Toxicity to HeLa cells of 205 drugs as determined by the metabolic inhibition test supplemented by microscopy

Toxicology, 17 (1980) 273--295 © Elsevier/North-Holland Scientific Publishers Ltd.

TOXICITY TO HeLa CELLS O F 205 D R U G S AS D E T E R M I N E D BY THE METABOLIC INHIBITION TEST SUPPLEMENTED BY MICROSCOPY

BJORN EKWALL Department o f Anatomy, University of Uppsala, Biomedical Centre, Box 571, S- 751 23 Uppsala (Sweden)

(Received September 19th, 1979) (Revision received July 10th, 1980) (Accepted July 22nd, 1980) SUMMARY The toxicity of 205 drugs to HeLa cells was evaluated by the Metabolic Inhibition Test supplemented by microscopy. T w o end points of cytoinhibition were estimated; total and partial inhibition after 24 h, based on absence or scarcity of spindle-shaped cells, respectively, and total and partial inhibition after 7 days, based on different degrees of a basic pH change of the phenol red included in the cell medium. Direct drug-induced pH changes and precipitates in the cultures were also recorded. Many drugs were found to induce a culture zone with a particularly low pH after 7 days of incubation, at concentrations below the cyto-inhibitory concentration. Forty-three drugs regularly caused this hyperacid reaction, while 44 drugs caused the reaction irregularly. Since the reaction was always continuous with the cyto-inhibitory zone, it was provisionally judged to represent excitatory cell injury. Since many o f the drugs which regularly induced the reaction are also known to induce proliferation o f the endoplasmic reticulum in various cells, the reaction m a y be related to this and allied effects. Many drugs that are k n o w n to accumulate in cells displayed a high inhibitory toxicity, which might have been due to the paucity of cells in the test system. However, the high 7~iay inhibitory toxicity shown b y antineoplastic and some anti-inflammatory drugs, including triamterene and disulfirarn, may constitute a genuine antimetabolic drug action. Seven grand mal antiepileptics were only very slightly toxic as compared with their precipitating tendency, which may be of significance in their therapeutic action. INTRODUCTION Tissue culture has been used for half a century as an investigativetool in pharmacology and toxicology [1--4]. As such, its use has been limited to a

273

s t u d y o f effects of drugs on basal cellular functions. Owing to the so called in vitro-in vivo gap [3], it has been difficult t o extrapolate the results of these studies to human conditions. With these restrictions, the use of tissue culture for this purpose has reflected successive areas of current interest in h u m a n drug action, from the screening o f antibiotics and antitumour drugs [5,6] to the more recent studies of drug metabolism [7,8]. Tissue culture has also been e m p l o y e d to study the reaction of the cultured cell to drugs for its own sake, a field which m a y be called cell pharmacology [1]. Such studies referring t o c y t o t o x i c drug concentrations m a y be categorized as in vitro cytotoxicology, thus comprising a subdiscipline to toxicology which compiles knowledge a b o u t chemical injury to cell cultures, relevant parts of which might be of subsequent value in explaining human drug action. One purpose of in vitro c y t o t o x i c o l o g y is to determine the toxicity of various chemicals to various cells in culture. Since C.M. Pomerat and C.D. Leake reported their pioneer study o f the toxicity of 110 drugs to primary cultures of fetal chicken cells [9], in vitro c y t o t o x i c i t y has been determined for other large drug groups [10,11], solvents [12], pesticides [13], insecticides [14] and chemicals [15,16]. Numerous cytotoxicity tests have also been performed with single or a few drugs [ 1], often representing the use of tissue culture as an investigative tool in pharmacology. One problem o f in vitro cytotoxicology is the incomparability of results of different studies, d u e to the variation in tissue culture methods. It is therefore difficult to acquire comprehensive knowledge of drug cytotoxicity in vitro from the discussed results o f different investigators, and especially from the findings in the m a n y disparate studies with isolated or a few drugs. Probably the only answer to this problem is standardization of procedures, i.e. t h e use o f programmes in which m a n y chemicals are tested by the same method. By comparing the results of such series with each other and with those o f older large-scale studies, the influence o f m e t h o d variation on the observed c y t o t o x i c effects could be assessed. With the aim o f contributing towards such standardization, a large number o f drugs were tested with the same m e t h o d in the present study. The drugs were selected from most fields o f pharmacothempy, and included both water-soluble and insoluble drugs, as well as drugs which have n o t been tested on tissue cultures to any large e x t e n t previously, such as diuretics [1]. To facilitate comparison with other studies, some drugs tested in early investigations were included, the c o m m o n l y used HeLa cells were chosen as test cells, 2 end points o f cyto-inhibition were examined, drug-induced pH and precipitation were monitored, and several different forms of 23 drugs were tested. The s t u d y also comprises the first step of a tier testing programme designed to evaluate the in vitro c y t o t o x i c i t y of drug combinations as described in previous communications [17,18]. MATERIALS AND METHODS Drug c y t o t o x i c i t y was tested by the Metabolic Inhibition Test, supple-

