Toxicology, 5 (1975) 49--62 © Elsevier/North-Holland, A m s t e r d a m -- Printed in The Netherlands

E F F E C T S OF TOBACCO AND TOBACCO SMOKE CONSTITUENTS ON CELL M U L T I PL I C A T I O N IN V I T RO

AKE P I L O T T I b, K L A S A N C K E R a, E R I K A R R H E N I U S a'* and C U R T E N Z E L L b

aEnvironment Toxicology Group, Wallenberg Laboratory, Stockholm University, S-10405 Stockholm, and bChemical Research Department, Swedish Tobacco Co., S-I0462 Stockholm (Sweden) (Received O c t o b e r 14th, 1974) (Revision received March 24th, 1975) ( A c c e p t e d April 10th, 1975)

SUMMARY

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxi ci t y test system for t oba cco and t obacco smoke constituents. Some 250 c o m p o u n d s , representative of these materials, were examined by exposing cells to different concentrations of these constituents and measuring the inhibition of culture growth, which was related to corresponding effects e n c o u n t e r e d for positive standards. When employing the present cell to x icity test system possible effects of factors such as penetration, distribution and microsomal metabolism of the c o m p o u n d s studied, are n o t taken into account. The m os t active constituents were found to be unsaturated aldehydes and ketones, phenols and indoles. The good correlation observed between functional groups and toxicity permits, within the range of functionalities studied, prediction of the t oxi ci t y for a c o m p o u n d of know n structure.

INTRODUCTION

The toxic effects of a com pl e x mixture on higher organisms can be evaluated at various levels of com pl e x i t y, bot h with respect to the target organism and the material to which the target is exposed. In the case of t o b a c c o smoke effects on man, studies at a low level of c o m p l e x i t y involving effects of individual c o m p o u n d s on various cells and subcellular

* S u p p o r t e d by a research fellowship with the Swedish Natural Science Research Council, and a research grant f r o m the Swedish T o b a c c o C o m p a n y .

49

organelles have obvious advantages. Although n o t directly applicable to the human situation these systems provide more clear-cut information on the mechanisms involved in the interaction between specific smoke constituents and distinct biochemical cell functions, thus fulfilling a necessary condition for a p r o p e r understanding of the more complex situation. The aim of the present study was to obtain such basic information witho u t either taking into a c c o u n t such effects of penetration and distribution as are often e n c o u n t e r e d in more complex systems, or evaluating the additive, synergistic or antagonistic interactions frequently associated with complex mixtures. However, the very large n u m b e r of constituents in tobacco and to b acco smoke prevents a t hor oug h study of the effects of all of these on the various organelle functions. A pre-screening procedure is thus required for the selection of those constituents which affect at least one organelle function. Ascites t u m o u r cell cultures were used for this purpose; th ey were exposed to a n u m b e r of different substances at various concentrations. No specific cell function was studied and the toxic effect was measured only as the capacity of the studied c o m p o u n d to inhibit the growth rate of a cell culture. Although for reasons still to be discussed this system by no means pinpoints every biologically active substance, the information obtained nonetheless provides a basis for further studies of the effects p r o d u c e d on subcellular organelles by a limited n u m b e r of selected tobacco and t obacco smoke com pone nts.

METHOD

Test system The cell culture system used in the present e x p e r i m e n t is a furt her develo p m e n t [1] of an immunological c y t o t o x i c test system developed by Munroe et al. [ 2 ] . The cell strain Ascites sarcoma B P8 used here offers certain experimental advantages allowing the handling of larger experimental series. Thus these cells can be grown for many generations in test tubes w i t h o u t intermediate inoculation of mice, and t he y are therefore easily handled in in vitro systems [ 2 ] . Moreover the cells can easily be reinoculated in the abdominal cavity of mice, providing the possibility of comparing results obtained in vitro with those obtained in vivo, where the cells are simultaneously exposed to the metabolites of the studied c o m p o u n d produced within the mammalian host (cf. ref. 3). The p r o p e r t y of these cells n o t to adhere to the surface of the incubation vessel provides a further advantage as with high reproducibility they can be suspended by shaking and c o u n t e d with the aid of an electronic particle counter. Stem cell cultures originating from inoculated C3H mice were grown in test tubes in Hams F 10 medium sterilized by filtration (through Millipore 0.45 M), with fetal calf serum (15% w/w), penicillin (100 IU) and streptomy cin (100 IU) added. The test tubes were gassed with sterilized air con-

50

¢~

23

20 21 22

13 14 15 16 17 18 19

1 2 3 4 5 6 7 8 9 10 11 12

No.

