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Activation of 2-aminoanthracene to mutagenic intermediates by guinea pig kidney microsomal enzymes following tobacco smoke inhalation
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
41, 119-122 (1977)
Activation of 2-Aminoanthracene to Mutagenic by Guinea Pig Kidney Microsomal Enzymes Tobacco Smoke Inhalation
Intermediates following
M. H. BILIMORIA, J. JOHNSON,J. C. HOGG, AND H. P. WITSCHI’ Pathology Institute, McGill
University, Montreal, Canada
Received October 4, 1976; accepted January 23, 1977
Activation of 2-Aminoanthracene to Mutagenic Intermediates by Guinea Pig Kidney Microsomal Enzymes following Tobacco Smoke Inhalation. BILIMORIA, M. H., JOHNSON, J., H~GG, J. C., AND WITSCHI, H. P. (1977).Toxicol. Appl. Pharmcol. 41, 119-122.Male guineapigsand malerats wereexposedto the smokeof five cigarettesand killed at various time intervalsup to 48hr later. The activity of aryl hydrocarbon hydroxylase in kidney of both specieswassignificantlyhigherthan in controlsat 3,6,12, and 24 hr after the end of smokeexposure.The 9000gsupernatantfrom guineapig kidney homogenatesactivated 2-aminoanthraceneinto a compound(s)mutagenicfor Salmonella typhimurium strain TA 98; the 9000g supernatantfrom smoke-exposed animalshad a greateractivating capacity than preparationsfrom control animals.Preparationsfrom rat kidneyswere much lessactive and no differencewas seenbetweensmoke-exposedand control animals. Much effort in tobacco research centers around the development of a lesshazardous cigarette. Each innovation will eventually have to be tested in chronic, time-consuming, and expensive inhalation studies. This makes it desirable to develop short-term in vivo bioassaysfor preliminary screeningprocedures. It hasbeen recently suggestedthat induction of microsomal aryl hydrocarbon hydroxylase (AHH) in rodent lung might serve as such a test (Akin and Benner, 1976). A possible extension of this approach would be to test lung microsomal preparations for mutagenic activity. In attempts to do so, it was concluded that lung preparations are poor activators (Hutton and Hackney, 1975). Preparations from liver would be better activators except that tobacco smoke inhalation usually fails to induce hepatic AHH to an appreciable degree (Welch et al., 1972; Marcotte and Witschi, 1972; Abramson and Hutton, 1975). Recently it was reported that cigarette smoke inhalation also induces renal AHH activity in mice and rats (Van Cantford and Gielen, 1975). This led usto examine to what extent kidney might serve asan appropriate tissueto look for biochemical endpoints of tobacco smoke toxicity. This communication reports that AHH may be induced by ’ Universitede Montrkal, Montrkal, Canada. Copyright 0 1977by AcademicPress,Inc. All rights of reproduction in any form reserved. Printed in Great Britain
119
1SSN 0041XIOSX
120
BILIMORIA ET AL.
tobacco smoke in guinea pig and rat kidneys. It further presentsevidence that kidney 9000gsupernatant from smoke-exposedguinea pigshasa greater potential for activating 2-aminoanthracene into mutagenic intermediates than has 9000g supernatant from normal kidney. METHODS
Male Sprague-Dawley rats (2 months old) and male randomly bred guinea pigs (2-3 months old) were purchased locally. They were kept at least 1 week on a conventional laboratory diet prior to each experiment. Four guinea pigs or four rats were exposedto the smoke from five cigarettes made from flue-cured tobacco. Details of the apparatus and smoke exposure have been published (Simani er al., 1974). Food was then withdrawn and the animals were killed at appropriate intervals. The kidneys were homogenized in 4 vol of 0.15 M KCI-0.05 M Tris . HCl and AHH activity was measured in total kidney homogenates.Formation of product was linear with time of incubation and amount of protein present. DetaiIs of the assay procedure have been described (Jacquesand Witschi, 1973). The kidney homogenatesfrom four animals were pooled and centrifuged for 10 min at 9OOOg. Aliquots of the 9000gsupernatant (containing approximately 2 mg of protein) were used for the mutagenesisassay, using the procedure described by Ames et al. (1973a,b). Salmonella typhimuritlm strain TA 98,2 an indicator organism for detecting frame-shift mutations, was usedas the test organism. AHH activity was measuredin a separatesample of the 9000g supernatant.
