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Effect of tobacco smoke compounds on the plasma membrane of cultured human lung fibroblasts
Toxicology, 15 (1980) 203--217 © Elsevier/North-Holland Scientific Publishers Ltd.
E F F E C T O F TOBACCO SMOKE COMPOUNDS ON THE PLASMA MEMBRANE OF CULTURED HUMAN LUNG FIBROBLASTS
MONICA
THELESTAM,
a MARGARETA
CURVALL
b and C U R T
R. E N Z E L L b
aDepartment of Bacteriology, Karolinska Institute& 8-104 01 Stockholm and bResearch Department, Swedish Tobacco Company, P.O. Box 17007, S-104 62 Stockholm
(Sweden) (Received February 18th, 1980)
(Revision received April 3rd, 1980) (Accepted April llth, 1980)
SUMMARY
The ability of compounds derived from tobacco and tobacco smoke to increase the permeability o f the membranes of h u m a n lung fibroblasts has been studied by measuring the release o f an intracellular marker after short term exposure. Of the 464 c o m p o u n d s tested, about 25 per cent gave rise to severe membrane damage. The most active compounds, when divided according to functionality, were found within the groups o f amines, strong acids and alkylated phenols, whereas nitriles and polycyclic aromatic hydrocarbons were found completely inactive. A pronounced effect o f the chain length on the activity was observed for the aliphatic alcohols, aldehydes and acids, and all monocyclic aromatic compounds but benzonitriles and benzoic acids showed an increase in activity with increasing alkylsubstitution. It is concluded t h a t tobacco smoke contains a n u m b e r o f membrane damaging substances. These membrane active compounds could not only cause direct toxic reactions but also potentiate the toxic effect by promoting the cell membrane penetration o f other toxic substances in tobacco smoke.
INTRODUCTION Some 3000 chemical substances have been detected in cigarette smoke so far [1,2], but little is known about their biological activity. Various in vitro methods have been devised with a view to clarifying the mechanisms behind the in vivo effects of smoke, and most o f the in vitro test systems used are based on effects at the cellular and subcellular levels. Thus effects Address correspondence to: Monica Thelestam, PhD, Department of Bacteriology, Karolinska Institute, S-104 01 Stockholm, Sweden.
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of tobacco smoke or single smoke components on tissue culture cells have been investigated using criteria such as: cell morphology [3], beating of rat heart cells [4], cell multiplication [5], DNA-content [6], cell transformation to malignancy [7], phagocytic functions [8], cell aging [9], mutagenesis [10], and cell respiration [11, Pettersson, B. et al., unpublished]. Furthermore, the mutagenic activity of individual smoke components on bacteria has been studied recently [Florin, I. et al., unpublished]. The present work comprises a study of the effects of tobacco and tobacco smoke constituents on plasma membranes of human lung fibroblasts in vitro. The method used is based on the simple principle that leakage of intracellular substances indicates damage to the plasma membrane and that the molecular size of the leaked material serves as an indicator of the degree of membrane damage in terms of the size of the "holes" induced by the test compounds [12]. A low molecular weight marker, uridine nucleotides [13], and a short exposure time were employed in this study to achieve high sensitivity and avoid secondary effects arising from cytotoxic damage. MATERIALS AND METHODS Biochemicals Eagle's minimal essential medium, new born calf serum and crude trypsin were purchased from Flow Laboratories, Ltd., Irvine, Scotland, Hank's balanced salt solution from the National Bacteriological Laboratory, Stockholm, Sweden, [5-3H]uridine and Aquasol ® Universal Cocktail from NEN Chemicals GmbH, Frankfurt, F.R.G. and tris(hydroxymethyl)-aminomethane, analytical grade, from E. Merck, Darmstadt, F.R.G. Compounds examined 464 compounds, most of them abundant in tobacco and tobacco smoke, have been examined. Those substances, which were not commercially available were either isolated from tobacco, (e.g. the terpenoids) or synthesized from available starting compounds, using adopted methods of synthesis, (e.g. ethers, esters, indoles and nicotine analogues). All compounds were checked for purity using thin-layer chromatography, gas chromatography and NMR. Compounds containing more than 3 per cent impurity were purified using liquid chromatography, recrystallization and distillation. The structures of the compounds studied were confirmed by [1H]- and [laC]NMR, e.g. the substitution pattern of multisubstituted aromatic substances and the branching pattern of methylsubstituted long chain alkyl derivatives. Cultivation and labelling of cells Human diploid embryonic lung fibroblasts (line MI~C-5) were cultivated in Eagle's medium [14] in polystyrene wells to a cell density of 10 s cells/ cm 2 (~ 7 × l 0 s cells/well). The cells in confluent monolayers were labelled with [3H]uridine to obtain a low molecular weight cytoplasmic marker, consisting of uridine nucleotides [ 13]. 204
Testing procedure and calculation o f results Labelled cultures were washed 3 times with Hank's balanced salt solution and then incubated for 30 rain at 370C with test c o m p o u n d s diluted to 25 mM in Tris-buffered saline, i.e. 0.15 mM NaC1 with 0.02 M tris--HC1 (pH 7.0). Then the solution containing leaked radioactive marker was removed and centrifuged (1000 g, 10 min, 4°C) and radioactivity in 0.1-ml aliquots of the s u p e m a t a n t was measured b y liquid scintillation [13]. A maximal release of the radioactivity was obtained b y treating control cells for 30 min with 0.06 M sodium borate buffer (pH 7.8) and scraping with a rubber policeman. This treatment ruptured the cell membranes leaving the nuclei intact [13]. The following expression was used to calculate the relative leakage of the radioactive marker: experimental release - spontaneous release× 100 maximal release - spontaneous release The spontaneous release of the nucleotide marker during incubation for 30 min at 37°C was 3--7 per cent of the maximal release [15]. RESULTS
In order to simplify the interpretation of the results, which are summarised in Table I, the c o m p o u n d s have been divided into 12 chemical classes according to functionality, and the degree of membrane damage classified as high (>70% nucleotide release), moderate (70--45%) and nil (<15%).
Alcohols The degree of membrane damage caused by the alcohols varied considerably. Thus, out of a homologous series o f 15 primary aliphatic alcohols, the C6--C10 representatives were highly active, whereas the CI--Cs and C12--C20 alcohols did n o t induce any significant membrane damage. All the isoprenoid alcohols, cyclic or acyclic, saturated or unsaturated were also highly potent. By contrast,-, the cyclohexanols were inactive, except for 4-isopropylcyclohexanol, which m a y be viewed as a nor-isoprenoid. Ethers Most of the ethers were found to be without-any effect on the cell membrane. Only 1-methoxynaphthalene was strongly active. All the anisoles were inacti*e, or weakly active showing a slightly increased activity u p o n increasing alkyl substitution. Enlargement of the aromatic ring system seems to cause an increased activity, while extension o f the alkyl unit appears to be without any effect. Acids Many members of this group caused severe damage to the cell membrane. The activity o f the n-alkyl acids varied with the length o f the carbon chain
205
TABLE I MEMBRANE DAMAGING ACTIVITY OF 464 TOBACCO SMOKE COMPONENTS No.
Compound
Nucleotide release
No.
Compound
(%) ALCOHOLS 1 Methanol 2 Ethanol 3 2-Propanol 4 1-Butanol 5 1-Pentanol 6 1-Hexanol 7 1-Heptanol 8 1-Octanol 9 1-Nonanol 10 1-Decanol 11 1-Dodecanol 12 1-Tridecanol 13 1-Pentadecanol 14 1-Heptadecanol 15 1-Oetadecanol 16 1-Eicosanol. 17 2-Buten-l-ol 18 4-Penten-l-ol 19 2-Hexen-l-ol 20 2,4-Hexadien-l-ol 21 Tetrahydrolinalool 22 Linalool 23 Tetrahydrogeraniol 24 Geraniol 25 Farnesol 26 Cyclohexanol 27 2-Methylcyclohexanol 28 2,6-Dimethylcyclohexanol 29 4-Isopropylcyclohexanol 30 Menthol 31 Piperitol 32 ~-Terpineol 33 ~-Ionol 34 Propylene glycol 35 Furfurylalcohol 36 Benzylalcohol 37 2-Hydroxybenzylalcohol 38 3-Hydroxybenzylalcohol 39 4-Hydroxybenzylalcohol 40 2-Phenylethanol ETHERS 41 Anisole 42 3-Methylanisole
206
0 2 0 0 0 100 100 100 100 100 12 8 24 0 0 0 6 8 5 4 81 100 100 100 100 0 0 3 96 99 89 85 100 0 95 7 3 3 4 0
4 12
Nuclotide release
(%)
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
4-Methylanisole 2,6-Dimethylanisole 3,5-Dimethylanisole 2,3,5-Trimethylanisole 2,4,6-Trimethylanisole 3,4,5-Trimethylanisole 4-Ethylanisole 4-n-Propylanisole 4-(2-Propenyl)anisole Ethylphenylether Diphenylether 1-Methoxynaphthalene 2-Methoxynaphthalene 2-Ethoxynaphthalene 1,2,3-Trimethoxybenzene
ACIDS 58 Methanoic acid 59 Ethanoic acid 60 Propanoic acid 61 Butanoic acid 62 Pentanoic acid 63 Hexanoic acid 64 Heptanoic acid 65 Octanoic acid 66 Nonanoic acid 67 Decanoic acid 68 Undecanoic acid 69 Dodecanoic acid 70 Pentadecanoic acid 71 Eicosanoic acid 72 2-Methylpropanoic acid 73 2-Methylbutanoic acid 74 2-Methylpropenoic acid 75 2-Butenoic acid 76 Phenylethanoic acid 77 3-Phenylpropanoic acid 78 3-Phenylpropenoic acid 79 Benzoic acid 80 2-Methylbenzoic acid 81 3-Methylbenzoic acid 82 4-Methylbenzoic acid 83 2,4,6-Trimethylbenzoic acid
7 22 23 24 37 24 18 15 36 11 11 81 51 35 0
67 17 11 17 61 74 73 71 83 81 85 82 13 8 53 79 7O 72 79 82 100 86 84 8O 83 89
TABLE I (Continued)
No. Compound
Nucleotide release
No.
