Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells II. Drug toxicity in the MIT-24 system compared with mouse and human lethal dosage of 52 drugs

Toxicology

Letters,

5 (1980)

o Elsevier/North-Holland

309-317

309

Biomedical Press

PRELIMINARY STUDIES ON THE VALIDITY OF IN VITRO MEASUREMENT OF DRUG TOXICITY USING HeLa CELLS II. DRUG TOXICITY IN THE MIT-24 SYSTEM COMPARED WITH MOUSE AND HUMAN LETHAL DOSAGE OF 52 DRUGS

BJ6RN EKWALL Department of Human Anatomy, S-75123, Uppsala (Sweden)

University

of Uppsala,

Biomedical

Centre,

Box

571,

(Received April 17th, 1979) (Revision received December 12th, 1979) (Accepted December 20th, 1979)

SUMMARY

By a comparison of the 50% inhibitory concentration to HeLa cells in the microtitre Metabolic Inhibition Test supplemented by microscopy of cells after 24 h incubation (the MIT-24 test), of 52 drugs with their mouse i.v. LD50 and available approximate human lethal dosage, 7 were found to have a human LD considerably lower than HeLa IC50, indicating a lethal action to specialized functions of the human body not found in vitro, while 39 were found to have a gross similarity between the human LD and HeLa IC50. INTRODUCTION

The MIT-24 test has been used to test combined toxicity to HeLa cells in vitro of 75 drug pairs combined at random from 37 common drugs [l, 21. The methods have been proposed for screening [ 21. At present, little is known of the relevance of in vitro toxicity to human toxicity of drugs [3]. Since the value of the combined tests [l, 21 depends entirely on such a relevance, this series of papers tries to examine the correlation between in vitro and in vivo toxicity. Previously [ 41 the MIT-24 toxicity to HeLa cells of 13 combined [ 1, 21 and 15 other drugs has been compared with the in vitro toxicity of the same drugs to various other cells. A similarity of the toxicity to all cell types was found, indicating a similar basal cytotoxic action to all cells irrespective of their degree of differentiation, In the present paper, the MIT-24 toxicity to HeLa cells of the 37 combined drugs [l, 21 and the 15 other drugs [4] is compared with mouse i.v. LD50 and with available approximations of human lethal dosage. The need for a systematic comparison of in vitro and in vivo toxic dosage

310

for a varied drug choice is emphasized by the scarcity of previous studies [3]. Two studies have reported a good correspondence between in vitro cytotoxicity and mouse or human systemic toxicity for small groups of related anesthetic drugs [ 5, 61. Smith et al. [ 71 compared KB cell toxicity and i.p. LD50 for mice of 37 antibiotics and miscellaneous chemicals, and found a weak positive correlation between the variables. Variation of the relation between in vitro and in vivo toxic dosage was not, however, related to different systemic drug toxicity. METHODS

In Tables I and II several categories of data thought to be relevant for comparison of HeLa toxicity with systemic mouse and human toxicity of 52 drugs have been listed. Table I presents such data for the combined 37 drugs [l, 21. The selection is biased in respect to human cytotoxic drug action, since some of the drugs were once included in the combined tests because of a suspected or known cytotoxicity (drugs Nos. 1,2,18,19,21,23, and 37). Table II presents data for 15 drugs which were analysed on their comparative in vitro cytotoxicity in the foregoing study [4] . Together, the 52 drugs are a fairly random sample. The 50% inhibitory drug concentrations (IC50) to HeLa cells in the MIT24 system derive (see note b, Tables I and II) from published IClOO [l, 2, 41. IC50 is clearly a concentration, but may also be considered to be a drug dose (pg/g) to the MIT-24 system. Based on the imprecise peroral human lethal dosage and the mouse p.o./i.v. ratio, an approximated human i.v. LD was calculated, and from it a calculated i.u. lethal concen t&ion (c.i.v. LC) was derived by dividing the i.v. LD by the volume of distribution (Vd) of 0.15 l/kg for a drug distributed in human extracellular water (10.5 1). The c.i.v. LC may be correct for drugs dispersed in ECV (0 and Oe in TAcolumn, Tables I and II), but is for other drugs rather a dose of the drug to ECV and its cellular contents. To supplement the c.i.v.LC, actual lethal blood and liver concentrations from the practice of forensic medicine have been included. These often derive from a small number of cases and include deaths from unknown, variable or excessive dosage, with a varied time lag between intake and analysis [17]. Available information on human tissue accumulation and blood protein binding is presented, since these factors probably influence the comparability of in vitro with in vivo dosage. The MIT-24 is a static, one-compartment system, in which a medium containing 5% serum holds less than a promille cells/water volume [l, 21. If a drug is not metabolized by the few cells or otherwise broken down (hydrolysis), drug action lasts 24 h or 7 days. The human body is a dynamic, multicompartmental system, in which an extracellular water containing more than 20% serum holds about 50% cells/water volume. Drug action in the human