RESULTS
The time course of AHH induction in guinea pig and rat kidney following exposure to five cigarettes is given in Table 1. A significant increasein AHH was seenl-3 hr after exposure. Peak values were found in rats at 6 hr and in guinea pigs at 12hr. Forty-eight hours after smoke exposure, values had fallen to basal levels. In controls, prolonged fasting did decreasebasal activity in kidney. The capacity of kidney 9000gsupernatant to activate various substratesinto mutagens was then evaluated. Similar to what had been observed with lung preparations (Hutton and Hackney, 1975), kidney 9OOOgsupernatant did not activate benzo(u)pyrene or tobacco smoke condensates.However, 2-aminoanthracene was converted into a mutagenic compound(s). We observed a marked species difference. Although 9000g supernatant from normal guinea pig kidney had a lower AHH activity than had rat kidney preparations, guinea pig SOOOg supernatant was about five times more active in activating 2-aminoanthracene (Fig. 1). We then tested renal 9000g supernatant from smoke-exposedanimals at the time of peak induction of AHH (12 hr after exposure for guinea pigs and 6 hr for rats). Kidney supernatants from smoke-exposedguinea pigs were considerably more active than were supernatants from control animals. No difference wasobserved betweenrats exposedto tobacco smokeand controls, although the AHH activity in the SOOOg supernatant was 10 times as high (Fig. 1). ’ Originally obtained from Dr. Bruce H. Ames, University of California, Berkeley, California.
TOBACCO
SMOKE
AND
RENAL
121
AHH
TABLE I INDUCTION
OF
AHH
PIG
IN GUINEA
Nanograms Time after end of exposure 0-4
AND RAT KIDNEY
of 3-OH benzpyrene
AFTER
EXPOSURE TO CIGARETTE
per minute per milligram
Guinea pigs Smoke-exposed animals
0.08 + 0.02 (4)b
3 6 12 24 48
0.33 k 0.05'(8) 0.32 f 0.02" (8) 0.50 + 0.04' (8) 0.09 + 0.02'(12) 0.03 IL-0.01 (6)
0.21 io.05
0.09 kO.03 (4) 0.08 +0.02(4) 0.09 f0.02 (7) 0.09 k 0.01 (8)
(4)
of protein
Rats Controls
0 1
SMOKE’
0.11 + 0.01 (8) 0.05 + 0.01 (12)
0.04 + 0.02 (7)
Smoke-exposed animals
0.35 f 0.05 (4) 0.62 k 0.15'(4) 1.94 f 0.26" (8) 2.73 k 0.24'(S) 1.86 + O.lo’(8) 0.23 I!Z0.06' (8) 0.06 + 0.01 (4)
Controls
0.22 + 0.05 (3) 0.21 i 0.06(4) 0.28 i 0.06 (8) 0.22 + 0.05 (8) 0.34 k 0.03 (8) 0.08 + 0.02 (8) 0.06 + 0.01 (4)
L?Animals were exposed to the smoke produced by five cigarettes; controls were kept in an identical smoking machine with no cigarettes. Each value is the mean i- SE. b Value in parentheses is number of animals. c Values significantly different from controls (p < 0.05) by Student’s 1 test. ----(
Gumsa pg. smh exposed Gumea pig, control (0.20)
t---Rat, smoke .--. Rot, control
(O&21
exposed (3 33) (0 32)
8000
t
pg
2-ominoanthracene/pbte
FIG. 1. Reversion of Salmonella typhimurium TA 98 to histidine independence. Pooled 9OOOg supernatant from the kidneys of four smoke-exposed or four control animals were assayed as described by Ames et al. (1973a,b). The number of mutants for each preparation is plotted as a function of the amount of 2-aminoanthracene per plate. Guinea pigs were killed 12 hr after the end of smoke exposure and rats were killed after 6 hr. The values accompanying the symbols indicate the AHH activity measured in the 9ooOg supernatant as nanograms of 3-OH benzpyrene per minute per milligram of protein.