Compound
(%) 84 85 86 87 88 89 90 91 92 93 94 95 96
4-Ethylbenzoic acid 2-Isopropylbenzoic acid 2-Methoxybenzoic acid 3-Methoxybenzoic acid 2-Hydroxybenzoic acid 2,5-Dihydroxybenzoic acid 3,5-Dimethoxy-4-hydroxybenzoic acid 2-Acetoxybenzoic acid 3-Pyridine carboxylic acid Indole-3-acetic acid 5-Hydroxyindole-3-acetic acid 4-Hydroxy quinaldic acid p-Phthalic acid
76 70 96 84 50 91 75 67 27 100 72 0 11
ESTERS 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
Vinyl acetate Ethyl acetate Phenyl acetate Benzyl acetate 2-Methylphenyl acetate 3-Methylphenyl acetate 4-Methylphenyl acetate 2,6-Dimethylphenyl acetate 3,5-Dimethylphenyl acetate 2,3,5-Trimethylphenyl acetate 2,4,6-Trimethylphenyl acetate Methyl propenoate Methyl pentanoate Ethyl pentanoate Ethyl stearate Methyl benzoate Ethyl benzoate Benzyl benzoate Methyl 2-methylbenzoate Methyl 3-methylbenzoate Methyl 4-methylbenzoate Methyl 2,6-dimethylbenzoate Methyl 3,5-dimethylbenzoate Methyl 2,4,6-trimethylbenzoate Methyl 2-isopropylbenzoate Methyl 3-phenyipropenoate Ethyl 3-phenylpropenoate Benzyl 3-phenylpropenoate
0 0 5 1 6 49 52 76 82 86 58 0 0 0 0 20 31 30 65 100 92 85 93 100 67 13 19 14
Nuclotide release
(%) 125 126 127 128 129 130
Coumarin Diethyl malonate Diethyl phthalate Di-n-propyl phthalate Di-n-butyl phthalate Di-n-octyl phthalate
4 0 10 10 18 1
NITRILES 131 Ethanonitrile 132 Propanonitrile 133 Butanonitrile 134 Pentanonitrile 135 Hexanonitrile 136 Heptanonitrile 137 Octanonitrile 138 2-Methylpropanonitrile 139 3-Methylbut anonitrile 140 Propenonitrile 141 Phenylethanonitrile 142 3-Phenylpropanonitrile 143 3-Phenylpropenonitrile 144 Benzonitrile 145 2-Methylbenzonitrile 146 3-Methylbenzonitrile 147 4-Methylbenzonitrile 148 2,3-Dimethylbenzonitrile 149 2,4-Dimethylbenzonitrile 150 2,5-Dimethylbenzonitrile 151 2,6-Dimethylbenzonitrile 152 3,4-Dimet hylbenzonitrile 153 2,4,6-Trimethylbenzonitrile 154 2-Ethylbenzonitrile 155 3-Ethylbenzonitrile 156 4-Ethylbenzonitrile 157 1-Naphthonitrile 158 2-Naphthonitrile 159 Nicotinonitrile 160 Indolyl-3-acetonitrile
5 7 6 0 4 0 10 0 5 2 5 0 5 3 8 0 4 4 2 4 2 5 19 54 15 9 33 2 0 30
KETONES 161 162 163 164 165 166 167
2-Propanone 2-Butanone 2-Pentanone 2-Hexanone 2-Heptanone 2-Decanone 4-Methyl-2-pentanone
0 1 0 1 2 36 10
207
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
release
(%)
(%) 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
208
6-Methyl-2-heptanone 3-Buten-2-one 3-Methyl-3-buten-2-one 3-Penten-2-one 6-Methyl-5-hepten-2-one 6-Methyl-3,5-heptadien-2-one 2,3-Butanedione 2,3-Pentanedione Cyclopentanone Cyclohexanone 2-Methylcyclohexanone 3-Methylcyclohexanone 4-Methylcyclohexanone 2,6-Dimethylcyclohexanone 2,2,6-Trimethylcyclohexanone 4-Isopropylcyclohexanone 2-Cyclohexenone 3-Methyl-2-cyclohexenone 3,5-Dimethyl-2-cyclohexenone Isophorone Piperitone Carvenone Carvone Pseudoionone ~-ionone Acetophenone 2-Methylacetophenone 3-Methylacetophenone 4-Methylacetophenone 2,4-Dimethylacetophenone 2,5-Dimethylacetophenone 2,4,6-Trimethylacetophenone 1-Phenyl-l-propanone 1-(2-Methylphenyl)-l-propanone 1-(3-Methylphenyl)-l-propanone 1-(4-Methylphenyl)-l-propanone 1-Phenyl-2-propanone 1-Phenyl-l-butanone 3-Methyl-l-phenyl-1-butanone 1-Phenyl-2-butanone 1-Phenyl-l-pentanone 1-Phenyl-l-octanone 1-Indanone 9-Fluorenone 2,3,6-Trimethyl-l,4-napthoquinone ( 3-Pyridyl)-1-propanone
5 4 35 32
12 8 0 5 1 2 0 0 3 9 86 54 3 2 3 0 60 85 95 68 44 0 3 8 16 54 58 72 10 56 13 13 4 24 48 42 53 50 0 9
39 0
Nuclotide
ALDEHYDES 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
Propanal Butanal Pentanal Hexanal Heptanal Octanal Nonanal Decanal Undecanal Dodecanal Tridecanal Octadecanal 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal 2-Methylpentanal 2,2-Dimethylpropanal Glyceraldehyde Propenal 2-Methylpropenal 2-Buten~al 2-Methyl-2-pentenal 2-Hexenal 2,4-Hexadienal ~-Cyclocitral Safranal Phenylethanal 3-Phenylpropanal 3-Phenylpropenal Benzaldehyde 2-Methylbenzaldehyde 3-Methylbenzaldehyde 4-Methylbenzaldehyde 2,4-Dimethylbenzaldehyde 2,5-Dimethylbenzaldehyde 2,4,6-Trim ethylbenzaldehyde 4 -Isopropylbenzaldehyde 4-Methoxybenzaldehyde 3,4-Dimethoxybenzaldehyde 2-Hydroxybenzaldehyde 3-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde 3,4-Dihydroxybenzaldehyde 4-Hydroxy-3 -methoxybenzaldehyde 5-Methyl-2-furfural
5 83 78 82 68 77 92 76 70 55 25 0 6 82 25 63 1 10 32 15 5 8 2 20 87 78 66 72 11 5 5 6 14 100 79 48 45 8 1 46 4 3 12
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
(%)
(%) 259 260 261
5 -Hydroxymethyl-2 -furfural 3-Indolecarboxaldehyde 3-Pyridinecarboxyaldeh
0 16 4
PHENOLS 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302
Phenol 2-Methylphenol 3-Methylphenol 4-Methylphenol 2,3-Dimethylphenol 2,4-Dimethylphenol 2,5-Dimethylphenol 2,6-Dimethylphenol 3,4-Dimethylphenol 3,5-Dimethylphenol 2,3,5-Trimethylphenol 2,4,5-Trimethylphenol 2,4,6-Trimethylphenol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol 3-Ethyl-5-methylphenol 2-Isopropyl-4-methylphenol 2,6-Di-tert-butyl-4-methyl phenol 2-Methoxyphenol 3-Methoxyphenol 4-Methoxyphenol 2,6-Dimethoxyphenol Eugenol Isoeugenol Catechol 3-Methylcatechol 4-Methylcatechol 3-Isopropylcatechol Resorcinol 2-Methylresorcinol 4-Ethylresorcinol 4-Hexylresorcinol Hydroquinone Methylhydroquinone Pyrogallol 2-Hydroxyacetophenone 3-Hydroxyacetophenone 4-Hydroxyacetophenone a-naphthol ~-naphthol
0 28 32 39 81 89 84 79 81 68 81 88 100 100 87 87 86 100 19 0 0 0 0 87 90 0 3 12 96 0 0 69 78 20 9 3 0 0 2 72 65
Nuclotide release
HYDROCARBONS 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
n-Pentane n-Hexane n-Heptane n-Octane n-Decane n-Pentadecane n-Eicosane 1,3-Pentadiene 2-Hexene 2,4-Hexadiene Cyclopentane Cyclohexane Methylcyclohexane 1,2-Dimethylcyclohexane 1,4-Dimethylcyclohexane 1,2,4-Trimethylcyclohexane 1,3,5-Trimethylcyclohexane Cyclohexene 3-Methylcyclohexene 4-Methylcyclohexene 1,3-Dimethylcyclohexene Limonene a-Pinene g-Pinene Benzene Methylbenzene 1,3-Dimethylbenzene 1,4-Dimethylbenzene 1,2,3-Trimethylbenzene 1,2,4-Trimethylbenzene 1,3,5-Trimethylbenzene Ethylbenzene n-Propylbenzene Isopropylbenzene Styrene Phenylacetylene Indan Tetraline Indene Diphenylmethane Azulene Naphthalene 1-Methylnaphthalene 2-Methylnaphthalene 1,4-Dimethylnaphthalene 1,5-Dimethylnaphthalene
3 57 0 0 6 0 19 32 37 54 57 52 99 14 19 0 0 98 92 75 68 90 16 28 18 47 86 87 0 28 35 84 89 84 71 57 84 85 87 67 2 13 77 68 56 51
209
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
(%) 349 350 351 352 353 354 355 356 357 358 359 360 361 362
2,3-Dimethylnaphthalene 1,3,6-Trimethylnapthalene Anthracene Phenanthrene Benz [a ]anthracene Chrysene Pyrene Benz[a ]pyrene Perylene Picene Acenaphthalene Acenaphthylene Fluoranthene Coronene
20 8 5 0 4 0 0 0 0 1 19 28 59 2
FURANES, THIOPHENES 363 364 365 366 367 368 369
Tetrahydrofuran Furan 2-Methylfuran 2,5-Dimethylfuran 2,3-Benzofuran Dibenzofuran Thiophene
0 0 7 12 39 7 0
N-HETEROCYCLES 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
210
Pyrrolidine N-Methylpyrrolidine 3-Pyrroline Piperidine N-Methylpiperidine Pyrrole N-Methylpyrrole 2,4-Dimethylpyrrole Pyridine 2-Methylpyridine 2,6-Dimethylpyridine 3,5-Dimethylpyridine 2,3,6-Trimethylpyridine 2,4,6-Trimethylpyridine 3-Ethylpyridine 3-Pyridinole Nicotinamide N-Methylnicotinamide Quinoline Pyrazine 2-Methylpyrazine
100 0 42 84 80 0 0 0 0 1 3 0 6 5 6 3 0 0 5 4 0
Nuclotide release
(%) 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436
