Callings, C. K.: Effect of Periodontal Dressings ‘on Tissue Culture Cells, J. Periodont. 37: 48-53; i966. 4. De la Rosa, A. M.: Cytotoxicity of Mouthwashes in Tissue Culture Systems, Thesis submitted to Baylor University, Dallas, Texas, 1966. 5. Fisher, E. T.: Effects of Cortisone in Treatment of Gingivitis, Locally and Systemically Administered. With and Without Other Forms of Treatment, Northwestern Univ. Bull. 52: 13-20, 195’6. for the 6. Fry, A. E., Watkins! R. F., and Phatak, N. M.: T,opical Use of Corticosteroids Relief of Pain Sensitivity of Dentine and Pulp, ORAL SURG., ORAL MED. & ORAL PATH. 13: 594-597, 1960. 7. Bowers, G. M., and Elliott, J. R.: Topical Use of Prednisolone in Periodontics, J. Periodont. 35: 486488, 1964. and Corticosteroids in the Reduction ,of Post8. Stewart, G. G.: The Antihistamines operative Sequelae Following Endodontic Therapy, ORAL SURG., ORAL MED. & ORAL PATH. 9: 216-220, 1956. 9. Saad, L. J., and Swenson, H. M.: Corticosteroid and Periodontal Packs, J. Periodont. 36: 407-412, 1965. 10. Rendershot, L. C.: The Use of Adrenocorticosteroid Hormones in Dentistry, D. Clin. North America, pp. 503-512, July, 1963.
V. H.: The Gorticosteroids in the 1lanagement of Diseases of the Skin, Ann. 11. Witten, Xew York Acad. Se. 82: 9X3-992, 1959. 12. Bovik, E. G.: Aloe wera: Panacea or Oltl \Vives’ Tales? Texas 1). 1. 84: 13-16, 1966. Exploitation of Aloe Leaf Pulp, Econ. Bot. 15: 13. Morton, F.: Folk Uses and Commercial 311-319, 1961. 14. Collins. C. E.. and Collins. (1.: Roenteen 1)ermatitis Treated n’ith Fresh Whole Leaf of Bloc 2;&a, Am. J. Roentgenol. 33: X)6:97, 1935. 15. TVright, C. S.: Aloe rcrn in the Treatment of Roentgen Ulcers and Telangiectasis, .J. A: M. A. 106: 136:+136l, 19:{6. S6. Lovermarl, A. B.: J,eaf of ;lloc l‘c,‘e in Treatment of Roentgen Ray Ulcers; Report of Two Additional Cases, Arch. 1)ernmt. & Syph. 36: 838-843, 1937. 17. Fine, A., and Brown, S.: (lultivation ant1 (Xnical Application of ,410~ 2’6ro Leaf, Radiology 31: 735-736, 1938. 18. Mandeville, F. B.: AZoc I’C~(C in the Treatment of Radiation Ulcers of Mucous Membranes, Radiology 32: 598-599, 1939. 19. Rowe, T. I>.: Effect of Fresh Alec wcro Gel in Treatment of Third-Degree Roentgen Reaction on White Rats; Preliminary Report, J. ilm. Pharm. A. (Scient. Ed.) 29: 348350, 1940. 20. Rowe, T. B., Lovell, B. I<., and Parks, 1,. M.: Further Observations on the Use of Aloe we~a Leaf in the Treatment of Third-Degree X-ray Reactions, J. Am. Pharm. A. (Scient. Ed.) 30: 266-269, 1941. 21. Crewe, J. E.: The External Use of Aloes, Minnesota Med. 20: 670-673, 1937. 22. Lushbauah. C. C.. and Hale. B. R.: Exuerimental Acute Radiodermatitis Following Beta Irradiation. ‘V. Histopathological Stndy of the Mode of Action ‘of Therapy With Aloe wera, Cancer 6: 690-698, 1953. 23. Gey, G. O., Coffman, \V. I)., and Kubicek, M. T.: Tissue (:ulture Studies of the Proliferative Capacit,y of Cervical Carcinoma and Normal Epithelium, Cancer I&es. 12: 264-265, 1952. J. T., and Gey, G. 0.: Stutlies on Propagation In Vitro of 24. Scherer, W. F., Syverton, Poliomyelitis Viruses, ,T. Exper. Med. 97: 695-710, 1953. of Cell Viability, Proc. See. Exper. 25. Hanks, J. H., and Wallace, J. H.: Determination Biol. & Med. 98: 188-192, 1958. W. J.: The Efl’ect of Anti-inflammatory Agents on Tissue Culture Cells. 26. Brasher, Thesis submitted to Baylor University, Dallas, Texas, 1968.