274

mented by microscopic viability determination of cells after 24 h (the MIT-24 test). The Metabolic Inhibition Test has been described b y Paul [19], and a brief description of the MIT-24 test has been given in previous papers [17,18]. T w o independent stock cultures of HeLa cells ( t y p e Mandel, Wallenberg Lab., Uppsala) were grown continuously as a m o n o l a y e r in Parker's medium 199 (SBL, Stockholm), 107o calf serum (SBL), benzyl penicillin (200 IU/ml), and streptomycin sulphate (0.1 mg/ml), with the use of stationary Sani flasks (Brockway Co., New York). Every 3.5 days the cells were harvested with Versene (SBL, 0.2 mg EDTA/litre PBS), and inoculated into n e w flasks gassed with 5% CO2 in air. New stock cultures were sometimes established from frozen cells. The drugs were tested on arrival from the manufacturer, and retested within a year. Samples were stored in sealed brown glass flasks in a cool place. Drugs sensitive to storage were stored frozen or were requested again for re-tests. Pure drug samples were dissolved in saline, dimethylsulphoxide (DMSO), NaOH or alcohol, or suspended in glycerol. Twelve drugs were incubated with cells in a batch of 6 microtitration plates, with 2 tests on each plate (see Figs. 1 and 2). The batch was then inspected for cyto-inhibition after 2 4 h and 7 days of incubation. The aim was to test every drug at least twice at an original concentration suited to place the toxic end points within the limits o f the test area, b u t outside the toxic range of the solvent, which means a drug toxicity 5 times (1 dilution step) less than the minimal solvent toxicity. Standard methods of tissue culture [19] were used. Water-soluble drugs were tested at a concentration near the maximum solubility. Insoluble drugs were first tested as a 100 mg/ml glycerol suspension. The choice of concentrations and solvents for subsequent tests depended on the previous results, a trial and error strategy. All drugs that were insufficiently soluble in saline could be sufficiently dissolved in DMSO or suspended in glycerol. Mechanical grinding of the drug was necessary in preparing glycerol suspensions. In spite of the fact that the microdiluter was invariably dipped exactly 2 mm into a freshly stirred glycerol suspension, as a standardization measure, the volume of the suspension distributed to test trays varied, because of variation in adhesion of the suspension to the diluter. Sodium h y d r o x i d e and alcohol solutions were therefore used to supplement some o f the glycerol tests. Every DMSO, NaOH, and alcohol solution precipitated in the first dilution of the test tray. After the dissolving procedures, the cells from t w o 3.5-day-old flasks of the stock culture were harvested and suspended in a new magnetically stirred culture medium kept at 35°C, the cell density of which was determined by 4 counts of unstained cells in a Buerker h a e m o c y t o m e t e r . The suspension was calibrated to a cell density of 1.0 × l 0 s cells/ml and was intermittently gassed with CO2 to maintain a pH o f a b o u t 8.0 for several hours. During that time the cells did n o t multiply; this was checked by n e w counts at the end of the incubation period. 275

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Fig. 1. An example o f th e experimental arrangements of a test o f caffeine (No. 60) and glutethimide (No. 131). Caffeine was tested as a 30 mg/ml " h o t " saline solution, and glutethimide as a 500 m g /m l glycerol suspension. Each drug was diluted (from above downwards in the records) as 5 parallel dilution series in a 5 x 8 cup area of a microtitration tray, separated from the other test area'by 2 x 8 reference cups. The limits for the separate toxicities o f the carrying agents are indicated by dotted lines. An approximate pH for some dilution steps o f interest is presented. The pH values of the 24-h record refer to the pH at the start o f incubation. T: total absence o f fusiform or spindle-shaped cells. D: deficient growth of fusiform cells as compared with reference cups. P: heavy precipitate making cells invisible, p: some precipitate. V: violet colour. R: red colour. B: brownish colour. Y: bright yellow colour. E m p t y circles: normal growth after 24 h of incubation, and normal orange colour of reference cups after 7 days of incubation.

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277

Parker's medium 199 with 0.6 mg/ml extra glucose was then adjusted to pH 6.0 (like the suspension pH, this was judged by the colour of the indicator phenol red included in Parker's medium) by gassing with 5% CO2 in air. Each of the ninety-six 0.4 ml cups of the test tray (IS FB 99, like other microtitration material from F l o w Lab., Solna) received 0.1 ml o f this medium. T w o drugs were diluted in steps o f 1 : 5 (1 : 10 between the initial test solution and the first dilution of the tray) by 0.025 ml microdiluters (M55) in t w o 5 × 8 cup areas as shown in Figs. 1 and 2. Five parallel dilution series of each drug was thus obtained in each test. All cups received 0.1 ml of the cell suspension and were sealed with liquid paraffin. The tray was sealed with transparent film (M 30 A) and was inspected immediately for drug-induced pH changes of the culture medium, after which it was incubated at 37°C for 7 days. At the start of incubation, the cultures had a mean pH of 7.5, with a variation between 7.0 and 8.0. Thus each culture finally contained 5 × 104 cells/ml in 0.2 ml Parker's medium 199, including 5% calf serum, 3 mg/ml glucose, 20 mg/ml phenol red, antibiotics at the same concentration as in stock cultures, and a varying drug concentration. After 24 +- 1 h, relevant cups of a tray were studied at r o o m temperature in an inverted microscope (Zeiss Standard UPI) at 100 × and 400 × magnification. Failure of the cells to spread was the sole criterion of toxicity. Cups without fusiform or spindle-shaped cells, i.e. cups with 100% round cells like the original inoculum from the suspension, were considered totally inhibited, while cups with less outgrowth of fusiform cells than normal reference cultures were considered as partially inhibited. The minimal drug concentration that resulted in a precipitate was recorded. Precipitates could consist of either crystalline drug or of a protein-drug complex. After 168 -+ 4 h, the colour of the phenol red in each cup was recorded. Violet cups, with a pH of a b o u t 8.5 caused by a gradual outward diffusion of CO2 during incubation, were considered totally inhibited. Red cups, with a pH of a b o u t 7.5 caused b y a limited production of acid metabolites, counteracting the effect of CO2 diffusion, were considered partially inhibited. Orange-yellow cups, with a pH of 6 . 0 - 7 . 0 caused by a gradual accumulation o f acid products from an undisturbed cell metabolism, were considered normal. A n y persistence of the initially recorded pH change, induced by high concentrations of unbuffered drugs, was noted {e.g. see drug No. 29, Fig. 2). It was sometimes n o t .possible to discriminate a drug-induced basic shift from the 7-days total inhibition. A corresponding acid shift was usually less acid after 7 days than initially. Further, some drugs induced an acid shift of the medium at high drug concentrations, which progressed in proportion to the incubation time and had an intensity directly related to the drug concentration, which in this study is called time-progressive acidity of drugs (see note k, Table II). Like persisting, initial pH changes, this time-progressive acidity sometimes interfered with the 7-day toxicity evaluation. After 400 tests in which only inhibitory toxicity was recorded, it was realized that, at subinhibitory concentrations, some drugs regularly induced a culture zone with an acidity greater than that of reference cultures (e.g. see drug No. 131, Fig. 1). The intensity of this reaction, called here the hyper278

