Mutatwn Research, 260 (1991) 9-18 © 1991 Elsevier Science Pubhshers B.V. 0165-1218/91/$03 50 ADONIS 0165121891000742
MUTGEN 01635
The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine D a v i d J. D o o l i t t l e , C a r o l y n A. R a h n a n d C h i n K . L e e Cellular and Molecular Bwlogy Dwtston, Butldmg 630-2, R J Reynolds Tobacco Company, Wmston-Salerr~ NC 27102 (U S A.) (Received 24 July 1990) (Accepted 27 August 1990)
Keywords. Rat urine, N~cotme exposure; Carbon monoxide, C~garette smoke, C~garette smoke condensate; Carboxyhemoglobm
Summary Cigarette smokers have been reported to void urine which is more mutagenlc than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the mlcrosuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperltoneal injection at 0.8 m g / k g (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting m 68% carboxyhemoglobin) did not increase urinary mutageniclty. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 m g / k g when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutageniclty following the inhalation of tobacco smoke.
Mutagenicity testing of human urine has been widely used as a means for evaluating exposure to potentially genotoxic materials (for review see
Correspondence- David Doohttle, Ph D , DABT, Cellular and Molecular Biology Division, R.J Reynolds Tobacco Company, Winston-Salem, NC 27102 (U S A ).
Everson, 1986). Once an agent or its metabohtes has been identified as a probable urinary mutagen in humans, the use of inbred ammal models would greatly facilitate mechanistic studies on the role and interactions of the agent in altering urinary mutagenicity. Although the specific urinary mutagen(s) have not been identified, cigarette smokers have been reported to void urine which is
10 more mutagenic than that voided by non-smokers (IARC, 1986; Doolittle et al., 1989). The development of a responsive animal model would provlde for controlled mechanistic studies on the effect of c~garette smoke and its components on urinary mutagenicity. Two components of interest are nicotine and carbon monoxide. Carbon monoxide is known to inhibit cytochrome P-450 (Ortlz de Montellano and Reich, 1986), a critical enzyme in the metabolic actwat~on of m a n y mutagens, including some dietary mutagens (Kato, 1986). Thus, administration of carbon monoxide could potentially increase or decrease unnary mutagenlc~ty. Although nicotine and its major metabolites are not mutagenic m the Ames test (Riebe et al., 1982), it has been proposed, but not demonstrated, that mcotine could be converted into mutagenic mtrosamlnes in the body (Hoffmann and Hecht, 1985). Thus, the maximum tolerated dose of nicotine was administered to the animals and urinary mutageniclty assessed. Baboons have been used as an animal model to study smoking-related urinary mutagenicity (Marshall et al., 1983), but widespread use of this animal model is not feasible. We have recently characterized a sensitive assay for detecting urinary mutagemc~ty in rats which uses XAD-2 resin to concentrate the urine and a microsuspens~on modificahon of the Ames bacterial mutagenesis assay to detect mutagens (Doolittle et al., 1988). Since the methodology ~s essentially ~dentical to that commonly used in studies on the mutagemcity of human urine, interspec~es comparisons are facilitated. The objective of this study was to evaluate the effect of exposure to nicotine, carbon monoxade, cigarette smoke and c~garette smoke condensate on the mutagenicity of rat urine. Materials and methods
Chemwals and dosmg soluttons High purity XAD-2 resin was obtained from Alltech (Deerfield, IL). Methanol, acetone and methylene chloride ( H P L C grade) were obtained from Burdick and Jackson (Muskegon, MI). Nicotine (Kodak Chemical Co.) was dissolved in phosphate-buffered saline and rejected i.p. at 0.8 m g / k g (1 ml dosing solution/kg). Electrostatically precipitated cigarette smoke condensate
(CSC) was prepared from 1R4F research cigarettes (University of Kentucky Smoking and Health Institute, Lexington, KY) according to standard techniques (Coresta, 1969). The stock solution of CSC was 200 mg Total Particulate Matter ( T P M ) / m l dimethyl sulphoxide (DMSO, Sigma Chemical Co.). This solution was diluted, if required, and administered to rats at a constant volume of 1 m l / k g . PD01483A and PD01102A reference cigarettes were representative tobaccoburning cigarettes and were obtained from the Research and Development Department of R.J. Reynolds Tobacco Company.
