Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells I. Comparative in vitro cytotoxicity of 27 drugs

Toxicology Letters, 5 (1980) 299-307 o Elsevier/North-Holland Biomedical Press

299

PRELIMINARY STUDIES ON THE VALIDITY OF IN VITRO MEASUREMENT OF DRUG TOXICITY USING HeLa CELLS I. COMPARATIVE IN VITRO CYTOTOXICITY OF 27 DRUGS

BJGRN EKWALL and ALF JOHANSSON Department of Human Anatomy, S- 75123, Uppsala (Sweden)

University

of Uppsala, Biomedical

Centre, Box 571,

(Received March 26th, 1979) (Revision received December 12th, 1979) (Accepted December 20th, 1979)

SUMMARY

Combined drug toxicity to HeLa cells was studied in vitro with use of the microtitre MIT-24 test system. Whether drug toxicity to HeLa cells is representative of drug toxicity to other cultivated cells was investigated by a comparison of the MIT-24 toxicity of 27 drugs to HeLa cells with their toxicity to various permanent cell lines and more differentiated primary cell cultures as reported in the literature, together with original recordings of the MIT-24 toxicity of 9 of the drugs to human fetal kidney cells. A similarity of drug toxicity to all cell types was found. Thus the MIT-24 recordings may be representative of a basal drug cytotoxicity, probably corresponding to local drug irritation and to causal systemic drug toxicity.

In recent studies [l, 21 combined drug toxicity to HeLa cells was investigated in vitro by the Metabolic Inhibition Test supplemented by microscopy of cells after 24 h of incubation (the MIT-24 test). Two modifications of this test were used to assess the toxicity of 75 drug pairs, combined at random from 37 drugs. The methods were proposed for screening combined human systemic toxicity of drugs and chemicals with an MIT-24 toxicity relevant to human toxicity, as a supplement to costly whole animal tests. Since the value of the tests clearly depends on the relevance of the HeLa cytotoxicity of the drugs to their human toxicity, and since not much is known about this relevance [3], the present series of communications examines the relevance to human lethal action (the most precise endpoint of human systemic drug toxicity) of the HeLa cytotoxicity of combined drugs. As a first step of this examination, drug toxicity to HeLa cells in the MIT24 system was in this study compared with literature data on drug toxicity in vitro to various other cells and with MIT-24 recordings of drug toxicity to human kidney cells.

2000

40

1600

1500

1500

1000

1000

1000

14 TheophyUine

15 Phenol

16 Procaine

17 Tripelennamine

18 Colistimethate

20 Epinephrine

900

2000

13 Phenylbutasone

19 Thiotepa

200

2000

12 Nicotine

80

450

1500

1500

600

890

200

600

900

4200

3000

9 Nikethamide

4000

7000

4200

8 Lidocaine

1600

9300

5000

3700

10000

11 Caffeine

9300

8000

10000

5 Tubocurarine

7 Sulfisoxszole

19000

4 Alcohol

6 Benzyl alcohol

23000

3 Methotrexate

1300

89000

200000

32000

1 Gallamine

2 BenzyIpeniciIhn

168 h

IClOO

24 h

IClOO

24 h

720 900

1900

2800

1600

1700

1600

670 200 89 8.0 36

200

200

200

120

18

490

350

I70 cl00 490

440

53

890 cl00

400

380

120c100

50

0.5

1300

400

4200

4500

850

1700

2200

3100

5.7

8.5

27000 580

-

IC50

100

cl200

Cl000

cl 2000

lclooe

168 h 156 hd 24 h

IC50

6400

40000

IC50

25

c5000

0.01

IC50

72h

1700

870

IClOO

120h

1469

Mouse

KB

KB liver

Chang HeLa liver,

HeLa

Schmidt

SYSTEMSa

Smith

TISSUE CULTURE .._. Eagle Nitta

IN VARIOUS

MIT-24’

IN pglml OF 27 DRUGS

10 Chloramphenicol

-

No. Drug nameb

CYTOTOXICITY

TABLE I

60

cl00

cl00

IClOO

120 h

HeLa

Toplin

-

190

IClOOf _

18 h

cytoma

Masto-

Karrel

1000

1300

2000

3300

cl500

250000

50

cl500

10000

- 10000 150000

IClOO

48h

Heart

IClOO

36 h

Spleen

chicken embryos

Primary explants,

Pomerat

250

100

830

5000

6300

1500

1300

1Cl00

36 h

Cord

Hum.