4 - O x o n o n a n o i c acid 2 , 5 - D i h y d r o x y b e n z o i c acid 3,5-Dimethoxy-4-hy droxybenzoic acid N i c o t i n i c acid b

Acids

Anisole Diphenylether 4-Methylanisole 3-Methylanisole 2-Methoxynaphthalene 2-Ethoxynaphthalene 1,2,3-Trimethoxybenzene

Ethers

Methanol a Ethanol 2-Propanol 1-Octadecanol 0~-Terpineol Geraniol Solanesol Farnesol P r o p y l e n e glycol Furfurylalcohol Benzylalcohol 2-Phenylethanol

Alcohols

Compound

0

2 7 4

8 100 43 40 46 100 14

3 10 7 8 37 99 0 100 2 28 6 7

1

!3 9

17

6

6

84

0.1

0.01

0.001

% i n h i b i t i o n at level (mM)

40 41 42 43 44 45 46 47 48

27 28 29 30 31 32 33 34 35 36 37 38 39

24 25 26

No.

Nicotinamide N-Methylnicotinamide Acetonitrile Propionitrile Butyronitrile Isobutyronitrile Valeronitrile Isovaleronitrilc Acrylonitrile

A m i d e s and Nitriles

Diethyl malonate Ethyl stearate Vinyl acetate Methyl acrylate Benzyl acetate Benzyl benzoate Benzyl cinnamate Coumarin Diethyl phthalate Di-n-propyl phthalate Dibutyl phthalate Dioctyl phthalate d P h t h a l i c acid a n h y d r i d e d

Esters and anhydrides

I n d o l y l - 3 - a c e t i c acid K y n u r e n i c acid b T e r e p h t h a l i c acid b

Compound

TABLE I E F F E C T S O F T O B A C C O A N D T O B A C C O S M O K E C O N S T I T U E N T S ON C E L L C U L T U R E G R O W T H R A T E

21 4 15

4 4 44 12 6

5 7 3 2 6 100 73 34 44 100 45 32 14

1

22 35 7 4 68

0.1

0

0.01

0.001

% i n h i b i t i o n at level (mM)

bo

Ketones

Benzylcyanide Nicotinonitrile Indolyl-3-acetonitrile

Compound

52 2 - P r o p a n o n e 53 2-Butanone 54 6 , 1 0 - D i m e t h y l u n d e c a n - 2 - o n e 55 2,3-Butanedione 56 2 , 3 - P e n t a n e d i o n e 57 3-Buten-2-one 58 3 - M e t h y | - 3 - b u t e n - 2 - o n e 59 3-Penten-2-one 60 6-Methyl-5-hepten-2-one 61 6 - M e t h y l - 3 , 5 - h e p t a d i e n - 2 - o n e 62 S o l a n o n e 63 P s e u d o i o n o n e 64 fi-Ionone 65 C y c l o p e n t a n o n e 66 C y c l o h e x a n o n e 67 2 , 2 , 6 - T r i m e t h y l c y c l o h e x a n o n e 68 Piperitone 69 Carvenone 70 Carvone 71 A c e t o p h e n o n e 72 1 - P h e n y l - 2 - b u t a n o n e d 73 1 - P h e n y l - l - b u t a n o n e 74 l q n d a n o n e 75 9 - F l u o r e n o n e 76 2 , 3 , 6 - T r i m e t h y t - 1 , 4 - n a p h t h o quinone

49 50 51

No.

TABLE I ( c o n t i n u e d )

5 4 71

ll 11 100 100 100 5 100 100 96 100 100 100 100 7 0 22 31 33 100 9 6 9 9 63 100

1

51 100

l 13

9 9

100 99 9 100 33 100 20

2 22

9

77

3

3

0.01

51 37 34

0.1

3

0.001

% i n h i b i t i o n at level (rrdtl)

78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

77

No.

