The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine

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

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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

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F~g. 2 The m u t a g e m c l t y of rat u n n e following a single m t r a p e n t o n e a l 0-P-) injection or a single oral (p o.) d o s e of ctgarette s m o k e c o n d e n s a t e at 200 m g per kg. U r i n e was collected a n d either used neat (open bars) or processed to extract A ( s o h d bars) as d e s c r i b e d m m a t e r i a l s and methods. 1, 5 or 10/.tl of the u n n e or u n n e extract was a d d e d to each bacterial p l a t e a n d m u t a g e n l c l t y assessed. These c o n c e n t r a t i o n s of urine extract c o r r e s p o n d to 0 2%, 1% a n d 2% of the 24-h u n n a r y o u t p u t The b a r represents the m e a n of the values from at least 2 a m m a l s (3 p l a t e s per a m m a l ) a n d an a s t e n s k indicates that the m e a n was at least twice b a c k g r o u n d .

cigarettes and dosed by gavage at 200 m g / k g (1 m l / k g DMSO) increased urinary mutagenicity (Fig. 1). Concentration of urine by XAD-2 resin (extract A) was necessary to detect the response. Extraction of the XAD-2 column eluate with methylene chloride (extract B) resulted in diminished mutagenlclty (Fig. 1). The mutagemclty of rat urine following i.p. injection or gavage of CSC was greatest when $9 was present and BG was absent and least when both $9 and BG were absent (Fig. 2). Thus, $9 is the most critical factor in the unnary mutagenic]ty of CSC, and BG appears to play only a minimal role at best. This observation suggests that most of the urinary mutagens produced by the administration of CSC are indirect-acting, unconjugated substances. Since

the addition of BG did not enhance urinary mutagenicity this test condition was omitted from later studies. The dose-response observed under the most sensitive assay conditions (extract A, + $9, -BG) following a single i.p. or p.o. administration of CSC is illustrated in Fig. 3. A minimum dose of 100 mg C S C / k g was required for a rat to void mutagenic urine. Results following 1.p. and p.o. administration were similar. Nicotine administered by i.p. injection at 0.8 m g / k g , the m a x i m u m tolerated dose, did not increase urinary mutagenicity (Table 1). Also, 14 days of exposure to 1800 p p m of carbon monoxide, the m a x i m u m tolerated dose as measured by carboxyhemoglobin (Ayres et al., 1989), did not alter urinary mutagenicity (Table 4).

13 TABLE 1 MUTAGENICITY OF RAT URINE FOLLOWING ACUTE EXPOSURE TO NICOTINE OR CIGARETTE SMOKE Treatment c

+ $9 a

- $9 b

0 0 #1 d

1.0 #1

5 0 p.1

10.0 gl

0 0 #1

1.0 p.1

5 0 pl

10.0 #1

30 3 e 50 7 42 3

32 5 48 2 42.3

37.2 53 9 39.3

34 7 55 3 41.5

40 8 36.6 37.5

33.8 39 1 45 9

35 0 43.0 48.2

35 4 40.8 47.5

40 5 46 4 45.7

44.1 52.2 52.9

43 7 61 4 58.9

35.3 40.2 43 8

33 7 43 8 40.9

35 0 40.3 45 0

34.8 40.2 51.9

38 8 49 3 40 2

37.4 52 0 43 2

43 7 26.6 47.9

36 7 35.3 46 9

33 5 40 5 43 2

33 9 40.4 45.8

35.6 44.2 45.4

46 0 56 9 46.2

42 8 52 5 49.1

36.4 36,5 39.9

34.7 43 3 39 2

36.4 46.4 42.0

34.7 48 5 49.2

Room air

Neat Extract A Extract B

Nicotine, 0 8 m g / k g l p

Neat Extract A Extract B

40.5 43 4 42.4

Sham mhalatton exposure, 1 h

Neat Extract A Extract B

37.0 44 7 37.4

I R 4 F ctgarette smoke, 1 07 mg TPM/1, 1 h

Neat Extract A Extract B

35 1 46 3 44.9

39.1 43 7 39.2

Positive control was 2-acetylarmnofluorene, 0 1 #g/plate = 179 revertants b Posmve control was 2-nltrofluorene, 0 1 #g/plate = 774 revertants c Rats were dosed, unne was collected for 24 h and either assayed neat or processed to either Extract A or Extract B prior to assay, as described m materials and methods. a Amount of urine or unne extract added to each bacterial plate. e Values represent the mean revertant number from 2-4 ammals (3 plates per ammal). a