420

1700

830

3300

~80000

10000

ICl.00

48h

Skin

expl.

8100

7600

IC50

kidney

monkey

cultures,

Primary

Metcalfe

30

0.2

26 Chlorpromazine

21 Ouabain

180 50

inhibition

0.018 0.005g

13

8.0

20

40

recorded

0.028

to use in Table III or previous reports [l,

19

gStrophantin

concentration.

K possessing 40% of Ouabain activity.

fThe described cytostatic

eThe described M.S.D. value, which totally inhibits propagation

period.

of subcultures from the tested cells.

toxicity. the maximal

(b). i.e. 0.2b. and the IClOO reported

mean value between

minimal injurious cont.

as the (geometrical)

62

16

name. IClOO or IC50, 100% or 50%

2 1. Drugs arranged in order of increasing 24 h MIT-24

step less than the reported

dA renewal of medium including drug three times within the incubation

IC50 = Jac = JO.Pbc.

not injuring cells (a), which is one dilution

(c), according to the formula:

5.2

32

210

are noted beneath investigator’s

45

values derived from Table III or previous reports Cl, 21. The IC50 was calculated

drug concentration

‘Toxicity

81 130 cl00

cut off by the researcher, i.e. 100 pg/ml or more.

names according

cont.; ~100, a toxicity

bAbbreviated.drug

inhibitory

0.04

9.5

10

20

60

180

180

time, and the percentage

0.04

30

20

80

89

400

aThe cell type used, the incubation

80

45

23 Quinine

24 Promethazine

200

22 Strychnine

25 Chloroquine

890

400

21 Methampyrone

16

302

In Table I the MIT-24 toxicity to HeLa cells of 27 drugs is compared with in vitro cytotoxicity of the same drugs recorded in 8 other studies [ 5-151, each of which was selected on grounds of a varied drug assortment which included at least three of the compared 27 drugs. In the eight studies selected, 13 of the combined 37 drugs [l, 2] were found (Nos. 4, 7, 9, 14, 16, 17, 18, 19, 21, 22, 24, 26, and 27). Prior to the combined testing [l, 21 about 50 drugs were separately tested in the MIT-24 system for their HeLa cytotoxicity, but were not used in the combined tests for a variety of reasons, e.g. similarity to an actually combined drug, expected human toxicity by drug interference with receptors certainly not found in tissue culture, precipitating tendency or a not suitably large difference between 24 h and 7 days’ toxicity. From this group 14 drugs were found to have been included either in one of the studies with more differentiated cells [ 12-151, and/or in two or more of the studies with permanent cell lines [ 5-111, and have been included in the analysis to widen the basis of comparison. Their toxicity to HeLa cells is found in Table III. In Table I studies of permanent cells have been placed to the left, while studies of one probably more differentiated cell line, i.e. the mastocytoma cells [4], and studies of primary cultures of explants have been placed to the right. In Tables I and II different variables in methods are presented to provide a basis for an evaluation of their influence on toxicity. Table IV shows the toxicity in the MIT-24 system of 9 randomly selected drugs to HeLa cells and third passage human fetal kidney cells. For all drugs except Nos. 6,9, 15,16, 24, and 26, increased incubation time in the MIT-24 system resulted in increased cytotoxicity (see Table I). Since all drugs tested by Nitta [ 6, 71 and by Karzel [ 121 belong to this category, the short incubation time used in their studies may have reduced the toxicity compared with that to equivalent cells recorded in other studies. The percentage inhibition estimated always affects toxicity. Drugs with an IClOO dissimilar to the IC50, e.g. drug No. 3, may be misinterpreted if IClOO [ll] is compared with IC50 [5,9]. The generally high toxicity recorded by Eagle and Foley [ 51 as well as by Smith et al. [ 91 may be explained by the recording of IC50 combined with a long incubation time (see drugs Nos. 10, 19, 23, and 27). This high toxicity may be further accentuated in Eagle’s and Foley’s study [ 51 by the repeated drug renewal, which may comparatively increase toxicity of drugs binding to cells. A small cell density of a method may also enhance toxicity of drugs binding to cells, such as Toplin’s [ 111 toxicity of drug No. 19. The serum content of the medium may protect cells from protein-binding drugs. The toxicity of protein interacting drugs, e.g. drugs Nos. 2, 4, 5, 7, 8, 10, 15, 18, 19, 20, 23, and 24, may be comparatively reduced in studies with a high serum contents of the medium [6, 7,10,14] . Cytotoxicity must also be influenced by a variation between studies of methods to determine cytotoxicity, endpoints of toxicity (inhibited outgrowth is not synonymous with metabolic inhibition or other injury), solvents, pH-adjustments, basal media, age of tested substances, and drug forms tested.