Methanal Ethanal Propanal Butanal Pentanal Hexanal Heptanal Octanal Nonanal Decanal Undecanal Dodecanal 2-Methylpropanal 3-Methylbutanal 2-Methylbutanal 2-Methylpentanal 2,2-Dimethylpropanal Glyceraldehyde d 2-Oxopropanal Propenal 2-Methylpropenal 2-Butenal 2-Hexenal 2,4-Hexadienal d 2-Methyl-2-pentenal ~-Cyclocitral Safranal Phenylethanal d

Aldehydes

(3-Pyridyl)- 1 - p r o p a n o n e

Compound

1

100 92 98 100 95 100 100 100 100 87 86 81 38 100 100 100 28 97 100 91 100 95 100 100 100 82 100 100

1

10 6 94 100 86 96 lO0 38 6 100 76

15 34 27 26 4 69 69 29 73 60 2 1 0 13 0 1

0.1

6 0

20 34 45 66 44 0

3 0

0 3

0.01

7

0.001

% i n h i b i t o n at level (raM)

123 124 125 126 127 128 129 130 131 132 133 134

121 122

Phenol o-Cresol m-Cresol p-Cresol 2,3-Dimethylphenol 2,4-Dimethylphenol 2, 5 - D i m e t h y l p h e n o l 2,6-Dimethylphenol 3,4-Dimethylphenol 3,5-Dimethylphenol 2,3,5-Trimethylphenol 2,4,5-Trimethylphenol

Phenols

3~Phenylpropanal d 3-Phenylpropenal Benzaldehyde 2-Methylbenzaldehyde 3-Methylbenzaldehyde 4-Methylbenzaldehyde 2-Hydroxybenzaldehyde 3-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde Anisaldehyde 3,4-Dihydroxybenzaldehyde Vanilline Veratrumaldehyde 5-Methylfurfural 5-Hydroxymethylfurfural 3-Indolecarboxaldehyde 3-Pyridinecarboxaldehyde

106 107 108 109 110 111 112 113 114 115 116 117 118 119

120

Compound

No.

TABLE I (continued)

25 56 31 93 78 99 74 79 75 44 81 88

100 100 4O 100 100 57 100 42 28 19 100 30 23 25 19 81 63

1

1

16

19 5 7 79

98

0 5 5 7 0 2

11

2

13

5 7 5

18

7

1 4

0.01

83 100

0.1

0.001

% i n h i b i t i o n at level (raM)

158 159 160 161 162 163

135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157

No.

cis-2-Pentene

Isopentane n-Eicosane 2-Methyl-l-butene 2-Methyl-2-butene Isoprene

Hydrocarbons

2,4,6-Trimethylphenol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol Thymol 3-Ethyl-5-methylphenol 2-Methoxyphenol 3-Methoxyphenol 4-Methoxyphenol 2,6-Dimethoxyphenol Eugenol Isoeugenol 2-Hydroxyacetophenone 3-Hydroxyacetophenone 4-Hydroxyacetophenone c~-Naphthol /3-Naphthol Catechol Resorcinol Hydroquinone Pyrogallo! 3-Methylcatechol 3-Isopropylcatechol

Compound

12 10 0 10 5 0

21 100 85 88 32 100 100 100 100

14

81 77 38 91 100 81 74 35 93 50 98 96 36

1

96 39 91 100

55

17 13

13

79 0 16

22 2 2 8

6 5

0.1

0 6

l

6

15

0.01

0.001

% i n h i b i t i o n at level (raM)

164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194

No.

1 , 3 - P e n t a d i e n e (cis/trans) Limonene Neophytadiene Cyclohexene (~-Pinene /3-Pinene Benzene Toluene m-Xylene p-Xylene Ethylbenzene n-Propylbenzene d Isopropylbenzene Styrene Phenylacetylene Indan Tetralin Indene Diphenylmethane Azulene Naphthalene 1-Methylnaphthalene 2-Methylnaphthalene 1,4-Dimethylnaphthalene Anthracene c Phenanthrene Benz [a] a n t h r a c e n e c Chrysene c Pyrene c Benzo[a]pyrene c Perylene c

Compound

TABLE I (continued)

6 100 2 3 100 100 4 9 40 32 43 100 100 25 34 100 100 100 100 85 100 100 100 100 12 22 27 9 33 6 0

1

17 7 6 10 12 35

5 7 9

4 10 3 3 3

3 10

11

0.1

0.01

0.001

% i n h i b i t i o n at level (raM)

207 208 209 210 211 212 213 214 215 216 217 218 219 220 221

201 202 203 204 205 206

195 196 197 198 199 200

No.