Animals Sprague-Dawley rats 56-90 days old (250-400 g), obtamed from Charles River Breeding Laboratories (Raleigh, NC), were given feed and water ad libitum and allowed to acclimatize for at least 1 week prior to use. The sex of the a ~ m a l does not affect the results obtained (unpublished observation). Females were used for the nicotine and 1R4F 1-day exposure studies; males were used m all other experiments. Smoke exposure of ammals Smoke exposure was performed in a Canon-type nose-only inhalauon chamber (Coggins et al., 1989). The cigarettes were puffed at standard Federal Trade Commission (FTC) conditions of 35 ml puff volume for a 2-sec duration at a frequency of once per minute. Smoke from the cigarettes was diluted with HEPA-filtered air to provide the desired concentrations. Animal exposure was 1 h per day. Sham-exposed animals were placed in the smoking machine for 1 h, but were exposed to HEPA-filtered air only. Carbon monoxide was monitored during the exposure with a Miran 80A infrared spectrophotometer, and carboxyhemoglobin was determined in a Model 282 CO-Oximeter (Instrumentation Laboratories, Hartford, CT). Total Particulate Matter (TPM) in the smoke was estimated by a RAM-1 instrument ( G C A Corporation, Lexington, MA) and by gravimetric measurement following collection of the T P M onto Cambridge filter pads. Nose-only exposure to 1800 p p m carbon monoxide was conducted as previously described (Ayres et al., 1989).
11
Urine collectton and extractton Animals were placed in Nalgene metabolic cages immediately following dosing in the i.p. injection and gavage studies or immediately after the termination of the final exposure period in the inhalation studies. Urine samples were collected for 24 h. The urine collection cups were surrounded with dry ice in a styrofoam bucket covered with foil so that the unne samples froze immediately upon contact. Samples were stored at - 7 0 ° C until extraction on XAD-2 resin as previously described (Doolittle et al., 1988). Briefly, urine samples (15-75 ml) were adjusted to p H 7.0, filtered through Whatman No. 1 filter paper, and then through a 0 . 2 / l m Acrodisc filter (Gelman). 1 ml of urine was retained, and the remainder placed onto a column containing 10 g XAD-2 resin at a flow rate of 2 - 3 m l / m i n . The column was washed with 10 ml water to remove residual urine from the column, dried with nitrogen, and eluted with 10 ml acetone. The acetone eluates were dried under nitrogen. Urine extract A was prepared by dissolving the dried eluate in 500 /~1 of water. U r m e extract B was prepared by dissolving the dried eluate in 20 ml water, followed by extraction with methylene chloride. The methylene chloride fraction was evaporated under nitrogen and the residue dissolved in 500 /~1 DMSO. All samples were stored at - 7 0 ° C until mutageniclty assays were conducted. Mutagemcay assay The microsuspenslon modification of the Ames assay was performed according to Kado et al. (1983). All mutagenicity testing was done using tester strain TA98 (obtained from Dr. B. Ames, University of California, Berkeley). An overnight culture of TA98 cells was concentrated 5 × and 0.1 ml of concentrated cells per plate was used in the rmcrosuspension assay. Rat liver $9 (Organon Teknika Corp., Charleston, SC) prepared from male Sprague-Dawley rats treated with Aroclor 1254 was used at a volume of 0.1 ml of $9 nuxture per plate (30 /~g protein). Potentially mutagenic materials may be excreted in the urine as glucuronide a n d / o r sulfate conjugates. The role of these conjugations was evaluated by adding flglucuronldase/aryl sulfatase (BG) to the Ames microsuspenslon assay and comparing to results in
the absence of these enzymes. The fl-glucuronid a s e / a r y l sulfatase mixture (Sigma G2887) was added at 1000 u n i t s / p l a t e . All samples were tested in 10-#1 volumes at a minimum of 4 concentrations with triphcate plates at each dose. A treatment was considered to be mutagenic if it doubled the r e v e r t a n t s / p l a t e compared to the solvent control and a dose-response was evident. Results