5 . lo4

Parker 199 5% serum

Microscopy pH-change

Human fetal kidney (Table IV)

1.2

Medium

Method(s) to determine cytoinhibition

Cell types used in the same system not described in Table I

Ref.

steps

Cells/ml at start of incubation

Dilution

5

saline DMSO glycerol medium suspension

Solvents

of drugs

52 varied

Number and type of drug

Adjustment to pH 7.4

MIT-24

CHARACTERIZATION

System

TABLE II FURTHER

5

5 other standard cell lines

Protein analysis (Folin)

5-10% serum

monolayer

10-5

-

medium solution suspension

190 anticancer

Eagle

OF STUDIES

6, 7

Microscopy Subculture

Hanks BSS 250 embryo extract 40% serum

monolayer

2

drug solutions

water alcohol methanol acetone isopropanol

32 antibiotic anticancer

Nitta

COMPARED

8. 9

-

Protein analysis (Folin) Microscopy

Eagle 10% serum

15 pg cell protein/ml

2

-

water alcohol DMF

112 antibiotic chemicals

Smith

IN TABLE

I

* 105

Connective tissue strain L

Purinepyrimidine analysis (McIntire) Microscopy

Earles BSS 20% embryo extract 40% serum

1.4

2

_

medium solution suspension

16 local anesthetics

Schmidt

Results in Table I based on many types

Ehrlich ascites permanent strain

Cell count (Coulter counter)

5 * lo5

Microscopy pH-change Subculture

lo3

Schindler 10% serum

*

drugs and medium

water, DMF medium solution HCl, NaOH ultrasound

18 antiinflammatory

Karzel

Eagle 10% serum

2.5

5

drugs and medium

saline HCl, NaOH alcohol methanol acetone

97 anticancer (=Eagle)

Toplin

Microscopy

BSS 25% embryo extract 50% plasma

explant

10-2

-

saline

110 varied

Pomerat

Microscopy DNA, RNA protein analysis (radio-label)

Eagle 10% serum

monolayer

DMSO DMF material evaporated on film

6 varied

Metcalfe

and

304 TABLE III TOXICITY

OF 15 DRUGS

Drug namea

TO HeLa

Minimal

CELLS

inhibitory

IN THE .~_

MIT-24

SYSTEM

drug concentrations,

in pg/mlb

Epinephrine bitartratee* f Caffeineg Hydroxyzine HCl, NF Methotrexate Nab Benzylpenicillin Na, M Chloramphenicolj Tubocurarine chloridek Gallamine triethiodide Lidocaine HCl Phenylbutazone, NFf,’ Quinine HCl, Mg Chloroquine phosphatef Nicotine, M Benzyl alcohol, Mm Phenol, M

Notes on not buffered or precipitating drugs

Microscopy after 24 h incubation

Indicator change, 7 days incubation

Full inhibition

Injury

Full inhibition

Injury

9.0*10Z 3.0*103 2.0*10’ 2.3 ~10~ 3.2~10~ 4.0~.10”

8.0 -10 3.0 *lo3 4.0-10 3.2 .lO-’ 6.4 *lo3 9.0*10*

8.0.10 6.0.10’ 2.0 *lo2 1.0 *lo4 1.3 *lo3 9.0 *lo2

80.10 1.2*102 9.0.10 3.2*10-’ 1.3s103 9.0 *lo*

7 -1

1.0*104 2.0~10~ 7.0.103 2.0.103 2.0 *lo*

1.0 *lo4 4.0s104 2.0v103 4.0~102 8.9 -10

5.0 *lo3 8.9 -lo4 2.0 *lo3 8.9.102 8.9 -10

5.0 alO3 4.0*104 2.0.10” 1.6.10 8.9.10

6.8

4.5.10 2.0*10” 9.3 -lo3 1.5*103

1.0~10 4.0*102 4.2~10~ 8.0*10’

2.0 -10 2.0*103 9.3 *lo3 1.5*103

8.0 2.0*103 9.3 -10” 8.0 ~10~

pHC

Max. cont. without precipitated

7.0 (5.5)’

