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Effect of cigarette smoke on drug metabolism in vitro
Pergamon Press
Life Sciences, Vol . 25, pp . 755-758 Printed in the U .S .A .
EFFECT OF CIGARETTE SMOKE (1N DRUG METABOLISM IN VITRO R.J .B . Garrett, * M .A . Jackson, * A .K . Filio, * and N .E . Garrett* College of Pharmacy, University of Kentucky, Lexington, Ky . 40506 Cellular Pathology and Biochemistry Section, Northrop Services, Inc ., Research Triangle Park, North Carolina 27709 ** (Received in final form July 20, 1979)
Surrma ry N-demethylation of aminopyrine and C-hydroxylation of aniline by hepatic microsomal enzymes were measured during in vitro exposure to cigarette smoke . Metabolism of aminopyrine during -smoTce exposure was not significantly altered . Metabolism of aniline during smoke exposure was inhibited 70-80~ (P < .001) thus indicating that an initial effect of exposure to cigarette smoke is a decreased rate of biotransformation via C-hydroxylation . This finding, coupled with the findings of other investigators who have shown that the delayed effect of exposure to cigarette smoke is induction of hydroxylase activity, suggests that cigarette smoke produces a biphasic alteration in certain hepatic biotransformation processes . Chronic cigarette smoking is believed to enhance biotransformation processes . Reportedly, propoxyphene, phenacetin, diazepam and nicotine are metabolized at a faster rate in the smoker than in the non-smoker (1-4) . Presumably this increased biotransforniation subsequent to chronic smoke exposure is due to induction of hepatic drug-metabolizing enzymes (2,4,5) . These results are consistent with the fact that following chronic administration of certain drugs i .e ., Phenobarbital, ethanol), biotransformation processes are accelerated ~6-9) . It is noteworthy, however, that after chronic administration of same commonly used drugs (i .e . disulfiram, allo urinol, nortriptyline) biotransformation processes are inhibited (10,11,12 rather than accelerated . Furthermore, certain compounds exert a biphasic effect on biotransformation processes (12,13) . For example, SKF 525A (6-diethylaminoethyl-diphenylpropylacetate HC1), 2,4-dichloro-6-phenylphenoxyethylamine, chlorcyclizine, glutethimide and phenaglycodol initially cause an inhibitory effect on drug metabolism ; however, the delayed effect of these compounds is one of accelerated biotransformation . Since short-term exposure to cigarette smoke inhibits protein synthesis in liver (14) and lung (15,16), and since the effect of long-term exposure is accelerated biotransformation (1-4,17) apparently due to synthesis of drug metabolizing enzymes, we undertook studies to determine the short-term effect of smoke on hepatic N-demethylation and C-hydroxylation reactions using aminopyrine and aniline, respectively as substrates . This work was supported in part by University of Kentucky Tobacco and Health Institute Project Number 091 to R.J .B . Garrett . Send reprint requests to N. E. Garrett, Northrop Services, Inc ., Research Triangle Park, N .C . 27709 . 0024-3205/79/090755 04$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
Vol . 25, No . 9, 1979
Smoke and Drug Metabolism In Vitro
756