122
BILIMORIA
ET AL.
DISCUSSION Epidemiologic data associate cigarette smoking with an increased mortality from bladder cancer (Clayson and Cooper, 1970). One possibility to account for this could be that carcinogenic compounds, inhaled with and absorbed from cigarette smoke, are probably eliminated by the kidneys. This may enhance the activity of renal AHH. In guinea pigs, but not in rats, increased AHH activity is accompanied by an enhanced capability of kidney 9000g supernatant to revert typhimurium mutant TA 98. Our observations indicate, therefore, that guinea pig renal tissue, after in viva exposure of the animal to tobacco smoke, has acquired a greater capability to transform foreign compounds and, possibly, might convert someof them into mutagens. In view of the general concern over the health hazards of smoking, the S. typhimurium assay with kidney preparations obtained from suitable animal speciesexposed to tobacco smoke in vivo could be a valuable addition to the present arsenal of short-term bioassayson tobacco smoke toxicity. ACKNOWLEDGMENTS
This work was supportedby the CanadianTobacco Manufacturers Council, Grant No. 27024.Excellent technicalassistance wasprovided by Miss LouiseBovert. REFERENCES ABRAMSON, R. K., ANDHUTTON,J. J. (1975).Effectsof cigarettesmokingon aryl hydrocarbon hydroxylaseactivity in lungsand tissuesof inbred mice. Cancer Res. 35,23-29. AKIN, F. J., AND BENNER, J. F. (1976).Induction of aryl hydrocarbon hydroxylasein rodent lung by cigarette smoke: A potential short-term bioassay.Toxicol. Appl. Pharmacol. 36,
331-337. AMES, B. N., LEE,F. D., AND DURSTON, W. E. (1973a).An improvedbacterialtest systemfor the detection and classificationof mutagensand carcinogens.Proc. Nat. Acad. Sci. USA 70,782-786. AMES,B. N.,DURSTON, W.E.,YAMASAKI,E.,ANDLEE,F. D. (1973b).Carcinogensaremutagens: A simpletest combiningliver homogenatesfor activation and bacteriafor detection.Proc. Nat. Acad. Sci. USA 70,2281-2285. CLAYSON, D. B., AND COOPER, E. H. (1970).Cancerof the urinary tract. Aduan Cancer Res. 13,271-381. HUTTON,J. J., AND HACKNEY,C. (1975).Metabolismof cigarettesmokecondensates by human and rat homogenatesto form mutagensdetectableby Sahnonella typhimrwirrm TA 1538. Cancer Res. 35,2461- 2468. JACQUES, A., AND WITSCHI, H. P. (1973).Beryllium effectson aryl hydrocarbon hydroxylase in rat lung. Arch. Environ. Health 27, 243-247.
MARCOTTE, J., ANDWITSCHI,H. P. (1972).Induction of pulmonary aryl hydrocarbon hydroxylase by marijuana.Res. Commun. Chem. Pathol. 4, 561-568. SIMANI,A. S., INOUE,S., ANDHocq J. C. (1974).Penetrationof the respiratory epitheliumof guineapigsfollowing exposureto cigarettesmoke.Lab. Invest. 31, 75-81. VAN CANTFORD, J., AND GIELEN, J. (1975).Organ specificity of aryl hydrocarbon hydroxylase by cigarettesmokein rats and mice.Biochem. Pharmacol. 24, 1253-1256. WELCH, R. M., CAVALLIYO, J., AND LOH,A. (1972).Effect of exposureto cigarettesmokein the metabolismof benzo(a)pyreneand acetophenetidinby lung and intestineof rats. Toxicol. Appt. Pharmacol.