acid reaction o f cultures was not directly related to the drug concentration and did not develop in proportion to the incubation time. The hyperacid cups were yellow to bright yellow (pH 6.5--5.5), and observations concerning this phenomenon were made in the last 700 tests of the study. Different drugs induced hyperacidity of different intensities. The final pH of reference cultures varied between batches (pH 7.0--6.0), which was partly due to variation of the initial pH and partly to other factors, such as varying growth rates of cells. The recording of the reaction was thus a matter of contrast between a varying reference and a reaction of varying strength, which might have resulted in many false negative recordings, especially for reactions of weak intensity. Most drugs were tested separately for their capacity to alter the pH of the medium. Relevant concentrations were serially diluted in 5 ml of Parker's medium 199 plus 5% serum, adjusted to pH 7.5, and the pH was measured (Beckman Zeromatic II). The results were identical to the phenol red recordings presented in Table II. RESULTS The classification of the tested compounds is shown in Table I, and the results are given in Tables II and III. The study of the inhibitory drug toxicity (1100 tests) resulted in 4 toxicity values, based on at least 2 tests, that were not directly influenced by solvent toxicity or cut off by the limits of the test area for each of 205 drugs and 7 solvents. In addition, the tables give the results of tests of different drugs forms for 23 drugs (Nos. 51, 59, 77, 78, 85, 86, 88--90, 93, 96, 98, 102, 128, 130, 133, 147, 149, 165, 172, 187, 193 and 194), as well as an account of the solvents used, of drug-induced pH and precipitates, and of the precision of repetitive testing. Toxicity data influenced or invalidated by pH, precipitates, or drug colours appear in brackets. For 41 drugs the 24-h cyto-inhibition was probably caused by drug-induced pH changes (Nos. 29, 34, 42, 51, 78, 85, 90, 93, 101 and 142) or influenced by precipitates (Nos. 3, 24, 31, 40, 45, 46, 48, 51, 53, 54, 77--79, 85, 87, 90, 94, 95, 109, 110, 112, 137, 140, 141, 151, 158, 163, 164--166, 178, 188, 199, 204 and 205). For 33 drugs the 7
TABLE I CLASSIFICATION OF COMPOUNDS

Type

Subgroups

Drug No.

Drugs u s e d in d i s o r d e r s of the respiratory tract

N o n - n a r c o t i c antitussives Antiasthmatic sympathicomimetics Nasal d e c o n g e s t a n t s

1 5

--4

9

- - 1 0

Glycosides Antiarrythmics Vasodilators E r g o t alkaloids

11 15 21 30

.... ----

Vasopressors

33

-- 3 6

Antihypertenaives

37

- - 44

Thiazides O t h e r s u l f a n i l a m i d e derivatives Miscellaneous d i u r e t i c s Xantincs

45 50 55 59

-----

61

-- 68

Cardiovascular drugs

Diuretics

Antihistamines

--8

14 20 29 32

49 54 58 60

G a s t r o i n t e s t i n a l drugs

B e l l a d o n n a alkaloids and c o n g e n e r s Miscellaneous g a s t r o i n t e s t i n a l drugs

69 76

- - 75 - - 77

Drugs acting o n t h e b l o o d

Iron preparations Anticoagulants

78 80

- - 79 - . 82

83

-~ 86 - - 91 - - 100 .... 103 --105

A n t i n e o p l a s t i c drugs Chemotherapeuties and antibiotics

Sulfonamides Antibiotics D r a g s u s e d against t u b e r c u l o s i s Urinary antiseptics

87 92 101 104

Endocrine drugs

Adrenal hormones A n a n t i t h y r o i d drug A n t i d i a b e t i c drugs

106 - - 107 108 109 - - 115

Drugs acting o n t h e central nervous system

N a r c o t i c analgesics General anaesthetics Hypnotics Sedatives ( m i n o r tranquillizers) N e u r o l e p t i c s ( m a j o r tranquillizers) Drugs against m a n o - d e p r c s s i v e d i s o r d e r s CNS-stimulating drugs Antiepileptics Drugs u s e d in P a r k i n s o n ' s disease Central m u s c u l a r r e l a x a n t s

116 - - 1 2 2 123 - - 125 126 - - 138 139 - - 142 143 .... 152 153 - - 156

280

157

-- 1 6 2

163

-- 1 6 9

170

-- 1 7 3

174

--

177

TABLE I (Continued) Type Drugs a c t i n g o n t h e peripheraln e r v o u s

system

Anti-inflammatorydrugs

Subgroups

Drug No.

Drugs acting o n n e u r o m u s c u l a r transmission Local anaesthetics

178--181 1 8 2 - - 186

Antipyretic analgesics Antimalarials

187 -- 195 196 -- 198

Non-classified drugs

199 - - 205

Solvents u s e d

$1 -- $7

in this study

obscured a moderately extensive hyperacid zone. Of the remaining 181 drugs, 155 were tested at least twice, while 26 were tested only once. Of the 155 drugs tested twice or more, 43 induced the reaction in both of 2 tests or the majority of 4 tests or more, i.e. they were regular inducers (Nos. 1, 20, 28, 32, 35, 38, 41, 49, 50, 51, 65, 71, 72, 75, 77, 81, 82, 86, 9 4 - 9 6 , 105, 114, 115, 119, 125, 126, 128--132, 134, 137, 139, 151, 156, 165, 176, 177, 181, 182 and 193), 44 drugs induced the reaction in some tests but not in others, i.e. they were irregular inducers (Nos. 7, 17, 18, 19, 22, 27, 31, 48, 53, 56, 58, 61- 63, 67, 68, 73, 74, 76, 88, 98, 101, 104, 112, 113, 124, 133, 136, 141, 144, 146, 150, 157,158, 161, 164, 166, 169, 171, 173, 190, 191, 200 and 203); for 51 drugs the reaction was negative in both tests and for 17 it was negative in 4 tests or more, i.e. 68 drugs were regular non-inducers. Of the 26 drugs tested once, 11 induced the reaction, i.e. they were less documented inducers (Nos. 2, 30, 37, 43, 97, 100, 109, 143, 178, 179, 199), while 15 drugs were negative, i.e. were less documented non-inducers. Thus a majority of 98 drugs induced the reaction in at least 1 test, while 83 drugs did not. Since this study must have produced m a n y false negative results,it is probable that m a n y of the irregular and less documented inducers would prove to be regular inducers on more refined testing. DISCUSSION T h e test m e t h o d