Methods Microsomes were prepared from livers of female Sprague Dawley rats (200-300 9m body weight) . Livers were perfused with Solution A (NaCI, 137 mM ; KCL, 2 .68 mM ; Na HPO 8 .1 mM ; KH PO , 1 .47 mM ; sucrose, 0 .25 M ; pH 7 .4) at 4°C, A 33% homoge~tate4 ~w/v) was pr~paied in Solution A, 4°C, using a Teflon-glass homogenizer (2000 rpm, 5 complete strokes) . After differential centrifugation of the homogenate (1,000 x g, 10 min, 4°C ; 10,000 x g, 10 min, 4°C) the supernatant (designated microsomal fraction) was harvested and used in the determination of the effect of smoke on biotransformation . In some experiments the microsomal faction was exposed to 60 puffs of smoke prior to assay of enzymatic activity . In other experiments smoke exposure was initiated at the same time as the assay for enzyme activity . Samples were exposed to cigarette smoke from University of Kentucky reference cigarettes (R1) according to a regimen of 1 puff/min as described previously (15 . In brief, smoke was generated in a rectilinear puff by a single-port reverse type smoking machine as described by Griffith et al . (18) . In sham control experiments the microsomal fraction was exposed to atmospheric air rather than to cigarette smoke . Assay of enzymatic activity was initiated by addition of the cofactor mix and the drug . For assay of aminopyrine, the final composition of the incubation mixture was : Tris, pH 7 .4, 10 mM ; MgCI , 10 mM ; creatine phosphate, 2 .5 uM ; creatine phosphokinase, 0 .6 mg ; GTP, 0 .1 21 uM ; glutathione, 2 .5 uM ; glucose-6-phosphate, 7 mM ; NADP+, 0 .4 mM ; KC1, 0 .01 M ; nicotinamide, 8 .3 mM ; semicarbizide, 7 .5 mM ; ATP, 0 .50 uM ; glucose6-phosphate dehydrogenase, 12 units ; aminopyrine, 1 mM ; microsomal fraction, 8 ml ; and enough 1 .15% KC1 in Solution A to give a final incubation volume of 20 ml . For assay of aniline, the final composition of the incubation mixture was the same as that for assay of aminopyrine except that aniline, 1 mM, was substituted for aminopyrine . All incubations were carried out at 37°C in a shaking water bath (70 oscillations/min) . Formaldehyde, the metabolic product of N-demethylation of aminopyrine, was measured by the method of Nash (19 ) . P-aminophenol, the metabolic product of C-hydroxylation of aniline, was measured by the method of Mazel (20) . Results and Discussion Exposure to cigarette smoke did not significantly alter metabolism of aminopyrine (Fig . 1), but metabolism of aniline during smoke exposure was markedly inhibited (Fi . 2) . The depressive effect on aniline biotransformation was 70 80% (P < .001 . Inhibition of aniline metabolism was also observed when liver microsomes were not pre-exposed to cigarette smoke (Fig . 3) . In these experi-
FIG . 1 Effect of cigarette smoke on N-demethylation of aminopyrine by liver microsomes preexposed to 60 puffs of smoke . Formaldehyde, a metabolic product of aminopyrine, was measured . Data are reported as ug formaldehyde formed per gram wet liver (mean ± SEM) . Key : "---~, metabol i sm of l i ver mi crosomes under control conditions ; 0---0, metabolism by liver microsomes exposed to cigarette smoke (1 puff/min) .
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Smoke and Drug Metabolism In Vitro
Vol . 25, No . 9, 1979
FIG . 2
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Effect of cigarette smoke on C-hydroxylation of aniline by liver microsomes pre-exposed to 60 puffs of smoke . P-aminophenol, the metabolic product of aniline, was measured . Data are reported as ug p-aminophenol formed per gram wet liver (mean ± SEM) . Key : H , metabolism by liver microsomes under control conditions ; 0--a, metabolism by liver microsomes Burin exposure to cigarette smoke (1 puff/min .
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FIG . 3
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Effect of increased smoke dosage on C-hydroxylation of aniline by liver microsomes which received no pre-exposure to cigarette smoke . The metabolic product, p-aminophenol, was measured . Data are reported as u p-aminophenol formed per gram wet liver ~mean±SEM) . Key : F--~ , metabolism by liver microsames under control conditions ; 0--0, metabolism by liver microsomes during exposure to smoke (1 puff/min) .