23,749-758.
AND
APPLIED
PHARMACOLOGY
41, 119-122 (1977)
Activation of 2-Aminoanthracene to Mutagenic by Guinea Pig Kidney Microsomal Enzymes Tobacco Smoke Inhalation
Intermediates following
M. H. BILIMORIA, J. JOHNSON,J. C. HOGG, AND H. P. WITSCHI’ Pathology Institute, McGill
University, Montreal, Canada
Received October 4, 1976; accepted January 23, 1977
Activation of 2-Aminoanthracene to Mutagenic Intermediates by Guinea Pig Kidney Microsomal Enzymes following Tobacco Smoke Inhalation. BILIMORIA, M. H., JOHNSON, J., H~GG, J. C., AND WITSCHI, H. P. (1977).Toxicol. Appl. Pharmcol. 41, 119-122.Male guineapigsand malerats wereexposedto the smokeof five cigarettesand killed at various time intervalsup to 48hr later. The activity of aryl hydrocarbon hydroxylase in kidney of both specieswassignificantlyhigherthan in controlsat 3,6,12, and 24 hr after the end of smokeexposure.The 9000gsupernatantfrom guineapig kidney homogenatesactivated 2-aminoanthraceneinto a compound(s)mutagenicfor Salmonella typhimurium strain TA 98; the 9000g supernatantfrom smoke-exposed animalshad a greateractivating capacity than preparationsfrom control animals.Preparationsfrom rat kidneyswere much lessactive and no differencewas seenbetweensmoke-exposedand control animals. Much effort in tobacco research centers around the development of a lesshazardous cigarette. Each innovation will eventually have to be tested in chronic, time-consuming, and expensive inhalation studies. This makes it desirable to develop short-term in vivo bioassaysfor preliminary screeningprocedures. It hasbeen recently suggestedthat induction of microsomal aryl hydrocarbon hydroxylase (AHH) in rodent lung might serve as such a test (Akin and Benner, 1976). A possible extension of this approach would be to test lung microsomal preparations for mutagenic activity. In attempts to do so, it was concluded that lung preparations are poor activators (Hutton and Hackney, 1975). Preparations from liver would be better activators except that tobacco smoke inhalation usually fails to induce hepatic AHH to an appreciable degree (Welch et al., 1972; Marcotte and Witschi, 1972; Abramson and Hutton, 1975). Recently it was reported that cigarette smoke inhalation also induces renal AHH activity in mice and rats (Van Cantford and Gielen, 1975). This led usto examine to what extent kidney might serve asan appropriate tissueto look for biochemical endpoints of tobacco smoke toxicity. This communication reports that AHH may be induced by ’ Universitede Montrkal, Montrkal, Canada. Copyright 0 1977by AcademicPress,Inc. All rights of reproduction in any form reserved. Printed in Great Britain
119
1SSN 0041XIOSX
120
BILIMORIA ET AL.
tobacco smoke in guinea pig and rat kidneys. It further presentsevidence that kidney 9000gsupernatant from smoke-exposedguinea pigshasa greater potential for activating 2-aminoanthracene into mutagenic intermediates than has 9000g supernatant from normal kidney. METHODS
Male Sprague-Dawley rats (2 months old) and male randomly bred guinea pigs (2-3 months old) were purchased locally. They were kept at least 1 week on a conventional laboratory diet prior to each experiment. Four guinea pigs or four rats were exposedto the smoke from five cigarettes made from flue-cured tobacco. Details of the apparatus and smoke exposure have been published (Simani er al., 1974). Food was then withdrawn and the animals were killed at appropriate intervals. The kidneys were homogenized in 4 vol of 0.15 M KCI-0.05 M Tris . HCl and AHH activity was measured in total kidney homogenates.Formation of product was linear with time of incubation and amount of protein present. DetaiIs of the assay procedure have been described (Jacquesand Witschi, 1973). The kidney homogenatesfrom four animals were pooled and centrifuged for 10 min at 9OOOg. Aliquots of the 9000gsupernatant (containing approximately 2 mg of protein) were used for the mutagenesisassay, using the procedure described by Ames et al. (1973a,b). Salmonella typhimuritlm strain TA 98,2 an indicator organism for detecting frame-shift mutations, was usedas the test organism. AHH activity was measuredin a separatesample of the 9000g supernatant.