The MIT-24 test has not been used previously for testing drug cytotoxicity [1 -~ 4]. Toplin et al. [6] used a similar system, however, in which toxicity was determined by microscopic evaluation of cell damage after 5 days of incubation. In their studies the colour change of phenol red was used as supplementary evidence of toxicity, but it was not precisely estimated in terms of drug concentrations. The results of Toplin et al. show a good correlation between microscopically determined inhibition and pH-indicator change. In the present study 8 drugs (Nos. 49, 54, 137, 140, 141, 163, 164 and 165A) appeared to have a

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x x x x x x x x x x x x x x × x x x × x x ×

x

° o ~ o o o o o o o ~ o o ~ o ~ o o o ~ o o o o o o o o o o o o o o ~ o ~ o ~ M N ~ N N N 4 N ~ 4 M ~ 4 M ~ M ~ 4 N N 4 4 ~ 4 N ~ N N ~ N ~ N ~

'e

=

~ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o X X x x x x x x x x x × x x × x x x x x x x x × x x x x x x x x x x x x x x x x x x

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286

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Disulfiram, M

Nicotine, M Pilocarpine HCI, USP Probenecid, USP

Protamine sulfate, USP NaF, USP

200

201 202 203

204 205

S/DP S D,G N S/P S/DP

A,D,G

D,G,SP S Gm HS S S S

× 103 × 103 × 103 × 102 × 10 × 10 X 10s)p

2.0 × 103 4.5 x 103 (1.0 x 103)x (2.0 X 10~) p . r (1.0 × 10~) ! (8.0 × 102)l

3.5 x 10 ~

2.0 4.0 4.0 2.0 4.5 4.0 (1.5

102 103 103 10

x 10 x 104

x × × ×

4.0 x 102 2.0 x 103 (2.0 x 102) (4.0 × 103) 4.0 x 102 2.0 × 102

5.0 x 10 ~

4.0 4.0 8.0 8.9 4.0 2.0 3.0

× × × × × × x

103 102 10 ~ 10 10 10 l0 s

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3.5 x 10 ~

8.9 8.0 4.0 8.9 2.0 2.0 1.5

x × x ×

10 102 10 10

2.0 8.9 2.0 i 8.0 4.0 2.0

x 103 × 10 ] x 102 X 10 ~ x 102 x 102

4.0 x 10 2

1.6 1.6 8.0 8.9 8.0 8.0 --

1.0 3.2 X 10

1.3 x 10 2

3.0 x 10' 0/2 0/2 s 2/4 q.t 1/1 0/3 0/6

1/4 v

0/4 0/1 0/3 0/3 0/3 0/2 1/1

6.2 (8.0) w

8.5 5.8 7.0(7.5)k 9.0

6.5

7.0

8.9 x 10 ~

2.0 4.0 4.0 1.0

x 103 X 103 x 102 x 102

6.0 X 103

5.0 × 102

I II II -I I

I

II II III I II I II

a Names refer to the United States Pharmacopoeia (USP), the National Formulary of the United States (NF), the Merck Index (M), Martindale's Extra Pharmacopoeia (Md), the British Pharmacopoeia (BP), or the British Pharmaceutical Codex (BPC}. b A: 96% alcohol solution. AA: 10 mg/ml ecetamide and saline solution. AC: 25% glacial acetic acid end saline solution. D: dimcthyiaulfoxide (DMSO) solution. G: glycerol suspension. HS: hot saline solution. M: cell medium solution. MP: cell medium suspension. MP/V: cell medium susp. of a volatile drug. N: NaOH solution. S: saline solution. SP: saline suspension. S/P: saline sol. precipitating in test trays. S/CP: S/P with crystalline precipitate. S/DP: S/P with diffuse precipitate. W: vial solution of the pure substance in sterile water. c A value in brackets denotes a toxicity either caused by drug precipitate (notes I, p, x) or drug pH (note r), or invalidated (minimal value) by drug colour (notes m, o) or drug pH (notes k, s). d A dash denotes the same injurious toxicity as the total inhibitory toxicity, for a drug regularly inducing the hyperacid reaction (notes c, f). ¢ The minimal drug concentration inducing the hyperacid zone. f The number of tests with a drug-induced hyperacidity of cultures, compared with the number of tests in which observations concerning this reaction were made (see Methods). A dash denotes a drug not tested for the hyperacid reaction. g A drug not noted in these columns was fully buffered by the cell medium and did not precipitate within its toxic range. h An approximate pH, at the start of incubation, of the culture medium containing the 24-h total inhibitory drug concentration. A: dash : deficient pH record. i The maximal drug concentration at which no precipitate occurred. i The variability of results from all tests on cyto-inhibition. If not indicated by a dash (denoting a single test of the drug form), the number of testa was not below two, and was often 4--6, i.e. a number usually larger than the last figure in the column denoted f. I: The same values for both 24-h and 7-day total cyto-inhibition in all successive tests. II: A variation of one or both total inhibitory concentrations within the 0.45C--2.2C range, where C is the mean value of the concentrations. III: A variation within the 0.2C--5C range. k The drug developed an acidity at concentrations higher than the cyto-inhibitory concentration, which progreued in proportion to the incubation time and always had an intensity proportional to the drug concentration. This "time-progressive acidity" sometimes obscured the 7-day toxicity. (B): buffered. Denotes a time-progressive acidity at concentrations higher than the 24-h total inhibitory concentration. (7.5): Slight interference with the recording of the 24-h total inhibitory concentration by acidity. The 7-day inhibitory concentrations could be identified without difficulty. (4.5--5.5): the actual pH of the culture medium containing the 24-h total inhibitory concentration after 7 days' incubation. This pH prevented precise identification of the 7-day inhibitory toxicity, including that of a short zone of drug-induced hyperacidity of cultures. 