2,3-Dimethylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine 2,3,5 -Trim ethylpyrazine 2,3,5,6-Tetramethylpyrazine 2-Ethylpyrazine 2-Ethyl-3-methylpyrazine 2-n-Butyl-3-methylpyrazine 2-Acetylpyrazine 2-Acetyl-3-methylpyrazine 2-Methoxy-3-methylpyrazine 3 -Et hyl-2-methoxypyrazine 3-Isopropyl-2-methoxypyrazine 3-Isobutyl-2-methoxypyrazine
3-sec-butyl-2-methoxypyrazine 2-Ethoxy-3-methylpyrazine 2-Ethoxy- 3-ethylpyrazine 2 -Ethoxy-3 -isopropylpyrazine Quinoxaline Indole 1-Methylindole 2-Methylindole 3-Methylindole 5-Methylindole 7-Methylindole 1,2-Dimethylindole 1,3-Dimethylindole 2,3-Dimethylindole 2,5-Dimethylindole 3,5-Dimethylindole 1,2,3-Trimethylindole 2,3,4-Trimethylindole 2,3,5-Trimethylindole 2,3,6-Trimethylindole 2,3,7 -Trimethylindole Carbazole 9-Ethylcarbazole Benzimidazole Nornicotine Nicotine g-Nicotyrine Anabasine N-Methylanabasine Cotinine Harman Norharman
1 1 1 0 2 0 0 0 0 0 0 0 83 80 62 0 68 77 0 100 31 82 100 100 67 57 50
9 17 98 30 90 17 36 85 21 21 2 49 18 27 48 0 0 73 53
TABLE I (Continued) No.
Compound
Nucleotide
No.
Compound
release
release
(%) AMINES 437 n-Butylamine 438 n-Hexylamine 439 n-Octylamine 440 n-Decylamine 441 2-Methylpropylamine 442 1-Methylbutylarnine 443 2-Propenylamine 444 Aniline 445 2-Methylaniline 446 3oMethylaniline 447 4-Methylaniline 448 2,3-Dimethylaniline 449 2,5-Dimethylaniline 450 2,6-Dimethylaniline
82 100 100 79 81 88 100 0 8 7 4 30 18 15
Nuclotide
(%)
451 452 453 454 455 456 457 458 459 460 461 462 463 464
2,4,5-Trimethylaniline 2,4,6-Trimethylaniline 2-Ethylaniline 4-Ethylaniline N-Methylaniline N-Ethylaniline N,N-Dimethylaniline N:N-Diethylaniline Diphenylamine Benzylamine a-Naphthylamine ~-Naphthylamine 2,6-Diaminotoluene 3,4-Diaminotoluene
47 74 31 75 14 55 100 38 92 95 76 15 0 1
and showed a m a x i m u m for the C6 ~C,2 acids. Introduction o f a conjugated double b o n d or a side chain in the acyclic acids seemed to increase the degree of membrane damage. All the benzoic acids, unsubstituted or containing m e t h y l , h y d r o x y l or m e t h o x y l substituents, were strongly active, while terephthalic acid, having 2 carboxyl groups, was inactive. Among the N-heterocyclic acids studied, only the indolylic representatives displayed some effect. Esters The allkylalkanoates exhibited no membrane activity. The effects of the alkylsubstituted phenylacetates and alkylsubstituted methylbenzoates are in accordance with the effects observed for the corresponding phenols and benzoic acids. Thus, the activity of the phenylacetates increased with increasing m e t h y l substitution, and all methylbenzoates were active, but showed, in contrast to the corresponding benzoic acids, a slightly augmented activity with increasing substitution. The cinnamates and phthalates were inactive. Nitriles Practically all members o f this group were found to be non-toxic to the cell membrane. The activity of the aliphatic nitriles was n o t increased by introduction of double bonds or alkylsubstituents. Benzonitrile and all the alkylsubstituted benzonitriles were inactive except for the moderately active 2-ethylbenzonitrile.
211
Ketones Most o f the ketones were inactive or weakly active. Thus, acyclic aliphatic methylketones were without effect and this situation was n o t altered on introduction of either alkyl groups or double bonds. Similarly, all the cyclohexanones and cyclohexenones were inactive, except for 2,2,6-trimethylcyclohexanone, a nor-carotenoid isolated from tobacco. Also the other terpenoid ketones examined (pseudo-ionone, ~-ionone, piperitone, carvenone, carvone) caused some membrane damage, as did the terpenoid alcohols and aldehydes. The acetophenones did not induce any membrane damage but the activity increased somewhat upon m e t h y l substitution. Other alkylphenylketones were only weakly active.
Aldehydes As in the case of the alcohols and acids, an homologous series of n-alkyl compounds was examined. All the C4--C,2 aldehydes were strongly active exhibiting a m a x i m u m for nonanal. In contrast to the situation observed for the corresponding acids, the activity decreased on introduction of alkyl side-chains in this series. There was no clear effect of the introduction of double bonds into the alkyl chain, although the activity in most cases seemed to be reduced somewhat. Both the terpenic aldehydes, ~-cyclocitral and safranal, were strongly membrane damaging. Substitution of an aromatic hydrogen in benzaldehyde by a methyl group increased the activity, while substitution by h y d r o x y l or m e t h o x y groups was without effect. Both Nand O-heteroaromatic aldehydes were inactive.
Phenols Phenol and the cresols did not damage the cell membrane, but further methylsubstitution or increase in the number of carbons in the substituent yielded strongly active compounds. Introduction of other substituents such as m e t h o x y , hydroxyl, acyl and aldehyde groups either lowered the activity or was without effect. By contrast, the insertion o f a carboxyl group yielded strongly membrane active compounds in agreement with the effect observed for the benzoic acids mentioned above. Extension of the aromatic system of phenol to that of ~- and ~-naphthol also increased the activity.
Hydrocarbons As might be expected, the n-alkanes displayed low or no activity. Consistent with this, no effect of chain length or unsaturation was observed. Of the cyclic, non-aromatic hydrocarbons, the cyclohexanes were inactive except for methylcyclohexane. However, incorporation of a double bond had a pronounced effect since the corresponding cyclohexenes were highly active. Alkylation seemed to lower the effect of both the cyclohexanes and cyclohexenes, whereas alkylation o f benzene, itself rather inactive, augmented the activity. In fact, an augmentation was observed both on increasing the number of carbons in the sidechain and the n u m b e r of methylsubstituents, except in the case of the trimethyl benzenes, which were inactive.
212
Of the strongly activity. effect on
methylnaphthalenes, only the m o n o s u b s t i t u t e d members were active, while further methyl substitution seemed to decrease the The polyaromatic hydrocarbons larger than naphthalene had no the fibroblast membrane.
Furanes, thiophenes All the members of this group comprising only 7 c o m p o u n d s showed no or low activity. Introduction of methylsubstituents or enlargement o f the ring system was without effect.
N-heterocycles Of the monocyclic N-heterocycles, the non-aromatics were membrane active, while most o f the aromatic (pyrroles, pyridines and pyrazines) were inactive. Alkylsubstitution did n o t affect the activity o f the pyridines. A large number of pyrazines were examined, since they constitute an important group o f flavour compounds. Most of the substituted pyrazines were inactive, the exception being some of the alkoxy substituted alkylpyrazines. As the indoles include many biologically active compounds, a selection o f mono-, di- and trimethylsubstituted indoles was examined. They displayed a great variation in activity and no correlation b e t w e e n the membrane damaging effect and the n u m b e r or positions o f the methyl groups could be established. Of the t o b a c c o alkaloids containing 2 N-heterocyclic rings, only harman was strongly active, whereas nor-harman and the nicotine-analogues were inactive or weakly active.