Pyrrolidine 3-Pyrroline Piperidine Pyrrole CarbazoleC 9-Ethylcarbazole Benzimidazole Indole 2-Methylindole 3-Methylindole 2,3-Dimethylindole 2,3,5-Trimethylindole Pyridine 2-Methylpyridine 3-Ethylpyridine

N-He terocycles

Tetrahydrofuran Furan 2,5-Dimethylfuran 2,3-Benzofuran Dibenzofuran Thiophene

Furanes, thiophenes

Picene c Coronene c 3-MethylcholanthreneC, d Acenaphthene Acenaphthylene Fluoranthene b

Compound

0 0 16 6 8 38 12 54 78 89 100 100 3 0 12

0 3 100 87 53 4

27 0 16 46 76 38

1

5 3 2 0 5

6 6 19

20

0.1

0.01

0.001

% i n h i b i t i o n at level (raM)

5~

28 28 4 100

Amines 1-Aminobutane 1-Amino-2-methyl-propane 2-Aminopentane 3-Aminopropene

235 236 237 238

a b c d

3 3 3 2 0 36 0 0 5 2 28 24 100

2,6-Dimethylpyridine 3,5-Dimethylpyridine 2,3,6-Trimethylpyridine 2,4,6-Trimethylpyridine 3-Pyridinole Quinoline Pyrazine 2-Methylpyrazine 2,6-Dimethylpyrazine Nicotine ~-Nicotyrine Anabasine Harman

222 223 224 225 226 227 228 229 230 231 232 233 234

100

66

9

52

5

0.01

4

0.001

256

239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255

No.

Dimethyl sulfoxide

Benzylamine Aniline 2-Methylaniline 3-Methylaniline 4-Methylaniline 2,3-Dimethylaniline 2,5-Dimethylaniline 2,6-Dimethylaniline 2,4,6-Trimethylaniline 2-Ethylaniline 4-Ethylaniline N-Ethylaniline Diphenylamine (~-Naphthylamine ~-Naphthylamine 2,6-Diaminotoluene 3,4-Diaminotoluene Other c o m p o u n d s

Compound

3

I3 30 1 0 34 16 12 10 37 14 2 28 100 100 72 0 87

1

8

15 8 14

0.1

0.01

0.001

% i n h i b i t i o n at level (raM)

Values n o t statistically significant (p > 0.001 ) are given in italics. C o m p o u n d s a d d e d o n l y as DMSO s o l u t i o n s ; all o t h e r c o m p o u n d s were e x a m i n e d as e t b a n o l s o l u t i o n s , e i t h e r exclusively or in b o t h solvents. C o m p o u n d s n o t c o m p l e t e l y soluble in DMSO a n d t h e r e f o r e c o u n t e d m a n u a l l y . C o m p o u n d s n o t y e t f o u n d in t o b a c c o or t o b a c c o s m o k e .

1

0.1

% i n h i b i t i o n at level (raM)

Compound

No.

TABLE I (continued)

taining 5% carbon dioxide and capped air-tight to maintain a stable pH of a p p r o x i m a t e l y 7.3 (phenol red maintained at pink to yellow). The cell cultures were reinoculated to a cell density of 0.1 • 104 cells/ml every 5th day. The cell densities were calculated with an electronic cell c o u n t e r (Celloscope 401, Linson Instr. AB, Stockholm, Sweden). For the tests, the cell suspension was diluted with sterile medium to an initial cell density of 0.4 • 1 0 4 cells/ml. The c o m p o u n d s to be tested were dissolved in ethanol (10 ill) or dim e t h y l sulfoxide (10 pl) and added to this suspension (3 ml), each in amounts to give the final concentrations indicated in Table I. All c o m p o u n d s were incubated at 37 ° for 48 h. Solvent (10 t~l) was added to the controls. After gassing and capping, the tubes, enclosed in a gassed (air, 5% carbon dioxide) and sealed plastic box to prevent errors due to pH-changes caused by gas leaks into single tubes, were incubated obliquely. TESTING

AND EVALUATION

PRINCIPLES

The tests were run in duplicates and the cells were allowed to grow for 48 h. During this period the n u m b e r of cells in the controls increased exponentially, i.e. d N / d t = k N where N is the cell density and t is the time, indicating that none of the essential growth factors in the medium was limiting. The growth rate of an incubated cell culture was calculated and compared to the average value of 8--10 controls p e r f o r m e d in each series. The doubling time for co nt r ol cultures was approximately 24 h. No systematic distinction was made between viable and total cell count. The effect of the tested comp o u n d is given as the ratio between the growth rates of the incubated cell culture and the controls, expressed as a percentage. The growth rate of a cell culture, v, was calculated using ln(N/No ) t - i n 2 - (No = cell density at the start of the experiment).