1 In cups containing the 24-h total inhibitory concentration the bottom area was totally covered by a semi-transparent precipitate or partially covered by a non-transparent precipitate. This permitted accurate observation of most cells, but must have contributed to cyto-inhibition. rn The initially colourleas drug developed a yellow or brown colour during the incubation period, sometimes obscuring the 7-day toxicity. o From the start of incubation the drug had a colour which sometimes invalidated the 7-day toxicity recording. P In cups containing the 24-h total inhibitory concentration the bottom area was totally covered by a non-transparent precipitate, which probably interfered with cellular life. This reduced t h e toxicity to a minimal value. q A variation in all performed tests of the minimal drug concentration inducing hyperacidity beyond the 0.45C--2.2C range, where C is the mean value of the concentrations. Drugs not noted in this respect varied within the 0.45C--2.2C range. r The drug-induced initial pH was as aberrant as the pH of acids and alkalies (Table III). ' Recording of the 7-day toxicity, including that of a short zone of drug-induced hyperacidity, was sometimes disturbed by the initial acid-base reaction induced by the drug. The 7-day toxicity concentration is therefore either a minimal value or a positively identified (but more uncertain) value. The record of the minimal drug concentration inducing hyperacidity of cultures has been cut off, i.e. it may have been less. v The zone of 7-day inhibition in some testa corresponded to a zone of hyperacidity in others. WThe initial pH of the 24 h total inhibitory concentration of drugs dissolved in NaOH or acetic acid is given in brackets. x A value not far from (half a dilution step, i.e. 0.45 x conc.) the toxicity border of the solvent. In spite of this the drug was not tested at a higher concentration because of the effect of the drug precipitate (notes 1 and p). Y Halothane including 0.01% (w/w) thymol.

Phenylbutazone, NF Phenylbutazone Na, M lndomethacin, NF Quinine HCI. M Chloroquine phosphate, OSP Hydroxychloroquine phosphate, M Dextran, M (Dextran 70)

194A 194B 195 196 197 198 199

t~ 00 00

Alcohol, USP Acetic Acid Glacial, M Dimethyl eulfoxide, M Glycerol, M NaCI, M NaOH, M HCI, USP

$1 $2 $3 $4 $5 $6 $7

*See Table II for footnotes.

Drug a,*

No.

TOXICITY OF 7 SOLVENTS TO HeLa CELLS

TABLE IH

M M M M M SIDP S

Solver,.tb

X X X X X

104 102 104 10 s 10"

7.1 × 10 2

3.7 4.5 2.2 1.3 1.1

2.0 X 102

104 103) r 10 s 10 s 104

(7.1 X I0~) r

X X X X X

"(2.0 X 101)r

1.9 (1.0 1.1 1.3 1.1

X 10' X 10~)s X 10" X 10 s X 104

. (7.1 X I0~) j

(2.0 X 101),

t 3.7 (1.0 2.2 1.3 1.1 7.1 X I0 ~

2.0 X 10 ~

3.7 X 10 ~ 2.0 X 103 2.2 X 104 2.5 X 104 1.1 X 104

Injuryd

Total inhibition

Total inhibition

Injury

7 day indicator change

24 h microscopy

Minimal inhibitory concentrations (~glml) c

Minimal drug conc. lowering 7 days pH compared with pH of reference cups (~g/ml)" 0/3 0/2 0/3 0/3 0/3 0/3 0/2

Number of tests with an extra low 7 days pH f

9.5 5.0

5.0

pH h

1.6 X 10 =

Conc. free from prec. (~g/ml)l

Notes on not buffered and/ or precipitating drugsg

II I I I I II I

Test variabilityj

lower toxicity at 7 days than at 24 h due to a lack of visibility of cells at the 24 h microscopy, caused by precipitation. Of the remaining 197 drugs, only for 2 (Nos. 50 and 51) was some metabolic function, as indicated by phenol red, observed in cells that had been considered totally inhibited at 24 h. This supports the reliability of the 24 h toxicity criterion, i.e. deficient spreading of cells. The .dual toxicity recording of the MIT-24 method has 2 advantages. Firstly, the toxicity of most drugs may be precisely determined by at least 1 of the end points. Secondly, the toxicity determinations at 2 different incubation times may reveal traits of the cytotoxic drug action. Thus, drugs with the same 24-h and 7-day toxicity may be expected to penetrate cells well or otherwise have a direct action, while drugs with a higher toxicity at 7 days than at 24 h may be expected not to penetrate cells well (e.g. drugs Nos. 87 and 92). Such latter drugs might also be antimetabolites (No. 85) or be converted by cells to more toxic metabolites (No. 189). Cytotoxicity was often influenced by drug pH and precipitates. The effect of direct drug-induced pH changes of the medium is indicated by the varying toxicities of different drug forms (see drugs Nos. 51, 88, 89, 102, 128, 130 and 165). In other cytotoxicity studies pH and precipitates have generally not been checked, in spite of similar testing of unbuffered drugs, which irrespective of a good initial solubility in various solvents often must have precipitated in cell cultures [ 5,8,9,10,12--16]. Methods employing an automatized cytotoxicity recording might even leave the investigator unaware of drug precipitates [ 8,11,15,16 ]. The extent of pH changes and precipitates depends greatly on the design of the test system (buffering capacity, contact of cells and precipitates), and the cytotoxicity affected by these factors must therefore be expected to vary between studies. The observations on these factors in the present MIT-24 system may facilitate future comparisons with cytotoxicity valued obtained in other systems. In the present study drug toxicity was not clearly influenced by the toxicity of the solvent, since it was recorded at subtoxic solvent concentrations. It must be suspected, however, that subtoxic solvent concentrations will alter the toxicity of some drugs in a similar manner to the synergistic action of subcytotoxic drug concentrations found in previous studies [ 17,18]. The extent of this influence may be evaluated from the data in the last column of Table II. Of the 54 drugs tested in several solvents, 29 drugs in repetitive tests showed the same toxicity irrespective of the solvent used, while the toxicity of 16 drugs varied moderately within the 0.45C--2.2C range. The toxicity of another 9 drugs varied beyond this range and is therefore recorded separately in Table II (drugs Nos. 49, 50,149A, 163, 164, 165A, 166, 188 and 203). For 7 of these, glycerol seems to have decreased the drug toxicity, compared with the effect of the other solvents. Overall, this variation is not much larger than the general variation of the present test results (see below), which indicates that the observed toxicities of drugs suspended in glycerol or dissolved in DMSO, NaOH, alcohol, and other solvents are roughly comparable. 289