W e d~d n o t k n o w a p r i o r i w h e t h e r i n h a l e d mainstream cigarette smoke would be most mutag e n i c m n a i v e rats a f t e r a c u t e o r c h r o n i c e x p o s u r e . Thus, we examined urine mutagenlcity after both exposure modes. Cigarette smoke administered by i n h a l a t i o n e x p o s u r e at a n a v e r a g e o f 1.0 m g W T P M / l i t e r for 1 h / d a y for 1, 7, 14 o r 90 d a y s did not increase urinary mutagenicity regardless of t h e test c o n d i t i o n s ( T a b l e s 1, 2 a n d 3). T h e s e concentrations of inhaled smoke were the maximum tolerated doses based upon carboxyhem o g l o b i n c o n c e n t r a t m n s a n d 10% o r g r e a t e r w e i g h t loss in e x p o s e d a n i m a l s ( C o g g i n s et al., 1989).

Discussion T e s t i n g for u r i n a r y m u t a g e n i c i t y is a s i m p l e , n o n - i n v a s i v e t e c h m q u e t h a t has b e e n u s e d to in-

v e s t i g a t e e x p o s u r e to p o t e n t i a l l y g e n o t o x i c a g e n t s ( f o r r e v i e w see E v e r s o n , 1986). U r i n e is c o l l e c t e d a n d c o n c e n t r a t e d , a n d t h e m u t a g e n i c i t y o f the c o n c e n t r a t e is m e a s u r e d u s i n g in w t r o g e n o t o x i c ity assays. C i g a r e t t e s m o k e r s h a v e b e e n r e p o r t e d to v o i d u r i n e w h i c h is m o r e m u t a g e n i c , as m e a s u r e d in t h e A m e s b a c t e r i a l m u t a g e n i c i t y assay, t h a n u r i n e v o i d e d b y n o n - s m o k e r s ( I A R C , 1986). S i n c e rats a r e o f t e n u s e d as b i o l o g i c a l m o d e l s for h u m a n s it is i m p o r t a n t to p e r f o r m s i m i l a r s t u d i e s in b o t h species w h e n e v e r p o s s i b l e . A s s e s s m e n t o f u r i n a r y m u t a g e n i c i t y in s i m i l a r l y e x p o s e d r o d e n t s and humans may allow for comparison between rodent models and human beings. In additmn, the development of a responsive animal model could a l l o w for m e c h a n i s t i c s t u d i e s o n t h e r o l e o f various c o m p o n e n t s o f c i g a r e t t e s m o k e in a l t e r i n g urinary mutagenicity. The present results on components of cigarette smoke indicate that urinary

15 TABLE 3 M U T A G E N I C I T Y OF RAT U R I N E F O L L O W I N G R E P E A T E D I N H A L A T I O N E X P O S U R E TO C I G A R E T T E S M O K E F O R 90 DAYS Treatment

