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The genotoxic potential of nicotine and its major metabolites
Genetic Toxicology
ELSEVIER
Mutation Research 344 (1995) 95-102
The genotoxic potential of nicotine and its major metabolites David J. Doolittle a.*, Richard Winegar b, Chin K. Lee a, William S. Caldwell a, A. Wallace Hayes a,1, j. Donald deBethizy a a R.J. Rey'nolds Tobacco Company, Environmental and Molecular Toxicology, Research and Development, P.O. Box 1236, Winston-Salem, NC 27102, USA b SRI, Menlo Park, CA, USA
Received 7 March 1995; revised 30 May 1995; accepted 31 May 1995
Abstract Nicotine is a naturally occurring alkaloid found primarily in members of the solanaceous plant family, which includes tobacco. Nicotine is rapidly absorbed by humans and then metabolized, primarily by cytochrome P450's. Studies on the genotoxic potential of these metabolites are limited. Nicotine and four of its major metabolites: cotinine, nicotine-N'-oxide, cotinine-N-oxide, and t r a n s - Y - h y d r o x y c o t i n i n e were evaluated for genotoxic potential in the Salmonella mutagenicity assay (strains TA98, TA100, TA1535, TA1537, and TA1538) at concentrations ranging from 0 to 1000 /xg/plate and in the Chinese hamster ovary sister-chromatid exchange (SCE) assay at concentrations ranging from 0 to 1000 /xg/ml. All assays were conducted with and without $9 metabolic activation. None of the five compounds increased the frequency of mutations or the frequency of SCEs. These results indicate that nicotine and its major metabolites are not genotoxic in the assays conducted.
I. Introduction Nicotine is a naturally occurring alkaloid found primarily in members of the solanaceous plant family, but widely distributed in the plant kingdom through 12 families and 24 genera (Leete, 1983). Although nicotine exposure may come from eating common solanaceous vegetables such as potato, egg plant, green pepper and tomato (Davis et al., 1991; Domino et al., 1993), the principal source of human exposure is through the use of tobacco and nicotine replacement therapies such as the transdermal nico-
* Corresponding author. Present address: The Gillette Company, Boston, MA, USA.
tine patch and nicotine-containing gum (Surgeon General, 1988). Nicotine is rapidly absorbed via the respiratory tract following cigarette smoking, via the buccal cavity following oral tobacco and gum use, and via the skin following exposure to tobacco leaves during harvesting and when using the transdermal patch. Systemic exposure to nicotine absorbed via the gastrointestinal tract is low because of substantial hepatic metabolism and excretion. Daily intake of nicotine varies from individual to individual and among the different nicotine-containing products. The venous plasma concentration of nicotine in cigarette smokers ranges from about 10 to 70 n g / m l (Curvall and Kazemi-Vala, 1993). Recent advances in mea-