Initial experiments were conducted to verify that the assay system used could detect urinary mutagens from cigarette smoke condensate, and to optimize the assay system for their detection. In these studies, animals were given high doses of CSC by i.p. or p.o. administration. CSC prepared from the mainstream smoke of 1R4F research 16C
14C
+ $9/-BG
12C
10c o o_
g
8C
~ 6c tl)
} 4c 20
1
lO
Fig 1 The m u t a g e n l o t y of rat urine following a single oral adrmnlstratlon of oga re t t e smoke condensate at a dose of 200 mg per kg. U n n e was collected and either assayed neat (open bars) or processed to either extract A (dotted bars) or extract B (sohd bars) as described in materials and methods. 1, 5 or 10 g l of the unne or u n n e extract was added to each bacterial plate and mutagemclty assessed. These concentrations of urine extract correspond to 0.2%, 1% and 2% of the 24-h urinary output. The bar represents the mean of the values from 2 - 4 animals (3 plates per ammal), and an asterisk indicates that the mean was at least twice background
12 I~
~0
1oo
+891-BG
•"!11'o
MUTGEN 01635
The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine D a v i d J. D o o l i t t l e , C a r o l y n A. R a h n a n d C h i n K . L e e Cellular and Molecular Bwlogy Dwtston, Butldmg 630-2, R J Reynolds Tobacco Company, Wmston-Salerr~ NC 27102 (U S A.) (Received 24 July 1990) (Accepted 27 August 1990)
Keywords. Rat urine, N~cotme exposure; Carbon monoxide, C~garette smoke, C~garette smoke condensate; Carboxyhemoglobm
Summary Cigarette smokers have been reported to void urine which is more mutagenlc than that voided by non-smokers, but the specific urinary mutagen(s) have not been identified. Since mechanistic studies are best performed in animal models, the objective of this study was to determine if a model to study the role of cigarette smoke and its components in urinary mutagenicity could be developed in rats. XAD-2 resin was used to concentrate the urine and the mlcrosuspension modification of the Ames test used to quantify mutagenicity. Nicotine administered by intraperltoneal injection at 0.8 m g / k g (the maximum tolerated dose) or inhalation of carbon monoxide for 14 days at the maximum tolerated dose (1800 ppm, resulting m 68% carboxyhemoglobin) did not increase urinary mutageniclty. Cigarette smoke condensate (CSC) prepared by electrostatic precipitation of mainstream smoke increased urinary mutagenicity at doses of 100 and 200 m g / k g when administered acutely by either i.p. injection or gavage, verifying that the assay system was capable of detecting cigarette smoke-related mutagens in the urine. However, cigarette smoke administered by the appropriate route of exposure, nose-only inhalation, for 1, 7, 14 or 90 days (1 h per day) did not increase urinary mutagenicity. The smoke concentration administered was at or near the maximum tolerated dose as evidenced by carboxyhemoglobin concentrations of approximately 50%, and of 10% or more weight loss in exposed animals. Thus, although cigarette smoke condensate is mutagenic in vitro and mutagenic urine was observed when rats were given high doses of CSC by inappropriate routes of administration, acute or subchronic inhalation exposure to the maximum tolerated dose of whole cigarette smoke did not increase urinary mutagenicity in rats. These results indicate that the rat may be an inappropriate model to study urinary mutageniclty following the inhalation of tobacco smoke.
Mutagenicity testing of human urine has been widely used as a means for evaluating exposure to potentially genotoxic materials (for review see
Correspondence- David Doohttle, Ph D , DABT, Cellular and Molecular Biology Division, R.J Reynolds Tobacco Company, Winston-Salem, NC 27102 (U S A ).