The variation of the inhibitory test results is presented in the last column of Table II. O f the 2 4 2 different drug forms which were tested at least twice, 164 yielded the same values for both the 24-h and the 7~lay total inhibition in all successive tests, while 73 forms showed a variation of one or both of these values within the 0.45C--2.2C range. Only for as few as 5 forms did the results vary within the 0.2C--5C range. The results from successive testing of the 2 less objective toxicity values, i.e. the 24-h and 7-day p a r t i ~ inhibition, varied more ( n o t shown in the table). Every single toxicity value also has an uncertainty in the range o f 0.45C--2.2C, which should be added to the variation o f repetitive results. The variation in repetitive tests of the minimal drug concentration that induced the hyperacid reaction is n o t e d by q in column 7, Table II. Of the 53 drag forms which induced the reaction i n at least 2 tests and yielded a result that was n o t c u t off by the limit of the test area, 42 forms varied within the 0.45C--2.2C range, while 11 forms showed a larger variation. This variation is explained b y the prerequisites for the recording of the reaction. The MIT-24 test seems to yield reproducible c y t o t o x i c i t y results for most substances by simple and inexpensive means. Important factors influencing t h e inhibitory drug toxicity, such as pH, precipitates, and the hyperacid reaction are monitored. The test system m a y be supplemented b y microphotography and biochemical measurements at various incubation times. The hyperacid reaction This reaction has been described solely to shed further light on the inhibitory drug toxicity. It will be discussed in more detail in future reports on morphological studies and further screening of inducers. Besides t h e described features o f the hyperacid reaction which connect it with cellular or subcellular activity (induction by low, subinhibitory concentrations, intensity, independent o f drug concentration, development independent o f incubation time), this reaction seems to have definite relations t o cyto-inhibition: (I) the hyperacid zone was always continuous with the 7~day inhibitory zone; (II) the zone often directly followed the 7-day total inhibitory zone, with no intervening partially inhibited zone (32 of 41 regular inducers showed this pattern -- marked b y a dash in column 5, Table II); (III) the zone o f t e n corresponded to the 24-h partially inhibited zone, i.e. with deficient spreading of the cells; (IV) many irregular inducers (18 of 44 drugs -- marked by v in column 7, Table II) ind u c e d a hyperacid zone which in positive tests occupied the same concentration range as did the 7~lay partially inhibited zone in negative tests; and (V) most non-inducers had a higher 7-day than 24-h toxicity (14 of the 17 best d o c u m e n t e d non-inducers, i.e. drugs Nos. 11, 33, 59, 69, 85, 89, 90, 102, 127, 155, 172, 188, 192 and 194), while m o s t inducers had a b o u t the same toxicity at 24 h and 7 days. The continuity of the hyperacid zone with the zone of cyto-inhibition (I,II) and its reciprocal relationship with the zone of partial inhibition (III, IV, V) indicate that the zone might represent

290

another facet of cellular injury, i.e. excitatory cell injury. Similarly to cell inhibition, such excitatory cell injury must preliminary be viewed as the net result of several different drug effects. Many drugs induce proliferation of the endoplasmatic reticulum, accelerated protein synthesis, and an increase of drug-metabolizing microsomal enzymes in various human and animal cells [20--22], including cell cultures [7,8]. The hyperacid reaction may represent such an induction of the endoplasmatic reticulum of HeLa cells, since many regular inducers of hyperacidity in this study are also known to stimulate microsomal enzymes, such as most barbiturates (with the exception of secobarbital), glutethimide, methyprylon, methaqualone, carbromal, meprobamate, diazepam, chlorpromazine, phenytoin, carbamazepine, carisoprodol, antipyrine, and aminopyrine while many non-inducers of hyperacidity, such as paraldehyde, trimethadione, sulfanilamide, and aspirin are known not to stimulate microsomal enzymes [20,22]. Deficient induction of hyperacidity by known enzyme inducers, such as tolbutamide, thioridazine, imipramine, nikethamide, orphenadrine, and phenylbutazone, may be due to the well known differential enzyme induction in various species and various types of cells. Since cell excitation is probably the net result of different drug actions, there may be other causes of the hyperacid reaction than enzyme induction. Examples are the well known hypermetabolic effect of insulin [23], uncoupling of the oxidative phosphorylation [24,25], or increased glycolysis induced by drugs [26]. Whatever its nature, the hyperacid reaction may affect the comparability of the inhibitory toxicity. Is the toxic concentration of a non-inducer of the hyperacid reaction equivalent to the inhibitory concentration, or to the "excitatory" (hyperacid) concentration of an inducer? The hyperacid reaction may complicate a comparison of partial inhibition in the MIT-24 test with results of other cytotoxicity studies, especially concerning irregular inducers of the reaction. For some drugs the very low concentrations inducing the hyperacid reaction (Nos. 28, 30, 49, 56, 77, 81, 94, 95, 96, 104, 105, 112, 114, 128, 129, 151, 156 and 165) may approach therapeutic drug concentrations. Further studies in vitro of the reaction would therefore seem important. By the MIT-24 method it is possible to record the whole concentration range inducing the reaction in a few tests, and also to study the morphology of the reaction. This method could probably be made still more sensitive and reliable by improvement of the checking of the initial pH of cultures.