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D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
suring total nicotine uptake have permitted more accurate estimates of daily exposure to nicotine. For example, Byrd et al. (1992) reported that smokers of full-flavor low-'tar' cigarettes absorb about 20 mg of nicotine per day and Curvall and Kazemi-Vala (1993)
reported that daily nicotine absorption in oral snuff users varied from 10 to 40 rag/day. Studies on the genotoxic potential of nicotine generally show negative results although a few isolated positive results have been reported. Nicotine is
Table 1 Induction of his + revertants in Sahnonella typhimurium without metabolic activation Metabolite
Nicotine 0.0/zg/plate 62.5/xg/plate 125.0/xg/plate 250.0/zg/plate 500.0/zg/plate 750.0/zg/plate 1000.0 /xg/plate Cot±nine 0.0/xg/plate 62.5 /xg/plate 125.0/zg/plate 250.0 /xg/plate 500.0 /xg/plate 750.0 /zg/plate 1000.0 #g/plate Cot±nine-N-oxide 0.0 p,g/plate 62.5 /.tg/plate 125.0 p~g/plate 250.0 /zg/plate 500.0/zg/plate 750.0/zg/plate 1000.0/xg/plate Nicotine-N'-oxide 0.0/zg/plate 62.5 ~g/plate 125.0/xg/plate 250.0/xg/plate 500.0/zg/plate 750.0 /.~g/plate 1000.0/xg/plate Trans-3'-hydroxycotinine 0.0/zg/plate 62.5 /.Lg/plate 125.0 /xg/plate 250.0 /xg/plate 500.0 /xg/plate 750.0 /~g/plate 1000.0/.tg/plate Positice controls 2-Nitrofluorene 4.0 /xg/plate
Revertants per plate (mean _+ S.D.) TA98
TA 100
19.7 ± 3.5 25.3 + 2.5 20.7 ± 4.9 23.7 ± 5.8 24.7 ± 2.5 25.7 _+ 1.2 23.7 ± 7.5
80.7 99.0 73.3 86.0 71.0 90.0 72.3
± ± ± ± ± + ±
24.3 ± 22.7 ± 18.3 ± 22.0 + 24.0 ± 25.3 ± 22.3 ±
5.9 6.5 5.9 5.2 5.3 2.5 2.1
73.3 91.0 87.3 79.3 85.0 81.7 82.3
± ± ± ± ± ± ±
26.3 22.0 22.0 23.7 21.7 24.3 25.0
± ± ± ± ± ± ±
4.2 5.6 3.0 5.0 7.2 3.8 6.0
80.7 79.7 78.7 84.7 70.7 73.0 74.0
29.0 25.3 22.3 26.7 27.7 26.3 23.0
_+ 6.0 ± 3. I ± 2.9 ± 3.8 ± 7.8 ± 3.2 ± 3.6
28.3 24.3 29.7 25.0 27.0 25.3 23.3
_+ 10.8 ± 5.9 + 6.0 ± 8.2 ± 7.5 ± 4.6 _+ 4.5
3215.0 ± 205.5 2874.7 ± 163.1 1752.3 ± 99.5
TA 1535
TA 1537
TA 1538
10.1 4.0 5.7 8.2 6.2 5.2 4.2
19.3 ± 3.2 16.0 ± 1.0 13.3 ± 3.8 18.3 ± 5.1 17.0 ± 6.1 16.0 ___2.0 14.7 ± 2.5
12.3 ± 10.7 ± 11.7 ± 12.3 ± 13.7 ± 10.0 + 11.7 ±
3.5 2.3 1.2 3.1 2.9 3.0 2.3
18.0 ± 3.6 16.0 ± 2.6 17.3 _+4.2 19.0 ___ h0 15.0 ± 2.6 16.7 _+ 5.0 14.3 ± 3.1
6.5 7.2 15.3 7.8 4.0 10.0 10.6
17.3 ± 2.1 17.0 ± 2.0 17.3 _+ 1.5 14.0 + 3.6 17.0 ± 4.4 17.0 ± 1.0 15.7 ± 3.1
14.3 ± 3.5 11.7 ± 0.6 11.3 ± 2.9 13.3 + 1.5 11.3 _+ 3.2 14.3 ± 2.1 12.7 ± 3.8
19.3 ___4.0 15.7 ± 3.5 16.3 ± 3.8 16.0 + 3.0 15.7 ± 2.1 21.0 ± 5.3 18.3 ± 1.5
± 3.1 ± 11.6 _ 14.6 ± 12.0 _+ 12.2 ± 9.6 ± 9.2
14.3 ± 1.2 18.7 ± 6.0 15.3 ± 1.5 13.7 ± 2.9 15.7 _+ 2.9 15.0 ± 4.6 17.7 ± 1.5
14.0 ± 11.7 ± 10.7 ± 12.0± 11.3 ± 12.3 ± 11.0 ±
4.4 4.5 3.5 1.7 2.1 3.8 4.4
23.5 ± 2.1 22.0 ± 7.0 19.7 ± 6.0 19.0_+ 6.2 19.3 ± 1.2 15.7 ± 3.1 17.7 ± 1.5
131.3 ± 9.1 133.3 ± 10.3 138.7 _+ 9.2 136.3 ± 8.1 149.0 ± 18.7 145.3 ± I 1.7 148.0 _+ 3.6
19.0 ± 6.1 19.3 + 4.2 19.7 _+ 3.8 16.3 ± 3.5 16.3 ± 3.1 16.3 ± 1.2 16.7 ± 4.0
16.3 ± 15.0 ± 18.3 ± 14.3 ± 13.0 ± 16.0 ± 17.0 ±