As the growth rate may vary between the different series, each series also contained 3 or m or e internal standards in duplicate, i.e. fixed concentrations of substances having d o c u m e n t e d inhibiting effects. These substances were in most cases 2-aminonaphthalene, propranal, quinoline and isopentone. The relative effects of these internal standards were the same for the different series and i n d e p e n d e n t of growth rate. All substances were initially tested by exposing the cells to a final concentration of 1 mM (cell medium + solvent). When at this concent rat i on a comp o u n d inhibited growth rate to 50% or more, further experiments at lower concentrations were p e r f o r m e d (0.1, 0.01 mM, etc.) until a concent rat i on was reached which p r o d u c e d an inhibitory effect below 50%. The fact that many c o m p o u n d s were not tested at the lower c o n c e n t r a t i o n does not, of course, imply that all of them were inactive. In most cases when the inhibiting effect was within the 15--50% region, thus eliminating the need for

56

further testing at lower concentrations, the inhibiting effect was still statistically significant (P < 0.001). The compounds tested were primarily selected on the basis of their abundance in tobacco or tobacco smoke; availability as well as solubility in ethanol or dimethyl sulfoxide were limiting factors, however. Included as well were some tobacco and tobacco smoke constituents having d o c u m e n t e d biological effects. The purity of the compounds was tested by thin-layer chromatography, NMR or gas chromatography. To obtain additional information on ,steric or substitutional effects on the biological activity, substances which are chemically related to the compounds selected according to the above principles were tested irrespective of their occurrence in tobacco or tobacco smoke. Most of these compounds are, however, also tobacco or tobacco-smoke constituents. RESULTS The results are summarized in Table I. The substances are subdivided according to their functional groups, which does not, however, imply that the functional group is responsible for the biological activity. In spite of this, the effects of the compounds will be discussed essentially in that order below. Alcohols. Most of the alcohols (1--12) were found to be non-toxic; interesting exceptions were some of the isoprenoid alcohols (5--8) which are fundamental units in the biosynthesis of higher terpenoids. Since the allylic but not the benzylic alcohols displayed activity, the --C=C--C--OH unit J rather than the h y d r o x y l group itself appears to be the ~critical chemical entity. Ethers. The effect of anisol (13) is negligible but increases on alkyl substitution of the aromatic ring (15,16). Enlargement of the aromatic as well as of the alkyl unit also seems to increase the toxicity. These effects require further study. Acids. Of the acids (20--26), only indolyl-3-acetic acid (24) displayed some activity. In view of the results obtained for the unsaturated alcohols this group will later be extended to include polyunsaturated fatty acids (PUFA). Esters and anhydrides. All members of this group (27--39) which comprise an aromatic acid exhibit toxic effects except the anhydride (39). The magnitude varies with the alcohol moiety; most remarkable is the effect of the chain length in the case of the phthalic esters (35--38). A similar effect has been observed for gallic acid esters [4], where maximum disturbance of the intact function of the microsomal detoxication chain of liver cells is displayed by compounds having a chain length of 8 carbons. It is also of interest that certain esters are capable of spontaneously forming biologically active electrophilic derivatives under adequate pH conditions [5]. A m i d e s and nitriles. The amides and nitriles (40--51), with the excep-