311

body is not constant, since drugs may be eliminated, redistributed, and metabolized to less or more toxic compounds by the considerable number of cells (including transformations of drugs by liver cells probably not operating efficiently in HeLa cells). Of these incongruencies between the systems, differential cell density may be the most important. An equivalent toxicity of a drug dose accumulating in cells ought to be accentuated in the MIT-24 system by its lesser cell density compared to the human body. Also the smaller extracellular protein content of the MIT-24 system ought to accentuate otherwise equivalent toxicity of drugs preferentially binding to extracellular protein. RESULTS

Human LD and the calculated human i.v. lethal concentration are considerably lower than the 24 h and 7 days’ HeLa IC50 for 7 (Nos. 14, 20, 25, 38, 40, 46 and 48) of the 52 compared drugs (Tables I and II). Drugs Nos. 20, 38, 40, 46 and 48 also have a low mouse LD50 compared with their HeLa IC50. For the remaining 39 drugs with a reported human lethal dosage, there is a statistically significant (r = 0.46, P < 0.01) positive correlation between the human LD and the 7 days’ HeLa IC50, seen by the roughly parallel decrease of both variables in Tables I and II. While the human i.v. LD is generally 5-10 times smaller than HeLa IC50, the calculated human i.v. LC has about the same magnitude as HeLa IC50. For 47 drugs the mouse i.v. LD shows a good correspondence to HeLa IC50, including the 6 drugs without a reported human LD. Thus the comparison of in vitro and in vivo dosage separates two drug groups: One small group with a clear discrepancy of compared dosage, and one large group with a gross similarity of dosage. DISCUSSION

A good correspondence between increasing HeLa toxicity and increasing tissue accr_mulation (t,T, and TT) as well as blood protein binding (p,P and PP) is seen for most drugs, the exceptions being Nos. 22, 31, 47, and 52 in the case of tissue accumulation, and Nos. 12, 13, 19, 37, and 52 in the case of protein binding. This indicates that these factors influence or even cause cytotoxicity, perhaps by an intracellular protein denaturation. For the 45 drugs with a crude similarity between in vitro and in vivo dosage the successive increase of the HeLa toxicity, proportional to drug tissue accumulation and protein binding, is not exactly parallelled by the increase of mouse and/or human toxicity. Instead, a gradual increase of human LD compared to the HeLa IC50, proportional to increased toxicity and drug accumulation, is seen, which is probably an effect of the higher cell density of the human body compared with the MIT-24 system.

I

Tripelennamine

Phenformin

Methyldopa

Colistimethate

Thiotepa

Strychnine

Na fluoride

Methampyrone

Quinidine

Dihydralazine

Methadone

Noscapine

Verapamil

Alprenolol

Amitriptyline

16

17

18

19

20

21

22

23

24

25

26

21

28

29

Methoxamine

14

15

Chlordiazepoxide

Papaverine

Procaine

11

13

TheophyIIine

12

Phenobarbital

9

10

Nikethamide

6

Sulfisoxazole

Hexobarbital

5

PriIocaine

Methimazole

4

a

Methyprylon

3

1

Lithium

Alcohol

name*

THEIR

1

Drug

FOR

INHIBITION

2

NO.

TESTED

DRUG

TABLE

HeLa

for

23

45

45

60

89

100

45

180

180

180

200

200

200

200

200

280

310

320

350

440

540

1300

1600

1600

1800

2200

3400

3100

3700

24 h

-

in pg/mlb

IC50

HeLa

37

45

89

40

89

74

13

81

130

130

8.0

89

89

200

200

180

110

480

670

170

540

890

120

1900

400

660

3400

1100

2000

7 days

cells

TO

COMPARED

TOXICITY

CELLS

COMBINED

OF

TO CELLS

MOUSE

for

0.40

l.OE

31

26R

8.0

83

16

170E

69

500E

40E

650

1900

1OOE

23

30E

33

95

45

200E

200E

62

2500R

150E

145

400E

280

2000

500E

/Jg/gc

mice,

i.v.