1.2 3.0 5.1 2.3 1.0 3.6 3.5
25.0 27.0 22.7 25.0 21.3 23.7 23.7
± 7.2 _+ 1.7 ± 5.0 ± 4.4 ± 4.0 ± 2.1 ± 2.1
151.7 ± 135.0 ± 129.0 ± 134.7 ± 137.7 ± 132.3 ± 135.0 ±
19.0 ± 18.3 ± 18.0 ± 15.0 ± 19.7 ± 17.3 ± 16.3 ±
13.7 _+ 3.1 14.7 ± 1.5 13.3 ± 2.5 I 1.7 ± 1.2 9.0 ± 1.0 14.7 ± 0.6 13.7 ± 4.5
27.3 28.7 29.7 28.0 28.3 27.3 32.3
± 2.3 ± 5.1 ± 4.0 _± 2.0 ± 4.5 ± 4.9 ± 5.9
6.5 10.4 8.9 10.8 3.1 7.4 3.6
6.0 4.2 2.0 2.0 5.5 2.5 1.5
2097.7 ± 35.2 2835.7 ± 83.2 1483.0 ± 195.4
D,J. Doolittle et al. / Mutation Research 344 (1995) 95-102
97
Table 1 (continued) Metabolite
Revertants per plate (mean + S.D.) TA98
Sodium azide 1,0/xg/plate
TA 100
TA 1535
371.3 +_ 43.7 656,0 +_ 28.8 492.7 _+ 35.9
380.3 _+ 32.9 469.0 _+ 55.0 251.0 _+ 12.0
9-Aminoacridine 100.0/xg/plate
not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA1538 (Florin et al., 1980; Riebe et al., 1982; De Flora et al., 1984) but has been reported to be either positive (Riebe et al., 1982) or negative (De Flora et al., 1984) in Escherichia coli assays for inducible DNA repair. Riebe et al. (1982) speculated that the positive results they observed in the E. coli test for inducible DNA repair may be due to "an effect in the pol A - / p o l A system which does not lead to mutagenic alterations" since the authors did not observe mutations in the Salmonella strains. High concentrations of nicotine have been reported to increase SCEs (Riebe and Westphal, 1983; Trivedi et al., 1990) and chromosome aberrations (Trivedi et al., 1990) in CHO cells in the absence of metabolic activation. The slight increase in SCEs observed in the presence of high concentrations of nicotine was eliminated when a metabolic activation system was added (Riebe and Westphal, 1983). Nicotine has been reported to slightly increase the mean frequency of chromosome aberrations in bone marrow cells following administration of a high dose (Barnes and Eltherington, 1973) of 5 m g / k g , p.o. to Chinese hamsters (Munzner and Renner, 1989), but the administration of the maximum tolerated dose (0.8 m g / k g , i.p.) of nicotine to rats did not result in mutagenic urine (Doolittle et al., 1991). Nicotine is metabolized by mammalian enzymes to a number of major and minor metabolites, including cotinine, nicotine-N'-oxide, cotinine-N-oxide, and trans-3'-hydroxycotinine. Studies on the genotoxic potential of these metabolites are limited, although Riebe et al. (1982) reported that cotinine and nicotine-N'-oxide were not mutagenic in the Ames' Salmonella test and did not induce DNA repair in E. coli. The objective of the present study was to
TA 1537
TA 1538
172.3 _+ 28.7 129.3 _+ 31.0 216.7 _+ 18.5
evaluate nicotine and these four major metabolites for genotoxic potential, as measured in the Ames' bacterial mutagenesis assay and the mammalian sister-chromatid exchange assay. The concentrations of each metabolite tested were orders of magnitude higher than the concentrations observed in the plasma of smokers.