Amines All the aliphatic amines caused severe membrane damage, and the most active in the n-alkyl series were hexylamine and octylamine. In contrast, the activity of the anilines varied considerably. Thus aniline itself was completely inactive, while anilines carrying methyl or ethyl substituents in the aromatic ring or at the heteroatom exhibited an activity which increased essentially with the n u m b e r of additional carbons, the most active being.N,N-dimethylaniline, p-ethylaniline and 2,4,6-trimethylaniline. Introduction of another amino group into the aromatic ring had no effect. Of the 2 naphthylamines only the a-isomer was active. Such a positional effect was also observed for several other substituents, i.e. for the isomeric pairs o f cyano-, methoxy-, hydroxy- and methyl-naphthalenes. DISCUSSION
The aim of the present study was to specifically detect primary damage to the plasma membrane caused by tobacco and t o b a c c o smoke components. Since cytoplasmic leakage may arise also as a secondary effect of general c y t o t o x i c damage on prolonged exposure, it was necessary to use a short incubation time. As a consequence o f this, fairly high concentrations o f the test substances (25 mM) had to be used to ensure that none of the mem-
213
brane damaging c o m p o u n d s escaped detection in this first screening, which comprised 464 compounds. Despite the high concentration, no or only slight membrane damage was observed for 61 per cent o f the c o m p o u n d s examined, while 27 per cent o f the c o m p o u n d s were strongly active. Our results parallel to a large extent those of Pilotti et. al. [5] who used the inhibition of cell growth of ascites sarcoma cells to evaluate the toxicity of 250 o f the present compounds. Although the highest concentration e m p l o y e d in their study was only 1 raM, their exposure time was 48 h which allowed c y t o t o x i c effects to become of importance. If, for the purpose of comparison, a c o m p o u n d is regarded as active only when the growth rate inhibition exceeds 75 per cent at 1 mM concentration or when the nucleotide release exceeds 75 per cent at 25 mM concentration, we find the results for the 2 test systems to agree in 70 per cent o f the cases. An account for the correlation between chemical functionality and the degree of membrane damage is given in Table II. The most active c o m p o u n d s were found among the acids, phenols and amines, whereas the nitriles were found almost completely inactive despite the fact that these constitute a group of toxic compounds. The toxicity of nitriles is considered to depend on their conversion to cyanide ions [16] and their lack of activity in the present system may be ascribed either to the lack o f metabolic conversion b y the cultured lung fibroblast, or, if the process is slow, insufficient exposure time. The degree of membrane damage caused by the aliphatic alcohols, aldehydes and acids was dependent on the length of the carbon chain (Table III). In most cases the highest activity was encountered for the C5--Clo members and it seems clear that the membrane damage in these cases was due primarily to a detergent effect [17]. Thus, at low concentrations such amphiphilic molecules will be inserted into the lipid bflayer of the cell membranes giving rise to a membrane stabilizing effect [18], whereas at TABLE II MEAN MEMBRANE DAMAGING ACTIVITY (% NUCLEOTIDE RELEASE) OF THE TWELVE D I F F E R E N T CHEMICAL CLASSES Chemical class
Mean activity
Number of compounds
Alcohols Ethers Acids Esters Nitriles Ketones Aldehydes Phenols Hydrocarbons Furanes, Thiopenes N-Heterocycles Amines
41 24 65 35 8 23 35 47 39 9 30 51
40 17 39 34 30 53 48 41 60 7 67 28
214
TABLE III THE EFFECT OF CHAIN LENGTH ON THE MEMBRANE ACTIVITY OF SOME n-ALKYL ALCOHOLS, ALDEHYDES AND ACIDS % Nucleotide release Compound, X =
CH~OH
HX CH3X CH3CH2X CH3(CH2)2X CH3(CH2)3X CH~(CH2),X CH3(CH2).~X CH3(CH2)6X CH3(CH2)~X CH3(CH2)~X CH3(CH2)9X CH~(CH2),,,X CH3(CH2),, X CH3(CH2),3X CH3(CH~),6X CH3(CH2),sX
0 2 0 0 0 100 100 100 100 100 12 8 24 0 0
CHO
5 83 78 68 77 92 76 70 55
COOH 67 17 11 17 61 74 73 71 83 81 85 82 13 8
co n cen tr atio n s exceeding the critical miceUar concent rat i on, a m e m b r a n e solubilizing effect will p r e p o n d e r a t e [17]. The degree o f solubilization should d ep en d on t he hydrophilicflipophilic character o f the test substance [19,20]. A reason for the lack o f m e m b r a n e activity o f t he c o m p o u n d s possessing longer carbon chains might be steric hindrance t o i n c o r p o r a t i o n due to f o r matio n o f large aggregates at the high concentrations used. Our results are consistent with those o bt ai ned for t he partitions o f acids and alcohols of various chain length in e r y t h r o c y t e membranes [21,22]. Moreover, a similar correlation b e t w e e n chain length and functional properties o f isolated skeletal muscles, which are d e p e n d e n t on m e m b r a n e integrity, has been d e m o n s t r a t e d by several authors [23---25]. Most o f the aromatic c o m p o u n d s exhibited a m e m b r a n e damaging effect which increased with alkyl substitution o f the aromatic ring (Table IV). The reason for this is n o t clear. This e f fect is m ost obvious in the case o f phenols, phenylacetates, a c t o p h e n o n e s and methylbenzoates. It may be concluded that t o b a c c o smoke contains a n u m b e r of membrane damaging substances. These might cause direct toxic reactions after long term exposure, since even a subtle change in m e m b r a n e permeability will change the metabolism of affected cells [26,27]. F u r t h e r m o r e , membrane active c o m p o u n d s could also p o t e n t i a t e t he toxic effects o f t o b a c c o smoke by p r o m o t i n g the cell m e m b r a n e penet rat i on o f o t h e r t oxi c substances in the smoke.
215
O~
b~
X L
~
X
C3H7
-'~j"~C2H5
X
"CH:
X CHs~'~CN:
C,
~ L . ~ oH3
x
X
Compound, X =
87
84
21
87
47
18
H
91
90
80
33
0
OH
79
36
5
72
% N u c l e o t i d e release
15
18
28
23
10
OCH
S
72
56
CCH 3
45
48
90
CHO
70
76
89
82
86
COOH
67
100
89
86
20
COCH 3
THE E F F E C T O F A L K Y L S U B S T I T U T I O N ON THE M E M B R A N E A C T I V I T Y O F SOME A R O M A T I C C O M P O U N D S
TABLE IV
26
19
CN
53
61
21
NH~
ACKNOWLEDGEMENT
We are grateful to Dr. T. Nishida for recording the NMtt spectra and to Ms. E. Kazemi-Vala and Ms E. Br~innk~irr for skilful technical assistance. REFERENCES 1 C.R. Green, Recent Adv. Tobacco Sci., 3 (1977) 94. 2 R.A. Heckman and F.W. Best, Abstract, 32nd Tobacco Chemists' Research Conference, Montreal, Canada 1978. 3 C. Leuchtenberger and R. Leuchtenberger, Exp. Cell Res., 62 (1970) 161. 4 D.G. Wenzel, J.W. Wheatley and G.D. Byrd, Toxicol. Appl. Pharmacol., 17 (1970) 774. 5 /~. Pilotti, K. Ancker, E. Arrhenius and C. Enzell, Toxicology, 5 (1975) 49. 6 J.A. Styles, Br. J. Exp. Pathol., 57 (1976) 286. 7 C. Leuchtenberger and R. Leuchtenberger, Br. J. Exp. Pathol., 58 (1977) 625. 8 W.R. Thomas, P.G. Holt and D. Keast, Infect. Immun., 20 (1978) 468. O 9 J. Litwin, C. Enzell and A. Pilotti, Acta Pathol. Microbiol. Scand. Sect. A, 86 (1978) 135. 10 C. Chang, M. Castellazzi, T.W. Glover and J.E. Trosko, Cancer Res., 38 (1978) 4527. 11 B. Pettersson, Chem.-Biol. Interact., 29 (1980) 95. 12 M. Thelestam and R. MSllby, Biochim. Biophys. Aeta, 557 (1979) 156. 13 M. Thelestam and R. MSllby, Infect. Immun., 11 (1975) 640. 14 H. Eagle, Science, 130 (1959) 432. 15 M. Thelestam and R. MSllby, Med. Biol., 54 (1976) 39. 16 A. de Bruin, Biochemical Toxicology of Environmental Agents, Elsevier, Amsterdam, 1976, p. 111. 17 C. Tanford and J.A. Reynolds, Biochim. Biophys. Acta, 457 (1976) 133. 18 M.P. Sheetz and S.J. Singer, Proc. Natl. Acad. Sci., 71 (1974) 4457. 19 J.N. Umbreit and J.L. Strominger, Proc. Natl. Acad. Sci., 70 (1973) 2997. 20 J.T. Davies, 2nd Int. Congr. Surface Activity, Vol. 1 (1957) 426. 21 P. Seeman, S. Roth and H. Schneider, Biochim. Biophys. Acta, 225 (1971) 171. 22 P. Seeman, Pharmacol. Rev., 24 (1972) 583. 23 G. Caffier, F. KSssler and G. Kiichler, Acta Biol. Med. Germ., 34 (1975) 99. 24 F. KSssler, G. Caffier and G. Kiichler, Acta Biol. Med. Germ., 35 (1976) 1327. 25 F. KSssler and G. Kiichler, Acta Biol. Med. Germ., 36 (1977) 1085. 26 M.L.S. Ledbetter and M. Lubin, Exp. Cell Res., 105 (1977) 223. 27 M. Lubin, Nature, 213 (1967) 451.