Sham Neat Extract A

+ $ 9 / - BG

a

Sham Neat Extract A

1 0 pl

5.0 #1

10.0 #1

0.0 #1

1.0 #1

5 0 #1

10 0 #1

32 8 g 33.7

34.8 27.8

33 6 34 1

31 5 35 4

N.T. 25 7

N.T. 25.0

N.T 25.7

N.T. 28 2

30 8 30.4

29 4 33.4

28.9 33.1

N T. 27.3

N T 26 4

N.T. 26 8

N.T 28.5

+ $ 9 / + BG e

- $ 9 / + BG f

0.0 #1 c

1 0 #1

5.0 #1

10 0 ~1

0 0/.tl

1 0 #1

5.0 #1

10.0 #1

30 4 34.8

33.7 33 8

32 2 34.5

33 8 39.9

N T 25 1

N T 24 8

N.T 28 1

N T. 35 9

32 8 32 4

34.7 35.3

34.7 39 3

N.T. 27.7

N T 28 3

N T 32 3

N.T 33.8

Mainstream ctgarette smoke d Neat 34 5 Extract A 33 6 a b c d e t g h

$ 9 / - BG b

0.0 #1 ¢

Mainstream ctgarette smoke d Neat 34.8 Extract A 28.5 Treatment

--

Posltxve control was CSC, 100 # g / p l a t e = 85 revertants Posture control was 2-mtrofluorene, 40 # g / p l a t e = 208 revertants. A m o u n t of u n n e or urine extract added to each bacterial plate. PD01483A reference cigarettes, 0 80 mg W T P M / h t e r , 864 ppm CO, 1 h / d a y , carboxyhemoglobm was 40 8%. Positive control was benzo[a]pyrene, 0.015 # g / p l a t e = 71 revertants Posmve control was 2-mtrofluorene, 0 01 # g / p l a t e = 84 revertants. Values represent the mean revertant number from 3 animals (3 plates per ammal). Urine was collected for 23 h begmmng lmme&ately after the final inhalation exposure and either assayed neat or processed to Extract A prior to bloassay, as descnbed m materials and methods.

mutagenicity is not altered following 14 day exposure to the maximum tolerated dose of carbon monoxide or by acute i.p. administration of the maximum tolerated dose of nicotine. The results of this study show that the particulate matter from mainstream cigarette smoke can increase urinary mutagenicity in rodents when administered at very high doses by inappropriate routes. These data show that the assay system can detect cigarette smoke-related mutagens in the urine when present. The minimal effective dose of CSC required to elicit a significant increase in urinary mutagenicity in rats following i.p. or p.o. administration is between 50 and 100 m g / k g (Fig. 3), which is 10-100 times greater by weight than the amount of benzo[a]pyrene or 2-acetylaminofluorene needed for a similar response (Doolittle

et al., 1988). However, when rats inhaled mainstream cigarette smoke for as long as 90 days no change in urinary mutagenicity was noted, even though the smoke was administered at the maximum tolerated dose, as measured by carboxyhemoglobin and weight loss. We know of only one other study which evaluated the mutagenicity of rat urine following exposure to cigarette smoke. This study (Mohtashamipur et al., 1984) reported that Wistar rats exposed to cigarette smoke voided mutagenic urine. However, these results must be viewed with caution. The authors used a microtiter fluctuation protocol to evaluate mutagenicity, which may yield false positive resuits when urine is tested (Gibson et al., 1983), and there was no relationship between the amount of smoke exposure and urinary mutagenicity.

16

The lack of urinary mutagenlcity in rats following inhalation of cigarette smoke is in contrast to results in humans (IARC, 1986; Doohttle et al., 1989). The reason for this species difference is not known, but does not appear to relate to inhaled amounts of cigarette 'tar'. A 70 kg man smoking 30 cigarettes (ca. 10 mg F T C ' t a r ' per cigarette) per day can inhale up to 300 mg o f ' tar' per day or 4.3 mg ' t a r ' / k g / d a y . A rat exposed to cigarette smoke at 1.0 mg ' t a r ' per liter of air for 1 h per day will mhale 42 mg ' t a r ' / k g / d a y (average breathing rate = 350 m l / m i n , 500 g rat). Also, an acute exposure to 50 mg of ' t a r ' per kg body weight given i.p. or orally did not increase urinary mutagenicity in rats (Fig. 3). Thus, the rats in the present studies, which did not have mutagenic urine, were exposed to 10 x more dally ' t a r ' on a

The present results on cigarette smoke are strikingly similar to previous studies using diesel exhaust. Rats exposed by gavage or intraperltoneal injection to diesel exhaust particles voided mutagenic urine (Belisario et al., 1984) while rats exposed to diesel exhaust particulates for up to 2 years by the inhalation route did not void mutagenic urine (Ong et al., 1985). Thus, although urinary mutagentcity studies in rats may be useful for evaluating some compounds, especially pure ones (Durston and Ames, 1974; Wlllems and deRaat, 1985; Lawlor et al., 1987; Crebelh et al., 1987; Wtllems et al., 1987; Doohttle et al., 1988), these assays appear to be of llrmted value for evaluating urinary mutagenicity resulting from some complex mixtures given by relevant routes of administration.