RESULTS
The time course of AHH induction in guinea pig and rat kidney following exposure to five cigarettes is given in Table 1. A significant increasein AHH was seenl-3 hr after exposure. Peak values were found in rats at 6 hr and in guinea pigs at 12hr. Forty-eight hours after smoke exposure, values had fallen to basal levels. In controls, prolonged fasting did decreasebasal activity in kidney. The capacity of kidney 9000gsupernatant to activate various substratesinto mutagens was then evaluated. Similar to what had been observed with lung preparations (Hutton and Hackney, 1975), kidney 9OOOgsupernatant did not activate benzo(u)pyrene or tobacco smoke condensates.However, 2-aminoanthracene was converted into a mutagenic compound(s). We observed a marked species difference. Although 9000g supernatant from normal guinea pig kidney had a lower AHH activity than had rat kidney preparations, guinea pig SOOOg supernatant was about five times more active in activating 2-aminoanthracene (Fig. 1). We then tested renal 9000g supernatant from smoke-exposedanimals at the time of peak induction of AHH (12 hr after exposure for guinea pigs and 6 hr for rats). Kidney supernatants from smoke-exposedguinea pigs were considerably more active than were supernatants from control animals. No difference wasobserved betweenrats exposedto tobacco smokeand controls, although the AHH activity in the SOOOg supernatant was 10 times as high (Fig. 1). ’ Originally obtained from Dr. Bruce H. Ames, University of California, Berkeley, California.
TOBACCO
SMOKE
AND
RENAL
121
AHH
TABLE I INDUCTION
OF
AHH
PIG
IN GUINEA
Nanograms Time after end of exposure 0-4
AND RAT KIDNEY
of 3-OH benzpyrene
AFTER
EXPOSURE TO CIGARETTE
per minute per milligram
Guinea pigs Smoke-exposed animals
0.08 + 0.02 (4)b
3 6 12 24 48
0.33 k 0.05'(8) 0.32 f 0.02" (8) 0.50 + 0.04' (8) 0.09 + 0.02'(12) 0.03 IL-0.01 (6)
0.21 io.05
0.09 kO.03 (4) 0.08 +0.02(4) 0.09 f0.02 (7) 0.09 k 0.01 (8)
(4)
of protein
Rats Controls
0 1
SMOKE’
0.11 + 0.01 (8) 0.05 + 0.01 (12)
0.04 + 0.02 (7)
Smoke-exposed animals
0.35 f 0.05 (4) 0.62 k 0.15'(4) 1.94 f 0.26" (8) 2.73 k 0.24'(S) 1.86 + O.lo’(8) 0.23 I!Z0.06' (8) 0.06 + 0.01 (4)
Controls
0.22 + 0.05 (3) 0.21 i 0.06(4) 0.28 i 0.06 (8) 0.22 + 0.05 (8) 0.34 k 0.03 (8) 0.08 + 0.02 (8) 0.06 + 0.01 (4)
L?Animals were exposed to the smoke produced by five cigarettes; controls were kept in an identical smoking machine with no cigarettes. Each value is the mean i- SE. b Value in parentheses is number of animals. c Values significantly different from controls (p < 0.05) by Student’s 1 test. ----(
Gumsa pg. smh exposed Gumea pig, control (0.20)
t---Rat, smoke .--. Rot, control
(O&21
exposed (3 33) (0 32)
8000
t
pg
2-ominoanthracene/pbte
FIG. 1. Reversion of Salmonella typhimurium TA 98 to histidine independence. Pooled 9OOOg supernatant from the kidneys of four smoke-exposed or four control animals were assayed as described by Ames et al. (1973a,b). The number of mutants for each preparation is plotted as a function of the amount of 2-aminoanthracene per plate. Guinea pigs were killed 12 hr after the end of smoke exposure and rats were killed after 6 hr. The values accompanying the symbols indicate the AHH activity measured in the 9ooOg supernatant as nanograms of 3-OH benzpyrene per minute per milligram of protein.