217
E F F E C T O F TOBACCO SMOKE COMPOUNDS ON THE PLASMA MEMBRANE OF CULTURED HUMAN LUNG FIBROBLASTS
MONICA
THELESTAM,
a MARGARETA
CURVALL
b and C U R T
R. E N Z E L L b
aDepartment of Bacteriology, Karolinska Institute& 8-104 01 Stockholm and bResearch Department, Swedish Tobacco Company, P.O. Box 17007, S-104 62 Stockholm
(Sweden) (Received February 18th, 1980)
(Revision received April 3rd, 1980) (Accepted April llth, 1980)
SUMMARY
The ability of compounds derived from tobacco and tobacco smoke to increase the permeability o f the membranes of h u m a n lung fibroblasts has been studied by measuring the release o f an intracellular marker after short term exposure. Of the 464 c o m p o u n d s tested, about 25 per cent gave rise to severe membrane damage. The most active compounds, when divided according to functionality, were found within the groups o f amines, strong acids and alkylated phenols, whereas nitriles and polycyclic aromatic hydrocarbons were found completely inactive. A pronounced effect o f the chain length on the activity was observed for the aliphatic alcohols, aldehydes and acids, and all monocyclic aromatic compounds but benzonitriles and benzoic acids showed an increase in activity with increasing alkylsubstitution. It is concluded t h a t tobacco smoke contains a n u m b e r o f membrane damaging substances. These membrane active compounds could not only cause direct toxic reactions but also potentiate the toxic effect by promoting the cell membrane penetration o f other toxic substances in tobacco smoke.
INTRODUCTION Some 3000 chemical substances have been detected in cigarette smoke so far [1,2], but little is known about their biological activity. Various in vitro methods have been devised with a view to clarifying the mechanisms behind the in vivo effects of smoke, and most o f the in vitro test systems used are based on effects at the cellular and subcellular levels. Thus effects Address correspondence to: Monica Thelestam, PhD, Department of Bacteriology, Karolinska Institute, S-104 01 Stockholm, Sweden.
203
of tobacco smoke or single smoke components on tissue culture cells have been investigated using criteria such as: cell morphology [3], beating of rat heart cells [4], cell multiplication [5], DNA-content [6], cell transformation to malignancy [7], phagocytic functions [8], cell aging [9], mutagenesis [10], and cell respiration [11, Pettersson, B. et al., unpublished]. Furthermore, the mutagenic activity of individual smoke components on bacteria has been studied recently [Florin, I. et al., unpublished]. The present work comprises a study of the effects of tobacco and tobacco smoke constituents on plasma membranes of human lung fibroblasts in vitro. The method used is based on the simple principle that leakage of intracellular substances indicates damage to the plasma membrane and that the molecular size of the leaked material serves as an indicator of the degree of membrane damage in terms of the size of the "holes" induced by the test compounds [12]. A low molecular weight marker, uridine nucleotides [13], and a short exposure time were employed in this study to achieve high sensitivity and avoid secondary effects arising from cytotoxic damage. MATERIALS AND METHODS Biochemicals Eagle's minimal essential medium, new born calf serum and crude trypsin were purchased from Flow Laboratories, Ltd., Irvine, Scotland, Hank's balanced salt solution from the National Bacteriological Laboratory, Stockholm, Sweden, [5-3H]uridine and Aquasol ® Universal Cocktail from NEN Chemicals GmbH, Frankfurt, F.R.G. and tris(hydroxymethyl)-aminomethane, analytical grade, from E. Merck, Darmstadt, F.R.G. Compounds examined 464 compounds, most of them abundant in tobacco and tobacco smoke, have been examined. Those substances, which were not commercially available were either isolated from tobacco, (e.g. the terpenoids) or synthesized from available starting compounds, using adopted methods of synthesis, (e.g. ethers, esters, indoles and nicotine analogues). All compounds were checked for purity using thin-layer chromatography, gas chromatography and NMR. Compounds containing more than 3 per cent impurity were purified using liquid chromatography, recrystallization and distillation. The structures of the compounds studied were confirmed by [1H]- and [laC]NMR, e.g. the substitution pattern of multisubstituted aromatic substances and the branching pattern of methylsubstituted long chain alkyl derivatives. Cultivation and labelling of cells Human diploid embryonic lung fibroblasts (line MI~C-5) were cultivated in Eagle's medium [14] in polystyrene wells to a cell density of 10 s cells/ cm 2 (~ 7 × l 0 s cells/well). The cells in confluent monolayers were labelled with [3H]uridine to obtain a low molecular weight cytoplasmic marker, consisting of uridine nucleotides [ 13]. 204
Testing procedure and calculation o f results Labelled cultures were washed 3 times with Hank's balanced salt solution and then incubated for 30 rain at 370C with test c o m p o u n d s diluted to 25 mM in Tris-buffered saline, i.e. 0.15 mM NaC1 with 0.02 M tris--HC1 (pH 7.0). Then the solution containing leaked radioactive marker was removed and centrifuged (1000 g, 10 min, 4°C) and radioactivity in 0.1-ml aliquots of the s u p e m a t a n t was measured b y liquid scintillation [13]. A maximal release of the radioactivity was obtained b y treating control cells for 30 min with 0.06 M sodium borate buffer (pH 7.8) and scraping with a rubber policeman. This treatment ruptured the cell membranes leaving the nuclei intact [13]. The following expression was used to calculate the relative leakage of the radioactive marker: experimental release - spontaneous release× 100 maximal release - spontaneous release The spontaneous release of the nucleotide marker during incubation for 30 min at 37°C was 3--7 per cent of the maximal release [15]. RESULTS
In order to simplify the interpretation of the results, which are summarised in Table I, the c o m p o u n d s have been divided into 12 chemical classes according to functionality, and the degree of membrane damage classified as high (>70% nucleotide release), moderate (70--45%) and nil (<15%).
Alcohols The degree of membrane damage caused by the alcohols varied considerably. Thus, out of a homologous series o f 15 primary aliphatic alcohols, the C6--C10 representatives were highly active, whereas the CI--Cs and C12--C20 alcohols did n o t induce any significant membrane damage. All the isoprenoid alcohols, cyclic or acyclic, saturated or unsaturated were also highly potent. By contrast,-, the cyclohexanols were inactive, except for 4-isopropylcyclohexanol, which m a y be viewed as a nor-isoprenoid. Ethers Most of the ethers were found to be without-any effect on the cell membrane. Only 1-methoxynaphthalene was strongly active. All the anisoles were inacti*e, or weakly active showing a slightly increased activity u p o n increasing alkyl substitution. Enlargement of the aromatic ring system seems to cause an increased activity, while extension o f the alkyl unit appears to be without any effect. Acids Many members of this group caused severe damage to the cell membrane. The activity o f the n-alkyl acids varied with the length o f the carbon chain
205
TABLE I MEMBRANE DAMAGING ACTIVITY OF 464 TOBACCO SMOKE COMPONENTS No.
Compound
Nucleotide release
No.
Compound
(%) ALCOHOLS 1 Methanol 2 Ethanol 3 2-Propanol 4 1-Butanol 5 1-Pentanol 6 1-Hexanol 7 1-Heptanol 8 1-Octanol 9 1-Nonanol 10 1-Decanol 11 1-Dodecanol 12 1-Tridecanol 13 1-Pentadecanol 14 1-Heptadecanol 15 1-Oetadecanol 16 1-Eicosanol. 17 2-Buten-l-ol 18 4-Penten-l-ol 19 2-Hexen-l-ol 20 2,4-Hexadien-l-ol 21 Tetrahydrolinalool 22 Linalool 23 Tetrahydrogeraniol 24 Geraniol 25 Farnesol 26 Cyclohexanol 27 2-Methylcyclohexanol 28 2,6-Dimethylcyclohexanol 29 4-Isopropylcyclohexanol 30 Menthol 31 Piperitol 32 ~-Terpineol 33 ~-Ionol 34 Propylene glycol 35 Furfurylalcohol 36 Benzylalcohol 37 2-Hydroxybenzylalcohol 38 3-Hydroxybenzylalcohol 39 4-Hydroxybenzylalcohol 40 2-Phenylethanol ETHERS 41 Anisole 42 3-Methylanisole
206
0 2 0 0 0 100 100 100 100 100 12 8 24 0 0 0 6 8 5 4 81 100 100 100 100 0 0 3 96 99 89 85 100 0 95 7 3 3 4 0
4 12
Nuclotide release
(%)
43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
4-Methylanisole 2,6-Dimethylanisole 3,5-Dimethylanisole 2,3,5-Trimethylanisole 2,4,6-Trimethylanisole 3,4,5-Trimethylanisole 4-Ethylanisole 4-n-Propylanisole 4-(2-Propenyl)anisole Ethylphenylether Diphenylether 1-Methoxynaphthalene 2-Methoxynaphthalene 2-Ethoxynaphthalene 1,2,3-Trimethoxybenzene
ACIDS 58 Methanoic acid 59 Ethanoic acid 60 Propanoic acid 61 Butanoic acid 62 Pentanoic acid 63 Hexanoic acid 64 Heptanoic acid 65 Octanoic acid 66 Nonanoic acid 67 Decanoic acid 68 Undecanoic acid 69 Dodecanoic acid 70 Pentadecanoic acid 71 Eicosanoic acid 72 2-Methylpropanoic acid 73 2-Methylbutanoic acid 74 2-Methylpropenoic acid 75 2-Butenoic acid 76 Phenylethanoic acid 77 3-Phenylpropanoic acid 78 3-Phenylpropenoic acid 79 Benzoic acid 80 2-Methylbenzoic acid 81 3-Methylbenzoic acid 82 4-Methylbenzoic acid 83 2,4,6-Trimethylbenzoic acid
7 22 23 24 37 24 18 15 36 11 11 81 51 35 0
67 17 11 17 61 74 73 71 83 81 85 82 13 8 53 79 7O 72 79 82 100 86 84 8O 83 89
TABLE I (Continued)
No. Compound
Nucleotide release
No.