57

tion of valeronitrile (46) and indolyl-3-acetonitrile (51), are inactive. Part of the effect found for indolyl-3-acetonitrile may possibly be associated with the indole ring system as indolyl-3-acetic acid (24) was the only acid studied (20--26) showing some activity while all other indoles examined were found to be toxic. These findings are somewhat surprising since nitriles constitute a group including m a n y biologically active compounds. Thus besides the abovementioned indolyl-3-acetonitrile, which is a plant growth hormone [6], phthalonitrile and di- and trichloroacetonitriles have been used as insecticides and malononitrile is of psychopharmacological interest [7]. Moreover, fi-aminopropionitrile is known as the Lathyrus factor in sweet peas causing bone damage in newborn rats [8]. The toxicity of nitriles is considered to be dependent on their metabolism to cyanide ions; this type of toxicity will thus depend on where this metabolism takes place [7]. This group may thus represent a case in which the present test system, due to lack of detoxicating metabolism in the ascites cells, produces inadequate results. The nitriies should therefore be examined in a test system involving metabolic detoxication functions. Ketones. The ketones (52--77) display -- not unexpectedly in view of the fairly large number of representatives -- great variation in toxicity. Of the saturated acyclic monoketones (52--54), only 6,10-dimethylundecan-2one (54, tetrahydrogeranylacetone), a flavour compound, is active. In contrast, the corresponding diketones (55,56) and the unsaturated acyclic monoketones (57--63), except for 3-buten-2-one (57, methylvinylketone) are toxic. The situation is much the same for the cyclic compounds (64--77), where the presence of a double bond (68--70) or an additional oxo group (76) is also a requirement for toxicity. The effect of the double bond is evidently larger in conjugation with the oxo group. Since this effect is absent when the double bond is replaced by an aromatic nucleus (71,73-75,77) it appears that, as in the case of the unsaturated alcohols, the double bond has a considerable impact. A possible explanation for this is the capacity of a,fi-unsaturated ketones to be hydrated producing molecules which like the diketones should be prone to complex metals, thus directly or indirectly affecting metallo-enzymes (vide infra) [9]. Aldehydes. Virtually all aldehydes (78--122) are toxic, even the saturated aliphatic (78--94). The activity of the latter are only moderately affected by the different side-chains and the most pronounced effect, apparently associated with steric hindrance, is observed for 2,2-dimethylpropanal (94). A small dependence on chain length is also observable for the n-alkyl aldehydes (78--89); the C6-C10 representatives (83--87) are somewhat more toxic than the rest. Introduction of a conjugated double bond in the aliphatic aldehydes (97-102) clearly increases toxicity, but no such effect is observed on conjugation with an aromatic ring. The importance of the double bond observed in the case of the ketones thus reappear in that of the aldehydes. Both nonaromatic cyclic aldehydes (103,104) are toxic, roughly to the extent ex-

58

pected on the basis of the above-mentioned counteracting steric and conjugative effects. Substitution of ~n aromatic hydrogen in benzaldehyde (108) by a methyl group (109--111) increases activity, while substitution by hydroxyl or m e t h o x y groups (112--118), has no effect except when a chelating capacity is produced (112,118). The aromat:c aldehydes frequently occur in essential oils and plant extracts; they are used to a large extent in perfumes. N-tteteroaromatic aldehydes (121,122) appear more toxic than O-heteroaromatic aldehydes (119,120). Generally, aldehydes and ketones are slowly metabolised in the mammalian body. The main conversions are oxidation and reduction to acid or alcohol, respectively. This should be kept in mind when evaluating the toxicity of ketones and aldehydes as the toxicity in intact animals largely depends on the degree of conversion of these compounds to the relatively nontoxic alcohols and acids. Phenols. Of the phenols examined (123--157), all but 3-hydroxyacetophenone (148) are toxic. The moderate toxicity of phenol {123) is increased on the introduction of electron-donating substituents such as alkyl (124-140), h y d r o x y l (152--157) and m e t h o x y (141--146) groups, especially in o r t h o and para positions. Similarly, insertion of electron-withdrawing substituents such as acyl (147--149), aldehyde ( 1 1 2 - - 1 1 4 , 1 1 6 , 1 1 7 ) a n d carboxyl (21,22) groups either creates chelating configurations (112) or lowers the activity or is essentially without effect. Incorporation of alkyl substituents containing olefinic bonds (145,146) has a very minor effect, in agreement with the aforementioned assumption that chelating properties are obtainable only when the allylic carbon is oxygenated. In contrast, extension of the aromatic system of phenol to that of a- and ~-naphthol (150,151) increases the activity somewhat. The polyvalent phenols (152,154--157) which may form quinones are efficient growth inhibitors for the cell culture. Since the same effect is observed for the non-chelating hydroquinone (154), and its m o n o m e t h y l ether (143), it could depend on the electron donor capacity of these compounds and be due to their interference with some of the electron transport functions of the cell. Substituted phenols are of great interest in view of their well documented uncoupling effect on the mitochondrial oxidative phosphorylation, which may disturb several energy-consuming functions in the cell [10]. One such disturbance of great importance in tobacco toxicology is ciliotoxicity; mitochondrial respiration experiments as well as ciliotoxicity studies are presently being performed on the active compounds. Another, relatively new aspect of the toxicity of substituted phenols is the probable disturbance of the intact function of liver cell detoxication enzymes in in vitro systems through interference with the electron transfer between the flavoprotein enzyme and cytochrome P 450 [11]. This causes a change in the metabolism of, among others, aromatic amines which may favour the production of toxic and carcinogenic electrophilic metabolites. If this occurs in vivo, the phenols could act as carcinogens. Such studies of