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ments metabolism of aniline was also shown to be positively correlated with the dosage of smoke . No si nificant difference was observed after 5 min of incubation, but after 10 min g(i .e . 10 puffs of smoke, 1 puff/min) smoke inhibited p-aminophenol formation approximately 40% (P < .O1) . After 20 minutes of incubation p-aminophenol formation was reduced by approximately 60% (P < ,001) . These data show that enzymes involved in N-demethylation of aminopyrine were not affected by cigarette smoke under in vitro conditions . In contrast, the activity of enzymes involved in C-hydroxy aT~on of aniline was suppressed, suggesting that the initial effect of exposure to cigarette smoke is a decreased rate of biotransformation via C-hydroxylation . It has previously been shown that exposure to cigarette smoke results in accelerated drug metabolism (1 - 4,21) . The increase in drug metabolism is believed to result from induction of hepatic microsomal protein s) and is apparently due to an increase 1n synthesis of the metabolizing enzymes (6,9) . However, we have previously shown that the immediate effect of in vivo exposure to cigarette smoke is a depression of protein synthesis in ~TielTver (14 ) . In addition, we speculated that the smoke-associated biochemical response was biphasic in character (14) . A biphasic response to cigarette smoke has been reported (22) relative to pulmonary 3,4-BP-hydroxylase . Immediately after smoke exposure, BP-hydroxylase activity was depressed, but subsequently metabolism was accelerated . Since the
758
Smoke and Drug Metabolism In Vitro
Vol . 25, No . 9, 1979
long-term effect of exposure to cigarette smoke is induction of hydroxylase activity (17,23), our data which show that the initial effect of smoke is depression of C-hydroxylation activity suggest that smoke possibly should be classified among those pharmacologically active agents which produce a biphasic effect on certain drug metabolizing processes in the liver . References 1 . H . JICK, Med . Clin . North Am . 58 1143 (1974), 2 . E .J . PANTUCK, K .C . HSIAO, A . hV~GIO, K . NAKAMURA, R . KUNTZMAN and A .H . CONNEY, Clin . Pharmacol . Terap . 15 9 (1974) . 3 . BOSTON COLLABORATIVE DRUG SURVEILTANCE PROGRAM, New Eng . J, Med . 288 277 (1973) . 4 . A .H . BECKETT and E .J . TRIGGS, Nature (Lond .) 216, 587 (1967) . 5 . R .E . VESTAL, A .H . NORRIS, J .D . TOBIN, B .H . CONÉA, N .W . SHOCK and R . ANDRES, Clin . Pharmacol . Therap . 18 425 (1975) . 6 . A .H . CONNEY and J .J . BURNS Adv . i n Pharmacol . 1 131 (1962) . 7 . E .S . VESELL and J .G . PAGE, J . Clin . Invest . 48 X202 (1969) . 8 . E .S . VESELL, J .G . PAGE and G .T . PASSANANTI, Z`Tin . Pharmacol . Therap . _12 192 (1971) . 9 . A .H . CONNEY, Pharmacol . Rev . 19 317 (1967) . 10 . B . STRIPP, R,E . GREENE and J .1F. GILLETTE, J . Pharmacol . Exp . Ther . _170 347 (1969) . 11 . T . HONJO and K .J . NETTER, Biochem . Pharmacol . 18 2681 (1969) . 12 . E .S . VESELL and G .T . PASSANANTI, Drug Metab . Dr ~spos . 1 402 (1973) . 13 . R . KATO, E . CHIESARA and P . VASSANELLI, Biochem . Pharmacol . 13 69 (1964) . 14 . R .J .B . GARRETT and M .A . JACKSON, J . Pharm . Exp . Therap . _209 'fT5 (1979) . 15 . R .J .B . GARRETT, Environ . Res . 17 205 (1978) . 16 . R .J .B . GARRET T and M .A . JACKSOT~ Environ . Res . i n press . 17 . F .J . AKIN and J .F, BENNER, Tox . Appl . Pharmacol . 36 331 (1976) . 18 . R .B . GRIFFITH, J .F . BENNER, S .S . OWENS and R .L . HAACOCK, Proc . Univ . Tobacco and Health Research Institute, Conference Report , p . 9 ). 19 . oc em . 20 . P . MAZEL, Fundamentalsof Drug Metabolism and Decomposition, Eds ., B .N . Ladu, H .G . Mandel and E . L . Way, p 546, William and Wilks Co . (1971) . 21 . R .M . WELCH, J . CAVALLITO and D .D . GILLESPIE, Drug Metab . Dispos . _1 211 (1973) . 22 . R .M . WELCH, A . LOH and A .H . CONNEY, Life Sci . 10 215 (1971) . 23 . R .M . WELCH, Y .E . HARRISON and A .H . CONNEY, Science 160 541 (1968) .