5.0~102

6.5 6:2

8 05

6.0.10’

aDrug names refer to the United States Pharmacopoeia, the Merck Index (M), or the National Formulary of the United States (NF). bDrugs tested in the MIT-24 system by methods described previously [ 1, 21. Drugs not specifically indicated were tested twice in a 5 x 8 cup area as pure substances dissolved in saline, and produced identical toxicity in both tests. ‘The actual pH at the beginning of incubation for the minimal full inhibitory 24 h cont. Not indicated drugs were buffered by the medium (pH = 7.5) within the toxic concentration range. dThe maximal drug cont. free from precipitate for precipitating drugs. eA brown colour of the medium which progressed with time, indicating drug oxidation. fA variation of one or both values of IClOO between repetitive tests within the 0.45 X C2.2 x C range, where C is the mean value of IClOO between successive tests. sDrug dissolved in hot saline. hThe injurious cont. may be less than 0.0032 fig/ml. ‘A drug fully buffered by the medium at start of incubation, which got acid with time. jDrug tested as a glycerol suspension. See note 1. kDrug tested as glycerol and saline suspensions. See note 1. ‘Drug tested as dimethylsulphoxide (DMSO) solution. The glycerol and DMSO were proven to be non-toxic at toxic drug cont. m Drug tested as a medium suspension.

305 TABLE IV TOXICITY TO HUMAN EMBRYONAL IN THE MIT-24 SYSTEM Drug namea

Tested cellb

KIDNEY CELLS AND HeLa CELLS OF 9 DRUGS

Minimal inhibitory concentrations, Microscopy,

24 h

Full inhib. Injury

in pg/mlc

pH-change, 7 days Full inhib.

Injury

A. Epinephrine

HK HeLa

20 220

20 45

10 45

10 45

B. Theophylline

HK HeLa

3500 700

700 700

200 700

200 700

C. Hydroxyzine

HK HeLa

200 90

200 18

40 90

40 90

D. Papaverine

HK HeLa

800 360

800 6.4

160 360

160 360

E. Phenobarbital

HK HeLa

4000 1800

800 800

800 1800

800 800

F. Promethazine

HK HeLa

100 45

100 45

100 45

100 45

G. Imipramine

HK HeLa

50 50

50 50

50 50

50 50

H. Prilocaine

HK HeLa

2000 2000

400 2000

400 2000

400 2000

I. Phenylbutazone

HK HeLa

4000 4000

4000 800

800 800

800 160

aAbbreviated drug names according to usage in Table III or previous reports [ 1, 21. Drugs were tested as commercial vial solutions including preservatives and antioxidants proven to be nontoxic to cells at toxic drug concentrations. Drugs C, F, G, and H were tested as hydrochlorides, drug A as the bitartrate, drug B as the isopropanolamine, drug D as the sulphate, drug E as the free acid, and drug I as the sodium salt. bHK, Human embryonal kidney cells, tested as the third passage. The cells were taken from 5-month-old embryos and probably were to a high degree fibroblasts. ‘Each drug was tested once on each cell type according to methods described previously [ 11. Since the kidney cells do not round up in the suspension culture prior to incubation, they could not like HeLa cells be judged objectively on microscopic viability on grounds of fusiform appearance, but were arbitrarily judged on viability. Thus 24 h toxicity is not strictly comparable.

306

Highly specialized ceils appear to be more drug sensitive than less differentiated cells. In Pomerat’s study [ 141 cord cells show a higher toxicity than spleen cells. Mastocytoma cells are more sensitive than the other cell lines, as confirmed by the results from parallel tests in the same system by Karzel [12] of 18 drugs to these cells and to the probably less differentiated Ehrlich ascites cells, which indicated a 2-10 times increased drug toxicity to the mastocytoma cells. The relative toxicity of the compared drugs seems to be similar both to less (HeLa and spleen cells) and to more differentiated (mastocytoma and cord) cells, which indicates an equivalent drug action to both cell categories. On the whole there is a similarity of drug toxicity to all the various cells studied, which is more evident if the possible influence on toxicity from variations in methods is considered. This is consistent with the good correspondence of drug toxicity to HeLa cells with toxicity of the same drugs to human fetal kidney cells (Table IV). The similarity of drug toxicity to various permanent cell lines may be expected, since several investigators [ 5, 10, 161 have recorded approximately the same drug toxicity to different cell lines in the same system. Eagle and Foley [ 51 as well as Toplin [ 1 l] therefore used various standard cell lines in the: studies. Toplin [ 111 also found a good correspondence of his own results and those of Eagle and Foley for 90 drugs tested in the two different systems (Table II). The conformity of drug toxicity to permanent cells (HeLa) with drug toxicity to primary cultures of more differentiated cells, such as the human adult skin cells (Table I) and human fetal kidney cells (Table IV), does not appear to have been demonstrated before. The present evidence tentatively indicates a qualitatively similar toxic drug action to all cells, whatever their source or degree of differentiation, and would imply a relevance to local irritancy in the human body of the HeLa cytotoxicity. The finding therefore is in agreement with the reported high correlation between an agar overlay test for biomaterial cytotoxicity and the U.S. Pharmacopoeia rabbit muscle implant test for irritation [ 171. Schmidt [lo] found a good correlation between the toxicity to standard cell lines of 16 local anaesthetics and the rabbit intradermal irritant threshold for these drugs. ACKNOWLEDGEMENT