2. Materials and methods
2.1. Test compounds Nicotine was obtained from Eastman Chemical (Kingsport, TN) and distilled in vacuo over solid sodium hydroxide (bp = 89.0°-90.5°C at 2 - 3 Torr) and stored in amber vials under dry nitrogen at - 2 0 ° C until used. The purity of the nicotine used for this study was > 99.5% as determined by capillary GC-MS. (S)-(-)-Cotinine, was obtained from Aldrich Chemical (Milwaukee, WI) and was 98.7% pure as determined by capillary GC-MS. NicotineN'-oxide, cotinine-N-oxide, and trans-3'-hydroxycotinine were provided by Dr. Peter Crooks (University of Kentucky). Nicotine-N'-oxide was prepared by the method of Phillipson and Handa (1975) and was > 99.0% pure as determined by HPLC with UV detection at 230 nm. Cotinine-N-oxide was prepared by the method of Dagne and Castagnoli (1972) and was 97.7% pure as determined by HPLC with UV detection at 230 rim. Trans-3'-hydroxycotinine was prepared by the method of Crooks et al. (1992), and was 98.6% pure as determined by capillary GC-MS.
2.2. Ames' mutagenici~ testing Mutagenicity was assessed in the Salmonella/microsome assay (Maron and Ames, 1983) with the
98
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
p r e i n c u b a t i o n m o d i f i c a t i o n d e s c r i b e d b y Y a h a g i et
Sprague-Dawley
al. ( 1 9 7 5 ) . T h e l i v e r h o m o g e n a t e ( $ 9 ) w a s p r e p a r e d
mg/kg
according
c o n c e n t r a t i o n in t h e $ 9 m i x w a s 5 % ( v / v ) ,
to
Ames
et
al.
(1975)
from
male
injection,
rats that w e r e g i v e n a s i n g l e 5 0 0 i.p.,
of Aroclor
1254.
The
Table 2 Induction of his + revertants in Salmonella typhimurium with 5% metabolic activation Metabolite
Nicotine 0.0/zg/plate 62.5/zg/plate 125.0/zg/plate 250.0/.Lg/plate 500.0 p,g/plate 750.0/xg/plate 1000.0/xg/plate Cotinine 0.0/zg/plate 62.5 ,ttg/plate 125.0/zg/plate 250.0 ,u,g/plate 500.0/zg/plate 750.0/zg/plate 1000.0 p~g/plate Cotinine-N-oxide 0.0 /xg/plate 62.5 txg/plate 125.0 /.~g/plate 250.0/.tg/plate 500.0/zg/plate 750.0 /zg/plate 1000.0/~g/plate Nicotine-N'-oxide 0.0/xg/plate 62.5 /xg/plate 125.0/xg/plate 250.0/.zg/plate 500.0 /xg/plate 750.0 p,g/plate 1000.0/zg/plate Trans-3'-hydroxycotinine 0.0 txg/plate 62.5 /xg/plate 125.0 #g/plate 250.0/zg/plate 500.0/zg/plate 750.0 /xg/plate 1000.0 p,g/plate Positice controls 2-Aminoantbracene 0.5 /zg/plate
2-Aminoanthracene 1.0 /xg/plate
Revertants per plate (mean 5: S.D.) TA98
TA 100
TA 1535
TA 1537
TA 1538
24.0 5:8.9 31.7 5:5.1 24.7 5:0.6 29.0 5:9.2 36.0 5:6.2 36.7 __ 1.5 34.0 5:6.2
73.0 5:10.4 69.7 _+ 7.6 74.7 5:12.4 77.3 5:9.0 66.0 _+ 13.0 73.3 5:4.2 75.0 5:2.0
12.3 _+ 1.5 13.3 5:2.1 11.7 5:0.6 16.5 5:0.7 12.7 5:2.1 10.7 5:1.5 12.3 + 0.6