LD50

HeLa

7

7

20

10E

4

2

9

6E

2

5E

2

8R

5

6SE

10

6

2E

4

3E

4

3E

3

5

2E

p.0.li.v. d ratio

AND

in

10E

PO 2.0

PO 10E

po 4.OER

PO 10E

PO 0.10

PO 12

PO 20

PO 4.5

PO 0.12

iv 0.30E

iv 10E

-

po

PO 3.5

SC 0.60

PO 10E

iv 6.OE

iv 2.0

PO 5.0

iv 5. OE

PO 60E

PO 10E

PO 3.0

PO 15

PO 300

PO 60E

ge

dose

Total

Approximate

APPROXIMATE

4.1

20

2.7

14

0.36

19

48

32

0.34

4.3

140

18

10

1.4

24

86

29

37

71

210

48

11

71

860

430

!Jg/gf

1.~. dose

human

HUMAN

210

2.4

27

140

19

95

130

320

2.3

29

950

120

67

9.5

160

570

190

240

480

1400

320

71

480

5700

2900

/.lg/m1g

DOSAGE

9.0c

0.6CR

1.0

45c

2.0

26

3.0

1oc

26P

150CR

100

78C

500E

30R

!Jg/mlh

cont.

Blood

dosage

1.~. cont.

lethal

LETHAL

31

5.5R

3.4

250

220

80

83

1OP

123

5000

MP/P’

cont.

Liver

OF

T

P

P

T

P

P

P

P

P

0

0

PR

PP

P

P

P

P

P

0

0

PBk

P

and

PREVIOUSLY

T

TR

T

t

t

0

eB

T

t

e

Oe

OL

t

Oe

Oe

t

e

e

0

e

Oe

e

e

TAj

binding

protein

Tissue

DRUGS,

21 n

20

11

19

m

14

Ref.’

z

0

20

ProPran

Benztropine

Imipramine

Chlorpromazine

Metbotrimeprazine

32

33

34

35

36

37

50

column.

handbooks

of a peroral s.c., subcutaneous

for rats or rabbits. dosage.

unknown

dose compared

A dash denotes

S, a subcutaneous

extrapolated

[17,

iFrom

corresponds

dosage.

from Ciba Co., Easel.

from H&de Co., Gothenburg.

“Report

by R are only found in literature

mReport

1Data marked

indicated

drug dose. 0, no or slight binding

[16],

TA, tissue of cells or no penetra-

l/kg. e, even distribu-

binding (30-70%).

in bone.

to a vd between B, accumulation P, moderate

p.o./i.v. E. lethal corm.

and 0.6 l/kg. t, moderate corresponding

0.15

in liver only. (O-30%).

L, accumulation

of less than 0.15 Oe, slight penetration

(vd) by vd between

I/kg).

volumes

ratio. and the mouse

in Drug Treatment

column.

1.2 and 8.0 l/kg. T, high tissue accumulation,

column.

in this column.

R, see Reference

binding of a therapeutic

PP. 99% binding.

PB, blood protein

binding (70-98%).

[14-161.

column.

handbooks

P, pronounced

kFrom

R, see reference

0.6 l/kg (0.15-1.2

by distribution

to a vd of 10 l/kg or more.

by vd between

R, see References

A on drug data information

for some drugs indicated

by vd around

(membrane).

corresponding

for some drugs indicated

3.0 and 10 I/kg. TT, very high tissue accumulation,

cellular (tissue) accumulation,

surface

is Appendix

for some drugs indicated

for some drugs indicated

to cellular

water,

1, or a P.O. total dose divided by 10.5 to a dose.

to iv.

not

E, lethal dosage extrapolated

of the average total lethal dose. P, drug plus metabolites.

P. drug plus metabolites.

reference

to tbe blood cont.

column.

to an intake

R, see Reference

0, no cell penetration,

of 10.5

this ~oncentration~corresponds

of which the most common

with some binding

cells and extraceullular

tion of cells combined

tion between

dosage.

of therapeutic

12, 14-161,

The liver cont.

accumulation

Ill,

a survey I171.

handbooks

iFrom

volumes,

C, a corm. corresponding

in different

concentration.

181.

distribute

from a highly toxic

two survey articles

extrapolated

hFrom

ratio. Since the drugs actually

water volume

____

with an iv. dose. R. see reference

in the special

extracellular

column.

i.v., intravenous;

E, a similar ratio

of administration,

R, note of the reference

the maximal

body weight of 70 kg, or a p.o. total dose divided by 70 and the mouse p.o/i.v.