Life Sciences, Vol . 25, pp . 755-758 Printed in the U .S .A .
EFFECT OF CIGARETTE SMOKE (1N DRUG METABOLISM IN VITRO R.J .B . Garrett, * M .A . Jackson, * A .K . Filio, * and N .E . Garrett* College of Pharmacy, University of Kentucky, Lexington, Ky . 40506 Cellular Pathology and Biochemistry Section, Northrop Services, Inc ., Research Triangle Park, North Carolina 27709 ** (Received in final form July 20, 1979)
Surrma ry N-demethylation of aminopyrine and C-hydroxylation of aniline by hepatic microsomal enzymes were measured during in vitro exposure to cigarette smoke . Metabolism of aminopyrine during -smoTce exposure was not significantly altered . Metabolism of aniline during smoke exposure was inhibited 70-80~ (P < .001) thus indicating that an initial effect of exposure to cigarette smoke is a decreased rate of biotransformation via C-hydroxylation . This finding, coupled with the findings of other investigators who have shown that the delayed effect of exposure to cigarette smoke is induction of hydroxylase activity, suggests that cigarette smoke produces a biphasic alteration in certain hepatic biotransformation processes . Chronic cigarette smoking is believed to enhance biotransformation processes . Reportedly, propoxyphene, phenacetin, diazepam and nicotine are metabolized at a faster rate in the smoker than in the non-smoker (1-4) . Presumably this increased biotransforniation subsequent to chronic smoke exposure is due to induction of hepatic drug-metabolizing enzymes (2,4,5) . These results are consistent with the fact that following chronic administration of certain drugs i .e ., Phenobarbital, ethanol), biotransformation processes are accelerated ~6-9) . It is noteworthy, however, that after chronic administration of same commonly used drugs (i .e . disulfiram, allo urinol, nortriptyline) biotransformation processes are inhibited (10,11,12 rather than accelerated . Furthermore, certain compounds exert a biphasic effect on biotransformation processes (12,13) . For example, SKF 525A (6-diethylaminoethyl-diphenylpropylacetate HC1), 2,4-dichloro-6-phenylphenoxyethylamine, chlorcyclizine, glutethimide and phenaglycodol initially cause an inhibitory effect on drug metabolism ; however, the delayed effect of these compounds is one of accelerated biotransformation . Since short-term exposure to cigarette smoke inhibits protein synthesis in liver (14) and lung (15,16), and since the effect of long-term exposure is accelerated biotransformation (1-4,17) apparently due to synthesis of drug metabolizing enzymes, we undertook studies to determine the short-term effect of smoke on hepatic N-demethylation and C-hydroxylation reactions using aminopyrine and aniline, respectively as substrates . This work was supported in part by University of Kentucky Tobacco and Health Institute Project Number 091 to R.J .B . Garrett . Send reprint requests to N. E. Garrett, Northrop Services, Inc ., Research Triangle Park, N .C . 27709 . 0024-3205/79/090755 04$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
Vol . 25, No . 9, 1979
Smoke and Drug Metabolism In Vitro
756
Methods Microsomes were prepared from livers of female Sprague Dawley rats (200-300 9m body weight) . Livers were perfused with Solution A (NaCI, 137 mM ; KCL, 2 .68 mM ; Na HPO 8 .1 mM ; KH PO , 1 .47 mM ; sucrose, 0 .25 M ; pH 7 .4) at 4°C, A 33% homoge~tate4 ~w/v) was pr~paied in Solution A, 4°C, using a Teflon-glass homogenizer (2000 rpm, 5 complete strokes) . After differential centrifugation of the homogenate (1,000 x g, 10 min, 4°C ; 10,000 x g, 10 min, 4°C) the supernatant (designated microsomal fraction) was harvested and used in the determination of the effect of