Compound
(%) 84 85 86 87 88 89 90 91 92 93 94 95 96
4-Ethylbenzoic acid 2-Isopropylbenzoic acid 2-Methoxybenzoic acid 3-Methoxybenzoic acid 2-Hydroxybenzoic acid 2,5-Dihydroxybenzoic acid 3,5-Dimethoxy-4-hydroxybenzoic acid 2-Acetoxybenzoic acid 3-Pyridine carboxylic acid Indole-3-acetic acid 5-Hydroxyindole-3-acetic acid 4-Hydroxy quinaldic acid p-Phthalic acid
76 70 96 84 50 91 75 67 27 100 72 0 11
ESTERS 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124
Vinyl acetate Ethyl acetate Phenyl acetate Benzyl acetate 2-Methylphenyl acetate 3-Methylphenyl acetate 4-Methylphenyl acetate 2,6-Dimethylphenyl acetate 3,5-Dimethylphenyl acetate 2,3,5-Trimethylphenyl acetate 2,4,6-Trimethylphenyl acetate Methyl propenoate Methyl pentanoate Ethyl pentanoate Ethyl stearate Methyl benzoate Ethyl benzoate Benzyl benzoate Methyl 2-methylbenzoate Methyl 3-methylbenzoate Methyl 4-methylbenzoate Methyl 2,6-dimethylbenzoate Methyl 3,5-dimethylbenzoate Methyl 2,4,6-trimethylbenzoate Methyl 2-isopropylbenzoate Methyl 3-phenyipropenoate Ethyl 3-phenylpropenoate Benzyl 3-phenylpropenoate
0 0 5 1 6 49 52 76 82 86 58 0 0 0 0 20 31 30 65 100 92 85 93 100 67 13 19 14
Nuclotide release
(%) 125 126 127 128 129 130
Coumarin Diethyl malonate Diethyl phthalate Di-n-propyl phthalate Di-n-butyl phthalate Di-n-octyl phthalate
4 0 10 10 18 1
NITRILES 131 Ethanonitrile 132 Propanonitrile 133 Butanonitrile 134 Pentanonitrile 135 Hexanonitrile 136 Heptanonitrile 137 Octanonitrile 138 2-Methylpropanonitrile 139 3-Methylbut anonitrile 140 Propenonitrile 141 Phenylethanonitrile 142 3-Phenylpropanonitrile 143 3-Phenylpropenonitrile 144 Benzonitrile 145 2-Methylbenzonitrile 146 3-Methylbenzonitrile 147 4-Methylbenzonitrile 148 2,3-Dimethylbenzonitrile 149 2,4-Dimethylbenzonitrile 150 2,5-Dimethylbenzonitrile 151 2,6-Dimethylbenzonitrile 152 3,4-Dimet hylbenzonitrile 153 2,4,6-Trimethylbenzonitrile 154 2-Ethylbenzonitrile 155 3-Ethylbenzonitrile 156 4-Ethylbenzonitrile 157 1-Naphthonitrile 158 2-Naphthonitrile 159 Nicotinonitrile 160 Indolyl-3-acetonitrile
5 7 6 0 4 0 10 0 5 2 5 0 5 3 8 0 4 4 2 4 2 5 19 54 15 9 33 2 0 30
KETONES 161 162 163 164 165 166 167
2-Propanone 2-Butanone 2-Pentanone 2-Hexanone 2-Heptanone 2-Decanone 4-Methyl-2-pentanone
0 1 0 1 2 36 10
207
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
release
(%)
(%) 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213
208
6-Methyl-2-heptanone 3-Buten-2-one 3-Methyl-3-buten-2-one 3-Penten-2-one 6-Methyl-5-hepten-2-one 6-Methyl-3,5-heptadien-2-one 2,3-Butanedione 2,3-Pentanedione Cyclopentanone Cyclohexanone 2-Methylcyclohexanone 3-Methylcyclohexanone 4-Methylcyclohexanone 2,6-Dimethylcyclohexanone 2,2,6-Trimethylcyclohexanone 4-Isopropylcyclohexanone 2-Cyclohexenone 3-Methyl-2-cyclohexenone 3,5-Dimethyl-2-cyclohexenone Isophorone Piperitone Carvenone Carvone Pseudoionone ~-ionone Acetophenone 2-Methylacetophenone 3-Methylacetophenone 4-Methylacetophenone 2,4-Dimethylacetophenone 2,5-Dimethylacetophenone 2,4,6-Trimethylacetophenone 1-Phenyl-l-propanone 1-(2-Methylphenyl)-l-propanone 1-(3-Methylphenyl)-l-propanone 1-(4-Methylphenyl)-l-propanone 1-Phenyl-2-propanone 1-Phenyl-l-butanone 3-Methyl-l-phenyl-1-butanone 1-Phenyl-2-butanone 1-Phenyl-l-pentanone 1-Phenyl-l-octanone 1-Indanone 9-Fluorenone 2,3,6-Trimethyl-l,4-napthoquinone ( 3-Pyridyl)-1-propanone
5 4 35 32
12 8 0 5 1 2 0 0 3 9 86 54 3 2 3 0 60 85 95 68 44 0 3 8 16 54 58 72 10 56 13 13 4 24 48 42 53 50 0 9
39 0
Nuclotide
ALDEHYDES 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258
Propanal Butanal Pentanal Hexanal Heptanal Octanal Nonanal Decanal Undecanal Dodecanal Tridecanal Octadecanal 2-Methylpropanal 2-Methylbutanal 3-Methylbutanal 2-Methylpentanal 2,2-Dimethylpropanal Glyceraldehyde Propenal 2-Methylpropenal 2-Buten~al 2-Methyl-2-pentenal 2-Hexenal 2,4-Hexadienal ~-Cyclocitral Safranal Phenylethanal 3-Phenylpropanal 3-Phenylpropenal Benzaldehyde 2-Methylbenzaldehyde 3-Methylbenzaldehyde 4-Methylbenzaldehyde 2,4-Dimethylbenzaldehyde 2,5-Dimethylbenzaldehyde 2,4,6-Trim ethylbenzaldehyde 4 -Isopropylbenzaldehyde 4-Methoxybenzaldehyde 3,4-Dimethoxybenzaldehyde 2-Hydroxybenzaldehyde 3-Hydroxybenzaldehyde 4-Hydroxybenzaldehyde 3,4-Dihydroxybenzaldehyde 4-Hydroxy-3 -methoxybenzaldehyde 5-Methyl-2-furfural
5 83 78 82 68 77 92 76 70 55 25 0 6 82 25 63 1 10 32 15 5 8 2 20 87 78 66 72 11 5 5 6 14 100 79 48 45 8 1 46 4 3 12
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
(%)
(%) 259 260 261
5 -Hydroxymethyl-2 -furfural 3-Indolecarboxaldehyde 3-Pyridinecarboxyaldeh
0 16 4
PHENOLS 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302
Phenol 2-Methylphenol 3-Methylphenol 4-Methylphenol 2,3-Dimethylphenol 2,4-Dimethylphenol 2,5-Dimethylphenol 2,6-Dimethylphenol 3,4-Dimethylphenol 3,5-Dimethylphenol 2,3,5-Trimethylphenol 2,4,5-Trimethylphenol 2,4,6-Trimethylphenol 2-Ethylphenol 3-Ethylphenol 4-Ethylphenol 3-Ethyl-5-methylphenol 2-Isopropyl-4-methylphenol 2,6-Di-tert-butyl-4-methyl phenol 2-Methoxyphenol 3-Methoxyphenol 4-Methoxyphenol 2,6-Dimethoxyphenol Eugenol Isoeugenol Catechol 3-Methylcatechol 4-Methylcatechol 3-Isopropylcatechol Resorcinol 2-Methylresorcinol 4-Ethylresorcinol 4-Hexylresorcinol Hydroquinone Methylhydroquinone Pyrogallol 2-Hydroxyacetophenone 3-Hydroxyacetophenone 4-Hydroxyacetophenone a-naphthol ~-naphthol
0 28 32 39 81 89 84 79 81 68 81 88 100 100 87 87 86 100 19 0 0 0 0 87 90 0 3 12 96 0 0 69 78 20 9 3 0 0 2 72 65
Nuclotide release
HYDROCARBONS 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348
n-Pentane n-Hexane n-Heptane n-Octane n-Decane n-Pentadecane n-Eicosane 1,3-Pentadiene 2-Hexene 2,4-Hexadiene Cyclopentane Cyclohexane Methylcyclohexane 1,2-Dimethylcyclohexane 1,4-Dimethylcyclohexane 1,2,4-Trimethylcyclohexane 1,3,5-Trimethylcyclohexane Cyclohexene 3-Methylcyclohexene 4-Methylcyclohexene 1,3-Dimethylcyclohexene Limonene a-Pinene g-Pinene Benzene Methylbenzene 1,3-Dimethylbenzene 1,4-Dimethylbenzene 1,2,3-Trimethylbenzene 1,2,4-Trimethylbenzene 1,3,5-Trimethylbenzene Ethylbenzene n-Propylbenzene Isopropylbenzene Styrene Phenylacetylene Indan Tetraline Indene Diphenylmethane Azulene Naphthalene 1-Methylnaphthalene 2-Methylnaphthalene 1,4-Dimethylnaphthalene 1,5-Dimethylnaphthalene
3 57 0 0 6 0 19 32 37 54 57 52 99 14 19 0 0 98 92 75 68 90 16 28 18 47 86 87 0 28 35 84 89 84 71 57 84 85 87 67 2 13 77 68 56 51
209
TABLE I (Continued) No.