R, see reference

P.o., peroral,

LD50.

drugs, rat dosage.

mouse dosage from other routes

or for isolated

lethal dosage,

P

P

P

P

P

P

P

23, while IC50 in Table II

TT

TT

TT

TT

T

gThe iv. total dose in mg, divided by the average human

dosage.

[8, 11-171.

to an i.v. mouse

minimal

of administration,

based on mouse

58

317

155

OL

T

mean value between

to HeLa cells [l,

as a geometrical

141. X, may be less.

1.6

3oc

7.0

24 h HeLa cytotoxicity.

0.48

75

drug toxicity

was calculated

on combined

IC50

0.071

11

29 48

7.1

24

38

190

4.3

3.6

5.7

28

in order of decreasing

iv 0.005

PO 4.0

PO 10

PO 3.0

PO l.OE

PO 8.0

previously

[4].

PO 10 -

fThe i.v. total dose in mg, divided by the average human

from highly toxic

eFrom

column.

dRatio

reference

E, estimates

corm., as described

with rat dosage for all routes

18-101.

5

20

10

4

20

from reports

in vitro cytotoxicity

100% inhibitory

study of comparative

and the minimal

handbooks

cont.

dosage by a comparison

‘From

injurious

derives from a previous

5 3

Drugs arranged

5.OE [ 1, 2.41.

corm. ICBO-values in Table I derive.

bIC50

reports

to usage ln previous

aDrug names according

= 50% inhibitory

0.018

0.04

Ouabain

75

16

36

24

22

220E

8.9

13

22

18

18

4.8

8.9

9.5

17

18

18

20

Ethacrynie

31

acid

Prometbazine

30

g

w

4500

2800

Gallamine

Benzylpenicillin

Tubocurarine

Benzyl alcohol

38

39

40

41

120

Phenol

Nicotine

Phenylbutazone

Epinephrine

Quinine

Hydroxyzine

Chloroquine

Methotrexate

45

46

47

48

49

50

51

52

For fobtnotes see Table I.

300

Chloramphenicol

44

8.5 x

10

40

60

400

490

850

1300

43

1700

Lidocaine

Caffeine

42

6400

40000

24 h

5.7x

8.0

95

40

36

58

890

490

400

120

900

4200

2200

580

27000

I days

10E

40

80E

68R

1.8

120

7.1

130

250

68

22

480

0.18

430E

4.3

wgc

mice,

LD50 i.v. for

DNgnmlea

IC50 for HeLa cells

5

10

6

7

5

4

2

10

9

3

ratio d

P.0.li.v.

LETHAL

DOSES FOR 15 DRUGS

8.0

PO 2.OE

PO

PO 7.0

PO 12

PO 20 iv 0.020

PO 0.010

PO 4.0

PO 42

PO 15

iv 3.OE

iv 0.020

iv 40E

iv 0.20

in ge

dose

Total

5.1

11

17

24

57 0.29

0.25

29

60

24

43

0.29

570

2.9

&vgf

1.~. dose

38

76

110

160

380 1.9

1.7

190

400

160

290

1.9

3800

19

pg/mlg

47R

12c

400

29

70R

68 106C

pg/mIh

cont.

I.v. cone. Blood

Approximate human lethal dosage

TO MOUSE AND HUMAN

in pg/mlb

TO HeLa CELLS COMPARED

NO.

DRUG INHIBITION -.._

TABLE II

Liver

308R

220

250

57 116

@g/gi

COIIC.

eR

TT

tR

t

t

0

tR

t

e

t eR

0

Oe

0

TAj

binding

PR

P

pR

P

P

PP

P 0

P

P OR

P

P

PBk

Tissue and protein

..--

21

26

25

5

24

23

22

Ref.’