59

the active compounds using liver microsomal systems are presently under way. Hydrocarbons. The acyclic hydrocarbons (158--164,166), whether unsaturated or not, are non-toxic. Of the cyclic, non-aromatic hydrocarbons (165,167--169), the monoterpenes (165,168--169) are active. The latter are all readily auto-oxidized giving a,~-unsaturated alcohols and ketones [12], a fact which may serve to explain their activity along the lines mentioned previously. In fact, a-pinene has long been known for its strong toxic action on liver and kidney on oral administration resulting in albuminuria in sheep [13]. Benzene (170) and toluene (171) are non-toxic, but further substitution or increase in the number of carbons in the substituent yields active compounds (172--182). Here, as in the aforementioned aromatic compounds (145,146), introduction of olefinic bonds (177,181) is without effect. The toxicity of azulene (183) and the naphthalenes (184--187) is about the same as that of the active hydrocarbons containing only one aromatic ring (172--181). The results for the polycyclic aromatic hydrocarbons (188--200), which indicate a low activity, are less certain on account of experimental difficulties. Thus since some of the heavier compounds (4,7,28,188,190--197,211) did not dissolve completely in DMSO, the cells had to be counted manually with a notable decrease in accuracy and reproducibility. Furans, thiophene. Tetrahydrofuran (201), furan (202) and thiophene (206) are non-toxic, while the substituted furans ( 2 0 3 - - 2 0 5 ) s h o w e d inhibitory effects. The activity of these compounds parallel that of the corresponding N-heterocycles. N-Hereocycles. Except for the weakly active piperidine (209), all monocyclic, aromatic as well as non-aromatic, N-heterocycles (207,208,210,219-226,228--230) are non-toxic. Similarly, the tobacco alkaloids (231--233), which have two heterocyclic rings linked together by a single bond display no or very weak activity, while the bicyclic quinoline (227) and indoles (214--218) are toxic. As mentioned previously, the indole ring system per se seems to be toxic and this is supported by the fact that both carbazole (211) and benzimidazole (213) are non-toxic. In contrast to the phenols, neither pyridines nor indoles show any marked increase in toxicity on alkylation. Indoles include many biologically active compounds such as tryptophan, reserpine, auxines and lysergic acid derivatives and are thus of great interest. The results obtained here must, however, be checked against their possible effect on the level of SH amino acids in the cell cultures as Wellens [14--16] has shown that growth inhibition can occur in animals due to extensive sulphate conjugation of indole which in turn gives rise to a deficiency of SH amino acids. This effect can be counteracted by cysteine, cystine, methionine or glutathione, compounds which are to be added in future tests of the toxic activity of indoles in our system. Amines. Of the aliphatic amines (235--239) only 3-aminopropene (238,