smoke on biotransformation . In some experiments the microsomal faction was exposed to 60 puffs of smoke prior to assay of enzymatic activity . In other experiments smoke exposure was initiated at the same time as the assay for enzyme activity . Samples were exposed to cigarette smoke from University of Kentucky reference cigarettes (R1) according to a regimen of 1 puff/min as described previously (15 . In brief, smoke was generated in a rectilinear puff by a single-port reverse type smoking machine as described by Griffith et al . (18) . In sham control experiments the microsomal fraction was exposed to atmospheric air rather than to cigarette smoke . Assay of enzymatic activity was initiated by addition of the cofactor mix and the drug . For assay of aminopyrine, the final composition of the incubation mixture was : Tris, pH 7 .4, 10 mM ; MgCI , 10 mM ; creatine phosphate, 2 .5 uM ; creatine phosphokinase, 0 .6 mg ; GTP, 0 .1 21 uM ; glutathione, 2 .5 uM ; glucose-6-phosphate, 7 mM ; NADP+, 0 .4 mM ; KC1, 0 .01 M ; nicotinamide, 8 .3 mM ; semicarbizide, 7 .5 mM ; ATP, 0 .50 uM ; glucose6-phosphate dehydrogenase, 12 units ; aminopyrine, 1 mM ; microsomal fraction, 8 ml ; and enough 1 .15% KC1 in Solution A to give a final incubation volume of 20 ml . For assay of aniline, the final composition of the incubation mixture was the same as that for assay of aminopyrine except that aniline, 1 mM, was substituted for aminopyrine . All incubations were carried out at 37°C in a shaking water bath (70 oscillations/min) . Formaldehyde, the metabolic product of N-demethylation of aminopyrine, was measured by the method of Nash (19 ) . P-aminophenol, the metabolic product of C-hydroxylation of aniline, was measured by the method of Mazel (20) . Results and Discussion Exposure to cigarette smoke did not significantly alter metabolism of aminopyrine (Fig . 1), but metabolism of aniline during smoke exposure was markedly inhibited (Fi . 2) . The depressive effect on aniline biotransformation was 70 80% (P < .001 . Inhibition of aniline metabolism was also observed when liver microsomes were not pre-exposed to cigarette smoke (Fig . 3) . In these experi-
FIG . 1 Effect of cigarette smoke on N-demethylation of aminopyrine by liver microsomes preexposed to 60 puffs of smoke . Formaldehyde, a metabolic product of aminopyrine, was measured . Data are reported as ug formaldehyde formed per gram wet liver (mean ± SEM) . Key : "---~, metabol i sm of l i ver mi crosomes under control conditions ; 0---0, metabolism by liver microsomes exposed to cigarette smoke (1 puff/min) .
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Smoke and Drug Metabolism In Vitro
Vol . 25, No . 9, 1979
FIG . 2
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Effect of cigarette smoke on C-hydroxylation of aniline by liver microsomes pre-exposed to 60 puffs of smoke . P-aminophenol, the metabolic product of aniline, was measured . Data are reported as ug p-aminophenol formed per gram wet liver (mean ± SEM) . Key : H , metabolism by liver microsomes under control conditions ; 0--a, metabolism by liver microsomes Burin exposure to cigarette smoke (1 puff/min .
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FIG . 3
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Effect of increased smoke dosage on C-hydroxylation of aniline by liver microsomes which received no pre-exposure to cigarette smoke . The metabolic product, p-aminophenol, was measured . Data are reported as u p-aminophenol formed per gram wet liver ~mean±SEM) . Key : F--~ , metabolism by liver microsames under control conditions ; 0--0, metabolism by liver microsomes during exposure to smoke (1 puff/min) .