Everson, 1986). Once an agent or its metabohtes has been identified as a probable urinary mutagen in humans, the use of inbred ammal models would greatly facilitate mechanistic studies on the role and interactions of the agent in altering urinary mutagenicity. Although the specific urinary mutagen(s) have not been identified, cigarette smokers have been reported to void urine which is
10 more mutagenic than that voided by non-smokers (IARC, 1986; Doolittle et al., 1989). The development of a responsive animal model would provlde for controlled mechanistic studies on the effect of c~garette smoke and its components on urinary mutagenicity. Two components of interest are nicotine and carbon monoxide. Carbon monoxide is known to inhibit cytochrome P-450 (Ortlz de Montellano and Reich, 1986), a critical enzyme in the metabolic actwat~on of m a n y mutagens, including some dietary mutagens (Kato, 1986). Thus, administration of carbon monoxide could potentially increase or decrease unnary mutagenlc~ty. Although nicotine and its major metabolites are not mutagenic m the Ames test (Riebe et al., 1982), it has been proposed, but not demonstrated, that mcotine could be converted into mutagenic mtrosamlnes in the body (Hoffmann and Hecht, 1985). Thus, the maximum tolerated dose of nicotine was administered to the animals and urinary mutageniclty assessed. Baboons have been used as an animal model to study smoking-related urinary mutagenicity (Marshall et al., 1983), but widespread use of this animal model is not feasible. We have recently characterized a sensitive assay for detecting urinary mutagemc~ty in rats which uses XAD-2 resin to concentrate the urine and a microsuspens~on modificahon of the Ames bacterial mutagenesis assay to detect mutagens (Doolittle et al., 1988). Since the methodology ~s essentially ~dentical to that commonly used in studies on the mutagemcity of human urine, interspec~es comparisons are facilitated. The objective of this study was to evaluate the effect of exposure to nicotine, carbon monoxade, cigarette smoke and c~garette smoke condensate on the mutagenicity of rat urine. Materials and methods
Chemwals and dosmg soluttons High purity XAD-2 resin was obtained from Alltech (Deerfield, IL). Methanol, acetone and methylene chloride ( H P L C grade) were obtained from Burdick and Jackson (Muskegon, MI). Nicotine (Kodak Chemical Co.) was dissolved in phosphate-buffered saline and rejected i.p. at 0.8 m g / k g (1 ml dosing solution/kg). Electrostatically precipitated cigarette smoke condensate
(CSC) was prepared from 1R4F research cigarettes (University of Kentucky Smoking and Health Institute, Lexington, KY) according to standard techniques (Coresta, 1969). The stock solution of CSC was 200 mg Total Particulate Matter ( T P M ) / m l dimethyl sulphoxide (DMSO, Sigma Chemical Co.). This solution was diluted, if required, and administered to rats at a constant volume of 1 m l / k g . PD01483A and PD01102A reference cigarettes were representative tobaccoburning cigarettes and were obtained from the Research and Development Department of R.J. Reynolds Tobacco Company.
Animals Sprague-Dawley rats 56-90 days old (250-400 g), obtamed from Charles River Breeding Laboratories (Raleigh, NC), were given feed and water ad libitum and allowed to acclimatize for at least 1 week prior to use. The sex of the a ~ m a l does not affect the results obtained (unpublished observation). Females were used for the nicotine and 1R4F 1-day exposure studies; males were used m all other experiments. Smoke exposure of ammals Smoke exposure was performed in a Canon-type nose-only inhalauon chamber (Coggins et al., 1989). The cigarettes were puffed at standard Federal Trade Commission (FTC) conditions of 35 ml puff volume for a 2-sec duration at a frequency of once per minute. Smoke from the cigarettes was diluted with HEPA-filtered air to provide the desired concentrations. Animal exposure was 1 h per day. Sham-exposed animals were placed in the smoking machine for 1 h, but were exposed to HEPA-filtered air only. Carbon monoxide was monitored during the exposure with a Miran 80A infrared spectrophotometer, and carboxyhemoglobin was determined in a Model 282 CO-Oximeter (Instrumentation Laboratories, Hartford, CT). Total Particulate Matter (TPM) in the smoke was estimated by a RAM-1 instrument ( G C A Corporation, Lexington, MA) and by gravimetric measurement following collection of the T P M onto Cambridge filter pads. Nose-only exposure to 1800 p p m carbon monoxide was conducted as previously described (Ayres et al., 1989).