The inhibitory drug cytotoxicity Calculated on a total number of 7.5 × 1013 cells and an extracellular water volume of 14 litres [27], the human body contains 5 × 109 cells/ml, while the cell density in the MIT-24 system is only 5 X 104 cells/ml. This difference between the 2 "systems" must be kept in mind when comparing MIT-24 toxicity with systemic drug concentrations in vivo, and when inhibitory concentrations in the MIT-24 system are compared with one another.

291

S o m e of the drugs with a high inhibitory cytotoxicity are also k n o w n to accumulate in h u m a n tissues, such as the sympathicomimetics, glycosides, phenothiozines, tricyclic antidepressants, and quinine- or chloroquine-like drugs [23 ]. The high toxicity of these drugs in the MIT-test, as compared with the MIT-24 toxicity of drugs which do not accumulate in cells,m a y be an effect of the low cell count in the MIT-24 system. Thus the observed high toxicity to H e L a cells of these former and allieddrugs m a y not be significant to the action of such drugs in man. Other drugs with a high MIT-24 toxicity are k n o w n to be cytotoxic in the h u m a n body, such as antitumour and some other cyto-inhibitory drugs. The k n o w n cytotoxic action in m a n of these drugs might be related to the high toxicity to HeLa cells; alkylating action of thiotepa, antimetabolic action of fluorouracil and methotrexate, and enzyme inhibition by sodium fluoride and phenylbutazone [23]. T w o drugs of this study, triamterene (No. 57) and disulfirarn (No. 200), like the above drugs, have a high cytotoxicity to H e L a cells.Tri'amterene includes a pteridine component in its formula, as do folic acid and methotrexate. Therapeutically disulfiram inhibits the enzyme alcohol dehydrogenase, but it is k n o w n to inhibit other enzymes as well [23]. The high MIT-24 toxicity of these drugs might thus be tentatively explained by an antimetabolic action in the case of triamterene and an enzyme inhibition in the case of disulfiram actions which might be relevant to action of the drugs in man. Some drugs are known to be m e t a b o h z e d to more toxic metabolites by human or mouse liver cells, such as acetaminophen, phenacetin, isoniazid, and secobarbital [ 28,29]. These drugs were also time~lependent inhibitors of HeLa cells in this study, i.e. their 7-day toxicity was higher than that at 24 h. This m a y indicate a similar metabolism o f the drugs by HeLa cells. Some drugs have a low toxicity to HeLa cells, such as nitrites, ganglionic blockers, sulfonamides, penicillins, antiepileptics, and neuromuscular blockers. With the exception of nitrites and antiepileptics, these drugs are known to penetrate cells inefficiently [23], which may explain the low inhibitory cytotoxicity. For most drugs of the study with a precipitating tendency, this was correlated to c y t o t o x i c i t y , so that the minimal total inhibitory concentration was often a b o u t 5 times lower than the lowest drug concentration with a precipitate (drug crystals or drug-protein complex). Thus most drugs were water soluble at non-cytotoxic concentrations. The correlation may tentatively be explained by a cytotoxicity caused by hydrophobic drug properties, leading to a rapid penetration of the drug into cells and intracellular protein denaturation [32]. Some drugs had a very low c y t o t o x i c i t y combined with a strong tendency to precipitate, however. They formed crystalline precipitates within a wide concentration range, including non-cytotoxic concentrations. The strong tendency to precipitate might be due to a low dissociation of the drug combined with a very low water solubility o f the prevailing undissociated drug form. This low water solubility could lead to deficient penetration of the cell 292

interior (water phase) and thus explain the non~cytotoxicity. The latter drugs may be identified by a high ratio between their 7-day total inhibitory concentration and the maximal concentration at which no precipitate is formed (column 9, Table II). When such a ratio was determined for all drugs in this study, 12 drugs had a ratio above 100 (Nos. 49--51, 54, 137, 140, 141, 163--166 and 199). In spite of widely differing chemical formulas, 7 of these drugs, namely acetazolamide, chlordiazepoxide, diazepam, mephobarbital, primidone, phenytoin, and mephenytoin, have an anti-epileptic action in common. Since their mechanisms of action is not known [ 23,30] one may speculate on a possible connection between their anti-epileptic effect and their unique ability to precipitate at non~cytotoxic concentrations; perhaps the low water solubility of these drugs may lead to accumulation of non-cytotoxic (see above) drug form on or in synaptic or dendritic membranes of nerve cells, thereby stabilizing these membranes and counteracting excitability.