315

The practical consequence of the comparative accentuation of the MIT-24 toxicity for accumulating drugs is that a similarity of in vitro and in vivo dosage is more valid for drugs not accumulating in cells (0, Oe, and e) than for those that do. Another way to express the same thing is to say that the calculated i.v. concentrations are truly comparable with the HeLa IC50 only for drugs actually confined to ECV (0, Oe), while drugs distributing in total body water or accumulating in cells (e,t,T, and TT) reach a smaller concentration (comparable to HeLa IC50) obtainable by division of their i.v. LD by their actual distribution volume. Since distribution volumes (V,-J for the tissue binding drugs vary between 1 and 20 l/kg (note k, Tables I and II), and since heavily accumulating drugs have a high LD compared with the HeLa IC50, this correction will not appreciably change the found crude (l--10 times) similarity of in vitro and in vivo dosage for a large drug group. It might be concluded, that 7 of the drugs (Nos. 14, 20, 25, 38, 40, 46, and 48) on grounds of a much lower dosage in vivo would be expected to cause human death by other mechanisms than their HeLa toxicity. The remaining 39 drugs with a gross similarity of human and HeLa inhibitory dosage may because of this similarity cause human death by an equivalent interference with basic cellular structures and functions of human target cells and HeLa cells [4], and thus also may have a MIT-24 toxicity relevant to human lethal action. For 47 drugs the similarity between mouse and HeLa inhibitory dosage indicates a possible similarity of lethal drug action to both mice and HeLa cells. These conclusions are consistent with the known human lethal action of the drugs [ ll--161 : 6 of the 7 drugs with clear dose discrepancy, i.e. methoxamine, methadone, gallamine, tubocurarine, nicotine, and epinephrine, interfere therapeutically with highly specialized neuroreceptors, and probably cause human death by interference with them. On the other hand, the 39 drugs with a gross similarity of in vitro and in vivo dosage, with exception of benztropine (No. 33), are known or suspected to cause human death by interference with relatively nonspecific receptors in brain cells or other cells (Nos. 1, 2, 5, 8, 9, 11, 18, 19, 21, 22, 23, 30, 35, 37, 42, 44, 45, 47,49, 51, and 52) or to have an essentially unknown human lethal action mechanism. Another 6 drugs without a known human dosage, but with equivalent mouse and HeLa inhibitory dosage, have an essentially unknown mode of human lethal action. Since 34 of the 37 combined [l, 21 drugs belong to the group of 45 drugs with a similar mouse and/or human and HeLa dosage combined with an unknown or nonspecific human lethal action mechanism, a provisional relevance may be claimed for most of the combined test results. Thus the clinically well known hexobarbital-alcohol potentiative toxicity was recorded by us in vitro [l] . Since 45 of 52 randomly selected common drugs seem to belong to a category with a possible relevance of MIT-24 toxicity, methods to screen combined human systemic toxicity in vitro [ 1, 21 ought to be a realistic

316

supplement to whole animal tests. Drugs would probably be included in these tests only on grounds of a provisionally relevant MIT-24 toxicity, since potentiative synergisms or antagonisms have to be confirmed by whole animal testing [2]. The results of this study suggest that drug interference with basic functions of specialized cells may be a common cause of drug-induced human death. Some of the present lack of knowledge of precise modes of toxic drug action may therefore be elucidated by the use of tissue culture. ACKNOWLEDGEMENTS

I am indebted to Dr. Birgitta Werner, Stockholm, Dr. Johan Hogberg, Stockholm, and Dr. Anders Blomqvist, Uppsala, for valuable discussions. 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. 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 B. Ekwall and A. Johansson, Preliminary studies on the validity of in vitro measurement of drug toxicity using HeLa cells, I. Comparative in vitro cytotoxicity of 27 drugs, Toxicol. Lett., 5 (1980) 297-305. 5 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, Toxicol. Appl. Pharmacol., 1 (1959) 454-461. 6 Y. Goto, C.A. Dujovne, D.W. Shoeman, and K. Arakawa, Liver cell culture toxicity of general anesthetics, Toxicol. Appl. Pharmacol., 36 (1976) 121-130. 7 C.G. Smith, J.E. Grady, and J.I. Northam, Relationship between cytotoxicity in vitro and whole animal toxicity, Cancer Chemother. Rept., 30 (1963) 9-12. 8 NIOSH registry of toxic effects of chemical substances, Rockville, Maryland, 1976. 9 C.D. Barnes and L.C. Eltherington, Drug Dosage in Laboratory Animals, University of California Press, Berkeley, CA, 1973. 10 The Merck Index, Merck, Rahway, NJ, 1968. 11 P. Cooper, Poisoning by drugs and chemicals, plants and animals - An index of toxic effects and their treatment, Alchemist, London, 1974. 12 R.H. Dreisbach (Ed.), Handbook of Poisoning, Lange, Los Altos, CA, 1977. 13 F. Sandberg, Overdosering av hikemedel (Overdosing of drugs), in FASS 1979, Almqvist and Wiksell, Uppsala, 1979. 14 L.S. Goodman and A. Gilman, The Pharmacological Basis of Therapeutics, McMillan, London, 1971. 15 Ainly Wade (Ed.), Martindale’s Extra Pharmacopoeia, Pharmaceutical Press, London, 1977. 16 G.S. Avery (Ed.), Drug Treatment - Principles and Practice of Clinical Pharmacology and Therapeutics, 2nd ed., Adis Press, Sydney, 1979.

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