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allylamine) is toxic and in remarkably low c o n c e n t r a t i o n (0.005 mM). This situation parallels t hat e n c o u n t e r e d for the alcohols, aldehydes and ketones and may be explained along the same lines. It is also parallel in the sense that the anilines (240--255) show much less or no activity. Most active of the latter c o m p o u n d s are diphenylamine (251), a- and ~-naphthylamines (252,253) and 3,4-diaminotoluene (255). Of these, 3,4-diaminotoluene meets the criteria for a metalligand in having two properly spaced atoms possessing free electron pairs. The naphthylamines need metabolic activation in the microsomal d e t o x i c a t i o n e n z y m e chain in order to exert their carcinogenic ef f ect [17] ; these d e t o x i c a t i o n systems, however, are absent in all cell culture systems which have been cultivated for several generations. Thus the toxic ef f ect observed here must be due either to some other functional disturbances or to a non-enzymatic conversion of the amine leading to similar reactive metabolites as those p r o d u c e d enzymatically by the detoxication enzymes of liver cells [ 1 8 ] . No effects of alkyl substitution in the aromatic ring were observed for the anilines, but the toxicity increased s o mewh at with N-substitution (250,251), and in the presence of anot her amino group. GENERAL CONCLUSIONS It should be poi nt e d out that the test system has limitations which must be considered when evaluating the results. Thus as m e n t i o n e d above these cells do n o t possess the det oxi cat i on e n z y m e systems which in many cells of the intact animal convert foreign c o m p o u n d s to non-toxic excretable products or to reactive intermediates which may damage cell functions. The present study must therefore be c o m p l e m e n t e d by studies involving metabolic systems in order to identify those substances whose metabolic products (rather than the c o m p o n e n t s themselves) are active. Such test systems, based on a co mb in atio n of the present cell culture system and h e p a t o c y t e cultures [19,20] or microsomal preparations [ 2 1 ] , are being developed. T he results obtained here are presently utilized for furt her studies on subcellular organelle systems to obtain further information regarding the functional background of the observed toxic effects. ACKNOWLEDGEMENTS The authors are indebted to Miss Pia Nyberg for skillful technical assistance. REFERENCES 1 E. Arrhenius and K, Ancker, in J. Nilsson (Ed.), Fluorescent Whitening Agents, MVC-Report 2, Swedish National Science Research Council, 1973, p. 83. 2 T.S. Munro and D.D. Porteous, Int. J. Radiat. Biol., 21 (1972) 87. 3 B. Holmberg, Personal communication, material in manuscript.

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4 E. Arrhenius, Unpublished. 5 E.C. Miller and J.A. Miller, in Twenty fourth Annual Symposium on Fundamental Cancer Research 1971, Williams and Wilkins, Baltimore, 1972, p. 5. 6 H.B. Henbest, E.R.H. Jones and G.F. Smith, J. Chem. Soc., (1953) 3796. 7 R.T. Williams, Detoxication Mechanisms, Chapman and Hall, London, 1959, p. 390. 8 J.J. Clemmons and D.M. Angevine, Federation Proc., 15 (1956) 512. 9 J.L. Simonsen and L.N. Owen, The Terpenes I, 2nd ed., Cambridge Univ. Press, Cambridge, 1953, p. 154. 10 A.L. Lehninger, The Mitochondrion, Benjamin, New York, 1964. 11 E. Arrhenius, in D. Shugar (Ed.), Biochemical Aspects of Antimetabolites and of Drug Hydroxylation, FEBS Symposium, Vol. 16, Academic Press, New York, 1969, p. 209. 12 J.L. Simonsen and L.N. Owen, The Terpenes II, Cambridge Univ. Press, Cambridge, 1957, pp. 138,199. 13 J.M. Harvey, University of Queensland Papers, 1 (1942) 23. 14 G. Wellens, Bull. Soc. Chem. Biol., 35 (1953) 1341. 15 G. Wellens, Bull. Soc. Chem. Bi01., 35 (1953) 1353. 16 G. Wellens, Bull. Soc. Chem. Biol., 36 (1954) 1655. 17 D.B. Clayson, Chemical Carcinogenesis, Little, Brown, Boston, 1962. 18 E. Arrhenius, Carcinogenic Amines. Some Aspects of their Metabolism and Interaction with Microsomal Functions of Liver Cells, Dissertation, Dept. of Cell Physiol., Wenner-Gren Institute, University of Stockholm, Sweden, 1968. 19 P.T. Yipe, in L. Tomatis and R. Montessano (Eds.), Chemical Carcinogenic Essays, Publ. 10, Intern. Agency Res. Cancer Lyon 1974, p. 119. 20 E. Huberman, in L. Tomatis and R. Montesano (Eds.), Chemical Carcinogenic Essays, Publ. 10, Intern. Agency Res. Cancer Lyon 1974, p. 137. 21 B.N. Ames, W.E. Durston, E. Yamasaki and F.D. Lee, Proc. Natl. Acad. Sci. (U.S.), 70 {1973) 2281.

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Effects of tobacco and tobacco smoke constituents on cell multiplication in vitro.

Ascites sarcoma BP8 cells, cultured in suspension in vitro were used as a general toxicity test system for tobacco and tobacco smoke constituents. Som...
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