122
BILIMORIA
ET AL.
DISCUSSION Epidemiologic data associate cigarette smoking with an increased mortality from bladder cancer (Clayson and Cooper, 1970). One possibility to account for this could be that carcinogenic compounds, inhaled with and absorbed from cigarette smoke, are probably eliminated by the kidneys. This may enhance the activity of renal AHH. In guinea pigs, but not in rats, increased AHH activity is accompanied by an enhanced capability of kidney 9000g supernatant to revert typhimurium mutant TA 98. Our observations indicate, therefore, that guinea pig renal tissue, after in viva exposure of the animal to tobacco smoke, has acquired a greater capability to transform foreign compounds and, possibly, might convert someof them into mutagens. In view of the general concern over the health hazards of smoking, the S. typhimurium assay with kidney preparations obtained from suitable animal speciesexposed to tobacco smoke in vivo could be a valuable addition to the present arsenal of short-term bioassayson tobacco smoke toxicity. ACKNOWLEDGMENTS
This work was supportedby the CanadianTobacco Manufacturers Council, Grant No. 27024.Excellent technicalassistance wasprovided by Miss LouiseBovert. REFERENCES ABRAMSON, R. K., ANDHUTTON,J. J. (1975).Effectsof cigarettesmokingon aryl hydrocarbon hydroxylaseactivity in lungsand tissuesof inbred mice. Cancer Res. 35,23-29. AKIN, F. J., AND BENNER, J. F. (1976).Induction of aryl hydrocarbon hydroxylasein rodent lung by cigarette smoke: A potential short-term bioassay.Toxicol. Appl. Pharmacol. 36,
331-337. AMES, B. N., LEE,F. D., AND DURSTON, W. E. (1973a).An improvedbacterialtest systemfor the detection and classificationof mutagensand carcinogens.Proc. Nat. Acad. Sci. USA 70,782-786. AMES,B. N.,DURSTON, W.E.,YAMASAKI,E.,ANDLEE,F. D. (1973b).Carcinogensaremutagens: A simpletest combiningliver homogenatesfor activation and bacteriafor detection.Proc. Nat. Acad. Sci. USA 70,2281-2285. CLAYSON, D. B., AND COOPER, E. H. (1970).Cancerof the urinary tract. Aduan Cancer Res. 13,271-381. HUTTON,J. J., AND HACKNEY,C. (1975).Metabolismof cigarettesmokecondensates by human and rat homogenatesto form mutagensdetectableby Sahnonella typhimrwirrm TA 1538. Cancer Res. 35,2461- 2468. JACQUES, A., AND WITSCHI, H. P. (1973).Beryllium effectson aryl hydrocarbon hydroxylase in rat lung. Arch. Environ. Health 27, 243-247.
MARCOTTE, J., ANDWITSCHI,H. P. (1972).Induction of pulmonary aryl hydrocarbon hydroxylase by marijuana.Res. Commun. Chem. Pathol. 4, 561-568. SIMANI,A. S., INOUE,S., ANDHocq J. C. (1974).Penetrationof the respiratory epitheliumof guineapigsfollowing exposureto cigarettesmoke.Lab. Invest. 31, 75-81. VAN CANTFORD, J., AND GIELEN, J. (1975).Organ specificity of aryl hydrocarbon hydroxylase by cigarettesmokein rats and mice.Biochem. Pharmacol. 24, 1253-1256. WELCH, R. M., CAVALLIYO, J., AND LOH,A. (1972).Effect of exposureto cigarettesmokein the metabolismof benzo(a)pyreneand acetophenetidinby lung and intestineof rats. Toxicol. Appt. Pharmacol.
23,749-758.