We are indebted to Prof. Daniel Acosta, Austin, Texas, for valuable discussions of this report. REFERENCES 1 B. Ekwall and B. Sandstrom, Combined toxicity to HeLa cells of 30 drug pairs, studied by a two-dimensional microtitre method, Toxicol. Lett., 2(1978) 285-292. 2 B. Ekwall and B. Sandstrom, Improved use of the Metabolic Inhibition Test to screen combined drug toxicity to HeLa cells - preliminary study of 61 drug pairs, Toxicol. Lett., 2 (1978) 293-298.

307 3 R.M. Nardone, Toxicity testing in vitro, in G.H. Rothblat and V.J. Cristofalo (Eds.), Growth, Nutrition, and Metabolism of Cells in Culture, Vol. III, Academic Press, New York, 1977. 4 R. Schindler, Margaret Day, and G.A. Fischer, Culture of neoplastic mast cells and their synthesis of 5-hydroxytryptamine and histamine in vitro, Cancer Res., 19 (1959) 47-51. 5 H. Eagle and G.E. Foley, Cytotoxicity in human cell cultures as a primary screen for the detection of anti-tumor agents, Cancer Res., 18 (1958) 1017-1025. 6 K. Nitta, Studies on the effects of actinomycetes products on the culture of human carcinoma cells (strain HeLa), I. The effect of known antibiotics having no or slight tumor-inhibitory activity on HeLa cells, Jap. J. Med. Sci. Biol., 10 (1957) 277-286. 7 K. Nitta, Studies on the effects of actinomycetes products on the culture of human carcinoma cells (strain HeLa), III. Comparative studies on anti-Hela-cell effects of known synthetic antitumor substances, Jap. J. Med. Sci. Biol., 10 (1957) 419-428. 8 C.G. Smith, W.L. Lummis, and J.E. Grady, An improved tissue culture assay, I. Methodology and cytotoxicity of anti-tumor agents, Cancer Res., 19 (1959) 843-846. 9 C.G. Smith, W.L. Lummis, and J.E. Grady, An improved tissue culture assay, II. Cytotoxicity studies with antibiotics, chemicals, and solvents, Cancer Res., 19 (1959) 847-852. 10 J.L. Schmidt, F.C. McIntire, D.L. Martin, M. Anita Hawthorne, and R.K. Richards, the relationship among different in vivo properties of local anesthetics and toxicity to cell cultures in vitro, Poxicol. Appl. Pharmacol., 1 (1959) 454-461. 11 I. Toplin, A tissue culture cytotoxicity test for large-scale cancer chemotherapy screening, Cancer Res., 19 (1959) 959-965. 12 K. Karzel, Der Einfluss von Antiphlogistica auf Lebens- und Vermehrungsfihigkeit normaler und neoplastischer Zellen in vitro, Arch. Inst. Pharmacodyn., 169 (1967) 70-82. 13 C.M. Pomerat, Action of chemical agents on living cells, Meth. Med. Res., 4 (1951) 260-280. 14 C.M. Pomerat and C.D. Leake, Short term cultures for drug assays: General considerations, Ann. N.Y. Acad. Sci., 58 (1954) 1110-1124. 15 Susan M. Metcalfe, Cell culture as a test system for toxicity, J. Pharm. Pharmacol., 23 (1971) 817-823. 16 H. Eagle and G.E. Foley, The cytotoxic action of carcinolytic agents in tissue culture, Am. J. Med., 21 (1956) 739-749. 17 S.A. Rosenbluth, G.R. Weddington, W.L. Guess, and J. Autian, Tissue culture method for screening toxicity of plastic materials to be used in medical practice, J. Pharm. Sci., 54 (1965) 156-159.