The relevance of the inhibitory drug cytotoxicity The value of experiments on in vitro cytotoxicity depends on their relevance for human drug toxicity. Only a few systematic investigations of this problem have been carried out previously [1--4]. Preliminary studies on the validity of determinations of drug toxicity to HeLa cells in the MIT-24 system were therefore performed by the author's group [31--34]. In those studies the MIT-24 toxicity to HeLa cells of 50 drugs of the present study have been compared with: (i) the toxicity of the same drugs to other cultured cells [31]; (ii) the systemic toxicity of the same drugs in mouse and man as expressed by lethal dosage [32]; and (iii) the systemic toxicity of the same drugs in man as expressed by lethal drug concentrations [33]. Conclusions from these and allied studies [17,18] have been presented in a summarized form [34]. In the first study [31] the inhibitory toxicity of 25 of the drugs in the present study to HeLa cells in the MIT-24 system was found to be grossly similar to the toxicity of the same drugs to various cultured cells tested by 8 other methods, reported in the literature. Differentiated cultured cells were generally more sensitive than less differentiated, but exhibited the same differential sensitivity to the drugs indicating a qualitatively similar (basal cytotoxic) action in both cell types. In the second study [32] the MIT-24 toxicity to HeLa cells of 50 compounds tested in the present study (Table II) was compared with the mouse i.v. LDs0 and the approximate human i.v. lethal dosage, recorded in the literature. Seven of the drugs (Nos. 5, 35, 118, 157, 178, 179 and 201 in Table II) were found to have a human lethal dosage considerably lower than their MIT-24 toxicity; these drugs are also known to be poisonous to humans by interfering with specialized neuroreceptors not found in tissue culture. The remaining drugs (including Nos. 3, 11, 15, 17, 18, 20, 39, 44, 55, 59, 60, 63, 67, 77, 83, 86, 88, 92, 96, 99, 108, 114, 126, 130, 132, 140, 143, 144, 145, 153, 154, 155, 160, 173, 182, 183, 184, 192, 194, 196, 197, 293

205) had similar in vitro and in vivo (mouse and man) toxicities, indicating a basal c y t o t o x i c lethal action in man. In the third study [32], MIT-24 toxic concentrations of 43 of the 50 previously analyzed drugs [31] were compared with human lethal concentrations of the same drugs, and for most of them a correlation between in vitro and in vivo toxicity data was found. Since the 50 drugs had been selected fairly randomly from the 205 drugs in Table II, it is possible that most drugs of the present study have an inhibitory toxicity for HeLa cells which may be relevant for the poisonous action of the same drugs in man. The mechanism of their lethal action in man could consequently be explored by further studies on the morphology and biochemistry of the HeLa cell reaction to such drugs in the MIT-24 system. REFERENCES 1 Mary Dawson, Cellular Pharmacology, Thomas, Springfield, 1972. " 2 A.N. Worden, Tissue culture, in E. Boyland and R. Goulding (Eds.), Modern Trends in Toxicology, Vol. 2, Butterworth, London, 1974, pp. 216--249. 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.G. Tardiff, Annu. Rev. Pharmacol. Toxicol., 18 (1978) 357. 5 H. Eagle and G.E. Foley, Cancer Res., 18 (1958) 1017. 6 I. Toplin, Cancer Res., 19 (1959) 959. 7 H.W. Gelboin, Mechanisms of induction of drug metabolism enzymes, in B.N. La Du, H.G. Mandel and E. Leong Way (Eds.), Fundamentals o f Drug Metabolism and Drug Disposition, Williams and Wilkins, Baltimore, 1971, pp. 279--307. 8 Ida S. Owens and D.W. Nebert, Molecular Pharmacol., 11 {1975) 94. 9 C.M. Pomerat and C.D. Leake, Ann. N.Y. Acad. Sci., 58 (1954) 1110. 10 J.L. Schmidt, F.C. McIntire, D.L. Martin, M. Anita Hawthorne, and R.K. Richards, Toxicol. Appl. Pharmacol., 1 (1959) 454. 11 K. Karzel, Arch. Int. Pharmacodyn., 169 (1967) 70. 12 B. Holmberg and T. Malmfors, Environ. Res., 7 (1974) 183. 13 I. Desi, G. Dura, J. Szlobodnyik and I. Czuka, J. Toxicol. Environ. Health, 2 (1977) 1053. 14 T.D.C. Grace and J. Mitsuhashi, The effects of insecticides on insect cells grown in vitro, in E. Weiss (Ed.), A r t h r o p o d Cell Cultures and Their Application to the Study of Viruses, Springer, Berlin, 1971, pp. 108--112. 15 ~ Pilotti, K. Ancker, E. Arrhenius and C. Enzell, Toxicology, 5 (1975) 49. 16 E.O. Dillingham, R.W. Mast, G.E. Bass and J. Autian, J. Pharm. Sci., 62 {1973) 22. 17 B. Ekwall and B. SandstrSm, Toxicol. Lett., 2 (1978) 285. 18 B. Ekwall and B. SandstrSm, Toxicol. Lett., 2 (1978) 293. 19 J. Paul, Cell and Tissue Culture, Churchill-Livingstone, Edinburgh, 1975. 20 H. Returner and H.J. Merker, Ann. N.Y. Acad. Sci., 123 {1965} 79. 21 A.H. Conney, Pharmaco!, Rev., ~9 (1967) 317. 22 R. Snyder and H. Returner, Pharmacol. Ther., 7 (1979) 203. 23 L.S. G o o d m a n and A. Gilman, The Pharmacological Basis of Therapeutics, Macmillan, London, 1970. 24 T.M. Brody and J.A. Bain, J. Pharmacol. Exp. Ther., 110 (1954) 148. 25 P. Arese and Amalia Bosia, Drugs affecting the mitochondrial metabolism, in S. Dikstein (Ed.), Fundamentals of Cell Pharmacology, Thomas, Springfield, 1974. 26 B.R. Fink and G.E. Kenny, Anesthesiology, 32 (1970) 300.

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27 28 29 30 31 32 33 34

A.C. Guyton, Textbook of Medical Physiology, Saunders, Philadelphia, 1976. J.R. Gillette, J.R. Mitchell and B.B. Brodie, Annu. Rev. Pharmacol., 14 (1974) 271. J.R. Gillette, Isr. J. Chem., 14 (1975) 193. D.M. Woodbury and J.W. Kemp. Pharmakopsyehiatr./Neuro-Psychopharmakol., 3 (1970) 201. B. Ekwall and A. Johanmon, Toxicol. Lett., 5 (1980) 299. B. Ekwall, Toxicol. Lett., 5 (1980) 309. B. Ekwall, Toxicol. Lett., 5 (1980) 319. B. EkwaU, Combined drug toxicity to HeLa cells in the MIT-24 test system and its relevance to human drug toxicity, Aeta Univ. Upsal., No. 353, Almquist & Wiksell, Uppula, 1980.

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