Compound
Nucleotide release
No.
Compound
(%) 349 350 351 352 353 354 355 356 357 358 359 360 361 362
2,3-Dimethylnaphthalene 1,3,6-Trimethylnapthalene Anthracene Phenanthrene Benz [a ]anthracene Chrysene Pyrene Benz[a ]pyrene Perylene Picene Acenaphthalene Acenaphthylene Fluoranthene Coronene
20 8 5 0 4 0 0 0 0 1 19 28 59 2
FURANES, THIOPHENES 363 364 365 366 367 368 369
Tetrahydrofuran Furan 2-Methylfuran 2,5-Dimethylfuran 2,3-Benzofuran Dibenzofuran Thiophene
0 0 7 12 39 7 0
N-HETEROCYCLES 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390
210
Pyrrolidine N-Methylpyrrolidine 3-Pyrroline Piperidine N-Methylpiperidine Pyrrole N-Methylpyrrole 2,4-Dimethylpyrrole Pyridine 2-Methylpyridine 2,6-Dimethylpyridine 3,5-Dimethylpyridine 2,3,6-Trimethylpyridine 2,4,6-Trimethylpyridine 3-Ethylpyridine 3-Pyridinole Nicotinamide N-Methylnicotinamide Quinoline Pyrazine 2-Methylpyrazine
100 0 42 84 80 0 0 0 0 1 3 0 6 5 6 3 0 0 5 4 0
Nuclotide release
(%) 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436
2,3-Dimethylpyrazine 2,5-Dimethylpyrazine 2,6-Dimethylpyrazine 2,3,5 -Trim ethylpyrazine 2,3,5,6-Tetramethylpyrazine 2-Ethylpyrazine 2-Ethyl-3-methylpyrazine 2-n-Butyl-3-methylpyrazine 2-Acetylpyrazine 2-Acetyl-3-methylpyrazine 2-Methoxy-3-methylpyrazine 3 -Et hyl-2-methoxypyrazine 3-Isopropyl-2-methoxypyrazine 3-Isobutyl-2-methoxypyrazine
3-sec-butyl-2-methoxypyrazine 2-Ethoxy-3-methylpyrazine 2-Ethoxy- 3-ethylpyrazine 2 -Ethoxy-3 -isopropylpyrazine Quinoxaline Indole 1-Methylindole 2-Methylindole 3-Methylindole 5-Methylindole 7-Methylindole 1,2-Dimethylindole 1,3-Dimethylindole 2,3-Dimethylindole 2,5-Dimethylindole 3,5-Dimethylindole 1,2,3-Trimethylindole 2,3,4-Trimethylindole 2,3,5-Trimethylindole 2,3,6-Trimethylindole 2,3,7 -Trimethylindole Carbazole 9-Ethylcarbazole Benzimidazole Nornicotine Nicotine g-Nicotyrine Anabasine N-Methylanabasine Cotinine Harman Norharman
1 1 1 0 2 0 0 0 0 0 0 0 83 80 62 0 68 77 0 100 31 82 100 100 67 57 50
9 17 98 30 90 17 36 85 21 21 2 49 18 27 48 0 0 73 53
TABLE I (Continued) No.
Compound
Nucleotide
No.
Compound
release
release
(%) AMINES 437 n-Butylamine 438 n-Hexylamine 439 n-Octylamine 440 n-Decylamine 441 2-Methylpropylamine 442 1-Methylbutylarnine 443 2-Propenylamine 444 Aniline 445 2-Methylaniline 446 3oMethylaniline 447 4-Methylaniline 448 2,3-Dimethylaniline 449 2,5-Dimethylaniline 450 2,6-Dimethylaniline
82 100 100 79 81 88 100 0 8 7 4 30 18 15
Nuclotide
(%)
451 452 453 454 455 456 457 458 459 460 461 462 463 464
2,4,5-Trimethylaniline 2,4,6-Trimethylaniline 2-Ethylaniline 4-Ethylaniline N-Methylaniline N-Ethylaniline N,N-Dimethylaniline N:N-Diethylaniline Diphenylamine Benzylamine a-Naphthylamine ~-Naphthylamine 2,6-Diaminotoluene 3,4-Diaminotoluene
47 74 31 75 14 55 100 38 92 95 76 15 0 1
and showed a m a x i m u m for the C6 ~C,2 acids. Introduction o f a conjugated double b o n d or a side chain in the acyclic acids seemed to increase the degree of membrane damage. All the benzoic acids, unsubstituted or containing m e t h y l , h y d r o x y l or m e t h o x y l substituents, were strongly active, while terephthalic acid, having 2 carboxyl groups, was inactive. Among the N-heterocyclic acids studied, only the indolylic representatives displayed some effect. Esters The allkylalkanoates exhibited no membrane activity. The effects of the alkylsubstituted phenylacetates and alkylsubstituted methylbenzoates are in accordance with the effects observed for the corresponding phenols and benzoic acids. Thus, the activity of the phenylacetates increased with increasing m e t h y l substitution, and all methylbenzoates were active, but showed, in contrast to the corresponding benzoic acids, a slightly augmented activity with increasing substitution. The cinnamates and phthalates were inactive. Nitriles Practically all members o f this group were found to be non-toxic to the cell membrane. The activity of the aliphatic nitriles was n o t increased by introduction of double bonds or alkylsubstituents. Benzonitrile and all the alkylsubstituted benzonitriles were inactive except for the moderately active 2-ethylbenzonitrile.
211
Ketones Most o f the ketones were inactive or weakly active. Thus, acyclic aliphatic methylketones were without effect and this situation was n o t altered on introduction of either alkyl groups or double bonds. Similarly, all the cyclohexanones and cyclohexenones were inactive, except for 2,2,6-trimethylcyclohexanone, a nor-carotenoid isolated from tobacco. Also the other terpenoid ketones examined (pseudo-ionone, ~-ionone, piperitone, carvenone, carvone) caused some membrane damage, as did the terpenoid alcohols and aldehydes. The acetophenones did not induce any membrane damage but the activity increased somewhat upon m e t h y l substitution. Other alkylphenylketones were only weakly active.
Aldehydes As in the case of the alcohols and acids, an homologous series of n-alkyl compounds was examined. All the C4--C,2 aldehydes were strongly active exhibiting a m a x i m u m for nonanal. In contrast to the situation observed for the corresponding acids, the activity decreased on introduction of alkyl side-chains in this series. There was no clear effect of the introduction of double bonds into the alkyl chain, although the activity in most cases seemed to be reduced somewhat. Both the terpenic aldehydes, ~-cyclocitral and safranal, were strongly membrane damaging. Substitution of an aromatic hydrogen in benzaldehyde by a methyl group increased the activity, while substitution by h y d r o x y l or m e t h o x y groups was without effect. Both Nand O-heteroaromatic aldehydes were inactive.
Phenols Phenol and the cresols did not damage the cell membrane, but further methylsubstitution or increase in the number of carbons in the substituent yielded strongly active compounds. Introduction of other substituents such as m e t h o x y , hydroxyl, acyl and aldehyde groups either lowered the activity or was without effect. By contrast, the insertion o f a carboxyl group yielded strongly membrane active compounds in agreement with the effect observed for the benzoic acids mentioned above. Extension of the aromatic system of phenol to that of ~- and ~-naphthol also increased the activity.
Hydrocarbons As might be expected, the n-alkanes displayed low or no activity. Consistent with this, no effect of chain length or unsaturation was observed. Of the cyclic, non-aromatic hydrocarbons, the cyclohexanes were inactive except for methylcyclohexane. However, incorporation of a double bond had a pronounced effect since the corresponding cyclohexenes were highly active. Alkylation seemed to lower the effect of both the cyclohexanes and cyclohexenes, whereas alkylation o f benzene, itself rather inactive, augmented the activity. In fact, an augmentation was observed both on increasing the number of carbons in the sidechain and the n u m b e r of methylsubstituents, except in the case of the trimethyl benzenes, which were inactive.
212
Of the strongly activity. effect on
methylnaphthalenes, only the m o n o s u b s t i t u t e d members were active, while further methyl substitution seemed to decrease the The polyaromatic hydrocarbons larger than naphthalene had no the fibroblast membrane.
Furanes, thiophenes All the members of this group comprising only 7 c o m p o u n d s showed no or low activity. Introduction of methylsubstituents or enlargement o f the ring system was without effect.
N-heterocycles Of the monocyclic N-heterocycles, the non-aromatics were membrane active, while most o f the aromatic (pyrroles, pyridines and pyrazines) were inactive. Alkylsubstitution did n o t affect the activity o f the pyridines. A large number of pyrazines were examined, since they constitute an important group o f flavour compounds. Most of the substituted pyrazines were inactive, the exception being some of the alkoxy substituted alkylpyrazines. As the indoles include many biologically active compounds, a selection o f mono-, di- and trimethylsubstituted indoles was examined. They displayed a great variation in activity and no correlation b e t w e e n the membrane damaging effect and the n u m b e r or positions o f the methyl groups could be established. Of the t o b a c c o alkaloids containing 2 N-heterocyclic rings, only harman was strongly active, whereas nor-harman and the nicotine-analogues were inactive or weakly active.