TABLE 4 U R I N A R Y M U T A G E N I C I T Y F O L L O W I N G REPEATED, N O S E - O N L Y E X P O S U R E TO 1800 p p m C A R B O N M O N O X I D E F O R 14 DAYS Treatment ~

+ $9 a

- $9

0.0/~1 b

1 0/~1

5 0 ~1

10.0/.tl

15 0 ~1

0.0 ttl

1 0 ~1

5 0 ~1

10 0 / t l

15.0/.tl

Sham Day 6 Neat Extract A Extract B

29 9 c 43.7 32 7

30 3 30 2 40 0

32 2 42 3 29 7

35 5 38 7 30 5

29.7 42.0 22 2

25 8 34 8 28 5

26 0 29.7 32.8

23 5 29 8 34 0

29 3 29 9 31 5

26 3 30 4 18 2

Day 13 Neat Extract A Extract B

31 8 34 9 35 9

31.4 38 9 39 2

30.5 37 0 36 7

34 2 36.2 40 2

33 7 37 0 34.3

28.2 31.9 31 5

29.2 34 7 33.0

25.3 32.3 36 2

29 2 30.0 41.9

29 6 39 4 37.9

Neat Extract A Extract B

29 3 34 4 33 2

30.5 36 7 34 2

32.1 34.2 31.5

34.8 42.2 30.3

34 4 33 9 18.2

29.2 34.3 30.3

27 4 30 4 34 7

28 8 32 3 31 7

25.0 35.7 38.4

28 1 36.0 34.7

Day 13 a Neat Extract A Extract B

29 9 39 9 37 0

31.3 33 7 34 8

30 1 38 0 33.0

32 1 34 8 33 9

33.3 33 8 37 5

30 3 33 5 33 0

27 7 29 7 31 0

30.9 32.0 39 5

28.2 37.7 46.5

27.9 38.7 47 4

1800 ppm CO Day 6 d

a b c d e

Posltlve control was 1R4F cigarette smoke condensate, 0 1 m g / p l a t e = 80 revertants Amo unt of urine or urine extract added to each bacterial plate Values represent the mean revertant number from 2 - 4 ammals (3 plates per ammal) Carboxyhemoglobm was 68% 23-h unne samples were collected on the indicated day and either assayed neat or processed to Extract A or Extract B prior to assay, as described in materials and methods

17

References

+S9/-BG

(x3

& 03

<£ ul added

1510

1510

1510

1510

1510

1510

1510

Dose

DMSO

10

50

100

200

DMSO

10

1 rnl/kg

mg/kg,i p

1 mVkg

itl,itlit 1510

1510

1510

50

100

200

rng,/kg,p o

Flg 3 The m u t a g e m o t y of rat u n n e following a single mtraperltoneal 0 P ) rejection (left side of figure) or a single oral (p o.) dose (right side of figure) of cigarette smoke condensate U n n e was collected and either used neat (open bars) or processed to extract A (sohd bars) as described in materials and methods. 1, 5 or 10 pl of the u n n e or urme extract was added to each bacterial plate and m u t a g e n i o t y 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 at least 2 ammals (3 plates per ammal) and an asterisk m&cates that the mean was at least twice background

per kg body weight basis compared to humans in a previous study where mutagenic urine was observed (Doolittle et al., 1988). The observed species difference may be related to differences in diet, to differences in deposition of the smoke, or to differences in absorption, distribution, metabohsm or excretion of the mutagens in cigarette smoke. Additional research on the complex mechanisms governing the expression of urinary mutagenicity following cigarette smoke inhalation is needed to understand these species differences.

Acknowledgments The authors thank Leroy Gerald for help with animal care, Drs. Chris Coggins, Arnold Mosberg and Paul Ayres for conducting the inhalation exposures, Ms. Teresa Lyalls for help in preparing this manuscript, and Drs. Gary Burger and A. Wallace Hayes for kindly reviewing the manuscript prior to publication.

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