11
Urine collectton and extractton Animals were placed in Nalgene metabolic cages immediately following dosing in the i.p. injection and gavage studies or immediately after the termination of the final exposure period in the inhalation studies. Urine samples were collected for 24 h. The urine collection cups were surrounded with dry ice in a styrofoam bucket covered with foil so that the unne samples froze immediately upon contact. Samples were stored at - 7 0 ° C until extraction on XAD-2 resin as previously described (Doolittle et al., 1988). Briefly, urine samples (15-75 ml) were adjusted to p H 7.0, filtered through Whatman No. 1 filter paper, and then through a 0 . 2 / l m Acrodisc filter (Gelman). 1 ml of urine was retained, and the remainder placed onto a column containing 10 g XAD-2 resin at a flow rate of 2 - 3 m l / m i n . The column was washed with 10 ml water to remove residual urine from the column, dried with nitrogen, and eluted with 10 ml acetone. The acetone eluates were dried under nitrogen. Urine extract A was prepared by dissolving the dried eluate in 500 /~1 of water. U r m e extract B was prepared by dissolving the dried eluate in 20 ml water, followed by extraction with methylene chloride. The methylene chloride fraction was evaporated under nitrogen and the residue dissolved in 500 /~1 DMSO. All samples were stored at - 7 0 ° C until mutageniclty assays were conducted. Mutagemcay assay The microsuspenslon modification of the Ames assay was performed according to Kado et al. (1983). All mutagenicity testing was done using tester strain TA98 (obtained from Dr. B. Ames, University of California, Berkeley). An overnight culture of TA98 cells was concentrated 5 × and 0.1 ml of concentrated cells per plate was used in the rmcrosuspension assay. Rat liver $9 (Organon Teknika Corp., Charleston, SC) prepared from male Sprague-Dawley rats treated with Aroclor 1254 was used at a volume of 0.1 ml of $9 nuxture per plate (30 /~g protein). Potentially mutagenic materials may be excreted in the urine as glucuronide a n d / o r sulfate conjugates. The role of these conjugations was evaluated by adding flglucuronldase/aryl sulfatase (BG) to the Ames microsuspenslon assay and comparing to results in
the absence of these enzymes. The fl-glucuronid a s e / a r y l sulfatase mixture (Sigma G2887) was added at 1000 u n i t s / p l a t e . All samples were tested in 10-#1 volumes at a minimum of 4 concentrations with triphcate plates at each dose. A treatment was considered to be mutagenic if it doubled the r e v e r t a n t s / p l a t e compared to the solvent control and a dose-response was evident. Results
Initial experiments were conducted to verify that the assay system used could detect urinary mutagens from cigarette smoke condensate, and to optimize the assay system for their detection. In these studies, animals were given high doses of CSC by i.p. or p.o. administration. CSC prepared from the mainstream smoke of 1R4F research 16C
14C
+ $9/-BG
12C
10c o o_
g
8C
~ 6c tl)
} 4c 20
1
lO
Fig 1 The m u t a g e n l o t y of rat urine following a single oral adrmnlstratlon of oga re t t e smoke condensate at a dose of 200 mg per kg. U n n e was collected and either assayed neat (open bars) or processed to either extract A (dotted bars) or extract B (sohd bars) as described in materials and methods. 1, 5 or 10 g l of the unne or u n n e extract was added to each bacterial plate and mutagemclty assessed. These concentrations of urine extract correspond to 0.2%, 1% and 2% of the 24-h urinary output. The bar represents the mean of the values from 2 - 4 animals (3 plates per ammal), and an asterisk indicates that the mean was at least twice background
12 I~
~0
1oo
+891-BG
•"!11'o