Amines All the aliphatic amines caused severe membrane damage, and the most active in the n-alkyl series were hexylamine and octylamine. In contrast, the activity of the anilines varied considerably. Thus aniline itself was completely inactive, while anilines carrying methyl or ethyl substituents in the aromatic ring or at the heteroatom exhibited an activity which increased essentially with the n u m b e r of additional carbons, the most active being.N,N-dimethylaniline, p-ethylaniline and 2,4,6-trimethylaniline. Introduction of another amino group into the aromatic ring had no effect. Of the 2 naphthylamines only the a-isomer was active. Such a positional effect was also observed for several other substituents, i.e. for the isomeric pairs o f cyano-, methoxy-, hydroxy- and methyl-naphthalenes. DISCUSSION
The aim of the present study was to specifically detect primary damage to the plasma membrane caused by tobacco and t o b a c c o smoke components. Since cytoplasmic leakage may arise also as a secondary effect of general c y t o t o x i c damage on prolonged exposure, it was necessary to use a short incubation time. As a consequence o f this, fairly high concentrations o f the test substances (25 mM) had to be used to ensure that none of the mem-
213
brane damaging c o m p o u n d s escaped detection in this first screening, which comprised 464 compounds. Despite the high concentration, no or only slight membrane damage was observed for 61 per cent o f the c o m p o u n d s examined, while 27 per cent o f the c o m p o u n d s were strongly active. Our results parallel to a large extent those of Pilotti et. al. [5] who used the inhibition of cell growth of ascites sarcoma cells to evaluate the toxicity of 250 o f the present compounds. Although the highest concentration e m p l o y e d in their study was only 1 raM, their exposure time was 48 h which allowed c y t o t o x i c effects to become of importance. If, for the purpose of comparison, a c o m p o u n d is regarded as active only when the growth rate inhibition exceeds 75 per cent at 1 mM concentration or when the nucleotide release exceeds 75 per cent at 25 mM concentration, we find the results for the 2 test systems to agree in 70 per cent o f the cases. An account for the correlation between chemical functionality and the degree of membrane damage is given in Table II. The most active c o m p o u n d s were found among the acids, phenols and amines, whereas the nitriles were found almost completely inactive despite the fact that these constitute a group of toxic compounds. The toxicity of nitriles is considered to depend on their conversion to cyanide ions [16] and their lack of activity in the present system may be ascribed either to the lack o f metabolic conversion b y the cultured lung fibroblast, or, if the process is slow, insufficient exposure time. The degree of membrane damage caused by the aliphatic alcohols, aldehydes and acids was dependent on the length of the carbon chain (Table III). In most cases the highest activity was encountered for the C5--Clo members and it seems clear that the membrane damage in these cases was due primarily to a detergent effect [17]. Thus, at low concentrations such amphiphilic molecules will be inserted into the lipid bflayer of the cell membranes giving rise to a membrane stabilizing effect [18], whereas at TABLE II MEAN MEMBRANE DAMAGING ACTIVITY (% NUCLEOTIDE RELEASE) OF THE TWELVE D I F F E R E N T CHEMICAL CLASSES Chemical class
Mean activity
Number of compounds
Alcohols Ethers Acids Esters Nitriles Ketones Aldehydes Phenols Hydrocarbons Furanes, Thiopenes N-Heterocycles Amines
41 24 65 35 8 23 35 47 39 9 30 51
40 17 39 34 30 53 48 41 60 7 67 28
214
TABLE III THE EFFECT OF CHAIN LENGTH ON THE MEMBRANE ACTIVITY OF SOME n-ALKYL ALCOHOLS, ALDEHYDES AND ACIDS % Nucleotide release Compound, X =
CH~OH
HX CH3X CH3CH2X CH3(CH2)2X CH3(CH2)3X CH~(CH2),X CH3(CH2).~X CH3(CH2)6X CH3(CH2)~X CH3(CH2)~X CH3(CH2)9X CH~(CH2),,,X CH3(CH2),, X CH3(CH2),3X CH3(CH~),6X CH3(CH2),sX
0 2 0 0 0 100 100 100 100 100 12 8 24 0 0
CHO
5 83 78 68 77 92 76 70 55
COOH 67 17 11 17 61 74 73 71 83 81 85 82 13 8
co n cen tr atio n s exceeding the critical miceUar concent rat i on, a m e m b r a n e solubilizing effect will p r e p o n d e r a t e [17]. The degree o f solubilization should d ep en d on t he hydrophilicflipophilic character o f the test substance [19,20]. A reason for the lack o f m e m b r a n e activity o f t he c o m p o u n d s possessing longer carbon chains might be steric hindrance t o i n c o r p o r a t i o n due to f o r matio n o f large aggregates at the high concentrations used. Our results are consistent with those o bt ai ned for t he partitions o f acids and alcohols of various chain length in e r y t h r o c y t e membranes [21,22]. Moreover, a similar correlation b e t w e e n chain length and functional properties o f isolated skeletal muscles, which are d e p e n d e n t on m e m b r a n e integrity, has been d e m o n s t r a t e d by several authors [23---25]. Most o f the aromatic c o m p o u n d s exhibited a m e m b r a n e damaging effect which increased with alkyl substitution o f the aromatic ring (Table IV). The reason for this is n o t clear. This e f fect is m ost obvious in the case o f phenols, phenylacetates, a c t o p h e n o n e s and methylbenzoates. It may be concluded that t o b a c c o smoke contains a n u m b e r of membrane damaging substances. These might cause direct toxic reactions after long term exposure, since even a subtle change in m e m b r a n e permeability will change the metabolism of affected cells [26,27]. F u r t h e r m o r e , membrane active c o m p o u n d s could also p o t e n t i a t e t he toxic effects o f t o b a c c o smoke by p r o m o t i n g the cell m e m b r a n e penet rat i on o f o t h e r t oxi c substances in the smoke.
215
O~
b~
X L
~
X
C3H7
-'~j"~C2H5
X
"CH:
X CHs~'~CN:
C,
~ L . ~ oH3
x
X
Compound, X =
87
84
21
87
47
18
H
91
90
80
33
0
OH
79
36
5
72
% N u c l e o t i d e release
15
18
28
23
10
OCH
S
72
56
CCH 3
45
48
90
CHO
70
76
89
82
86
COOH
67
100
89
86
20
COCH 3
THE E F F E C T O F A L K Y L S U B S T I T U T I O N ON THE M E M B R A N E A C T I V I T Y O F SOME A R O M A T I C C O M P O U N D S
TABLE IV
26
19
CN
53
61
21
NH~
ACKNOWLEDGEMENT
We are grateful to Dr. T. Nishida for recording the NMtt spectra and to Ms. E. Kazemi-Vala and Ms E. Br~innk~irr for skilful technical assistance. REFERENCES 1 C.R. Green, Recent Adv. Tobacco Sci., 3 (1977) 94. 2 R.A. Heckman and F.W. Best, Abstract, 32nd Tobacco Chemists' Research Conference, Montreal, Canada 1978. 3 C. Leuchtenberger and R. Leuchtenberger, Exp. Cell Res., 62 (1970) 161. 4 D.G. Wenzel, J.W. Wheatley and G.D. Byrd, Toxicol. Appl. Pharmacol., 17 (1970) 774. 5 /~. Pilotti, K. Ancker, E. Arrhenius and C. Enzell, Toxicology, 5 (1975) 49. 6 J.A. Styles, Br. J. Exp. Pathol., 57 (1976) 286. 7 C. Leuchtenberger and R. Leuchtenberger, Br. J. Exp. Pathol., 58 (1977) 625. 8 W.R. Thomas, P.G. Holt and D. Keast, Infect. Immun., 20 (1978) 468. O 9 J. Litwin, C. Enzell and A. Pilotti, Acta Pathol. Microbiol. Scand. Sect. A, 86 (1978) 135. 10 C. Chang, M. Castellazzi, T.W. Glover and J.E. Trosko, Cancer Res., 38 (1978) 4527. 11 B. Pettersson, Chem.-Biol. Interact., 29 (1980) 95. 12 M. Thelestam and R. MSllby, Biochim. Biophys. Aeta, 557 (1979) 156. 13 M. Thelestam and R. MSllby, Infect. Immun., 11 (1975) 640. 14 H. Eagle, Science, 130 (1959) 432. 15 M. Thelestam and R. MSllby, Med. Biol., 54 (1976) 39. 16 A. de Bruin, Biochemical Toxicology of Environmental Agents, Elsevier, Amsterdam, 1976, p. 111. 17 C. Tanford and J.A. Reynolds, Biochim. Biophys. Acta, 457 (1976) 133. 18 M.P. Sheetz and S.J. Singer, Proc. Natl. Acad. Sci., 71 (1974) 4457. 19 J.N. Umbreit and J.L. Strominger, Proc. Natl. Acad. Sci., 70 (1973) 2997. 20 J.T. Davies, 2nd Int. Congr. Surface Activity, Vol. 1 (1957) 426. 21 P. Seeman, S. Roth and H. Schneider, Biochim. Biophys. Acta, 225 (1971) 171. 22 P. Seeman, Pharmacol. Rev., 24 (1972) 583. 23 G. Caffier, F. KSssler and G. Kiichler, Acta Biol. Med. Germ., 34 (1975) 99. 24 F. KSssler, G. Caffier and G. Kiichler, Acta Biol. Med. Germ., 35 (1976) 1327. 25 F. KSssler and G. Kiichler, Acta Biol. Med. Germ., 36 (1977) 1085. 26 M.L.S. Ledbetter and M. Lubin, Exp. Cell Res., 105 (1977) 223. 27 M. Lubin, Nature, 213 (1967) 451.
217