!a a :o z la lo a 0
757
o
a
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ments metabolism of aniline was also shown to be positively correlated with the dosage of smoke . No si nificant difference was observed after 5 min of incubation, but after 10 min g(i .e . 10 puffs of smoke, 1 puff/min) smoke inhibited p-aminophenol formation approximately 40% (P < .O1) . After 20 minutes of incubation p-aminophenol formation was reduced by approximately 60% (P < ,001) . These data show that enzymes involved in N-demethylation of aminopyrine were not affected by cigarette smoke under in vitro conditions . In contrast, the activity of enzymes involved in C-hydroxy aT~on of aniline was suppressed, suggesting that the initial effect of exposure to cigarette smoke is a decreased rate of biotransformation via C-hydroxylation . It has previously been shown that exposure to cigarette smoke results in accelerated drug metabolism (1 - 4,21) . The increase in drug metabolism is believed to result from induction of hepatic microsomal protein s) and is apparently due to an increase 1n synthesis of the metabolizing enzymes (6,9) . However, we have previously shown that the immediate effect of in vivo exposure to cigarette smoke is a depression of protein synthesis in ~TielTver (14 ) . In addition, we speculated that the smoke-associated biochemical response was biphasic in character (14) . A biphasic response to cigarette smoke has been reported (22) relative to pulmonary 3,4-BP-hydroxylase . Immediately after smoke exposure, BP-hydroxylase activity was depressed, but subsequently metabolism was accelerated . Since the
758
Smoke and Drug Metabolism In Vitro
Vol . 25, No . 9, 1979
long-term effect of exposure to cigarette smoke is induction of hydroxylase activity (17,23), our data which show that the initial effect of smoke is depression of C-hydroxylation activity suggest that smoke possibly should be classified among those pharmacologically active agents which produce a biphasic effect on certain drug metabolizing processes in the liver . References 1 . H . JICK, Med . Clin . North Am . 58 1143 (1974), 2 . E .J . PANTUCK, K .C . HSIAO, A . hV~GIO, K . NAKAMURA, R . KUNTZMAN and A .H . CONNEY, Clin . Pharmacol . Terap . 15 9 (1974) . 3 . BOSTON COLLABORATIVE DRUG SURVEILTANCE PROGRAM, New Eng . J, Med . 288 277 (1973) . 4 . A .H . BECKETT and E .J . TRIGGS, Nature (Lond .) 216, 587 (1967) . 5 . R .E . VESTAL, A .H . NORRIS, J .D . TOBIN, B .H . CONÉA, N .W . SHOCK and R . ANDRES, Clin . Pharmacol . Therap . 18 425 (1975) . 6 . A .H . CONNEY and J .J . BURNS Adv . i n Pharmacol . 1 131 (1962) . 7 . E .S . VESELL and J .G . PAGE, J . Clin . Invest . 48 X202 (1969) . 8 . E .S . VESELL, J .G . PAGE and G .T . PASSANANTI, Z`Tin . Pharmacol . Therap . _12 192 (1971) . 9 . A .H . CONNEY, Pharmacol . Rev . 19 317 (1967) . 10 . B . STRIPP, R,E . GREENE and J .1F. GILLETTE, J . Pharmacol . Exp . Ther . _170 347 (1969) . 11 . T . HONJO and K .J . NETTER, Biochem . Pharmacol . 18 2681 (1969) . 12 . E .S . VESELL and G .T . PASSANANTI, Drug Metab . Dr ~spos . 1 402 (1973) . 13 . R . KATO, E . CHIESARA and P . VASSANELLI, Biochem . Pharmacol . 13 69 (1964) . 14 . R .J .B . GARRETT and M .A . JACKSON, J . Pharm . Exp . Therap . _209 'fT5 (1979) . 15 . R .J .B . GARRETT, Environ . Res . 17 205 (1978) . 16 . R .J .B . GARRET T and M .A . JACKSOT~ Environ . Res . i n press . 17 . F .J . AKIN and J .F, BENNER, Tox . Appl . Pharmacol . 36 331 (1976) . 18 . R .B . GRIFFITH, J .F . BENNER, S .S . OWENS and R .L . HAACOCK, Proc . Univ . Tobacco and Health Research Institute, Conference Report , p . 9 ). 19 . oc em . 20 . P . MAZEL, Fundamentalsof Drug Metabolism and Decomposition, Eds ., B .N . Ladu, H .G . Mandel and E . L . Way, p 546, William and Wilks Co . (1971) . 21 . R .M . WELCH, J . CAVALLITO and D .D . GILLESPIE, Drug Metab . Dispos . _1 211 (1973) . 22 . R .M . WELCH, A . LOH and A .H . CONNEY, Life Sci . 10 215 (1971) . 23 . R .M . WELCH, Y .E . HARRISON and A .H . CONNEY, Science 160 541 (1968) .