11.7 + 0.6 11.7 5:0.6 12.7 5:3.2 10.0 + 1.7 11.7 5:2.5 8.7 5:2.1 12.3 5:1.5
21.3 21.3 23.0 23.0 22.0 20.0 21.0
25.7 24.7 27.0 25.0 32.3 22.0 28.3
5:1.5 5:0.6 5:3.0 5:7.0 5:1.2 5:1.7 5:4.2
71.0 5:10.0 76.0 + 14.7 71.3 5:10.2 77.0 5:10.4 81.3 5:17.2 74.7 + 12.3 81.0 5:5.3
12.7 5:4.0 12.0 5:2.6 11.0 5:1.7 15.3 __. 3.1 12.3 5:1.5 8.3 _ 2.5 I 1.0 5:2.0
14.7 5:2.1 13.7 5:2.5 I 1.3 5:2.1 13.3 5:4.5 10.3 5:3.8 8.3 5:4.2 12.7 5:2.9
22.7 5:6.0 21.0 5:3.6 21.7 5:6.7 25.0 5:10.0 18.7 5:4.5 19.7 5:7.4 18.7 5:6.1
26.7 29.3 33.3 27.3 23.7 22.0 23.3
5:3.2 5:4.5 5:4.7 _+ 3. I _ 4.7 5:6.6 _+ 8.4
74.7 89.7 75.0 66.3 73.7 64.0 73.3
11.3 _+ 2.9 I 1.3 5:2.1 11.0 _+ 3.5 11.7 5:5.1 12.0 5:3.0 14.7 5:0.6 13.0 5:2.0
12.0 5:4.4 12.7 5:1.5 11.7 5:3.2 10.0 5:2.6 11.3 5:2.5 8.7 5:3.8 12.7 __+1.5
20.0 20.0 24.3 20.0 24.7 27.0 26.7
5:4.0 5:3.0 5:4.7 5:4.4 5:2.3 5:1.0 5:2.1
25.0 28.7 33.0 35.0 33.7 36.3 32.7
5:1.0 _+ 2.5 5:4.4 5:3.5 + 9.3 5:6.0 5:3.8
132.7 5:10.1 114.0 5:10.4 114.3 5:19.9 129.0 5:1.7 108.0 5:9.5 137.0 _+ 10.4 131.3 _+_8.4
14.0 _+ 1.0 9.0 5:2.0 11.7 + 0.6 13.0 5:1.0 12.0 5:2.6 13.0 5:1.0 16.7 5:1.5
17.0 5:5.6 16.3 5:3.2 20.0 5:3.6 18.3 _ 0.6 13.3 5:1.5 13.0 5:1.7 13.3 5:1.5
21.7 21.0 24.7 29.0 26.0 23.0 29.0
5:4.7 5:2.6 5:1.5 5:5.6 5:3.6 5:5.2 5:3.5
30.7 27.7 24.0 32.0 33.0 28.3 31.3
5:9.0 + 7.2 5:1.0 ___6.2 5:3.6 5:3.8 5:5.1
115.7 + 6.7 119.0 5:13.9 132.0 _+ 4.0 115.7 5:16.8 105.0 5:1.7 131.7 5:4.9 131.3 5:5.0
16.7 5:1.5 14.0 _+ 4.4 15.0 5:3.5 12.3 5:0.6 11.7 5:3.2 13.3 _+ 2.5 14.0 5:2.0
14.0 ___ 1.7 15.0 5:1.7 13.3 _+ 0.6 14.3 5:0.6 15.0 _+ 2.0 14.7 5:0.6 13.3 + 3.2
22.7 25.0 23.7 28.0 23.7 22.3 22.0
5:4.0 +_ 3.6 5:3.2 5:3.6 5:4.9 5:2.1 5:6.2
381.3 5:11.4 598.3 5:25.7 463.3 5:48.2
_+ 12.5 5:10.1 5:8.0 5:6.7 5:6.1 ___4.0 5:9.3
330.7 5:15.3 772.3 5:24.7 739.0 5:4.4
_+ 5.0 5:4.9 5:1.0 5:8.5 5:2.6 5:2.6 5:5.3
370.0 + 14.4 437.3 5:13.6 401.7 5:27.2 98.0 5:3.6 134.3 + 16.2 114.3 + 17.9
99.0 + 9.5 97.7 _ 4.0 93.7 5: 8. I
$9
a n d 0.5
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
ml of the $9 mix was added per plate. Components of the assay were added to a 15 × 85 mm test tube in the following order: the test sample, the $9 mix or 0.2 M phosphate buffer, and the test bacteria. The mixture was shaken and allowed to incubate for 20 min at 37°C prior to the addition of 2 ml molten top agar containing 0.5 mM histidine-biotin. The contents of the tube were then poured onto minimal glucose agar and incubated at 37°C for 48 h. Concurrent negative and positive controls were performed with all experiments (see Tables 1 and 2). All testing was done by using triplicate plates at each concentration. A sample was considered to be mutagenic if it induced a concentration-dependent in-
99
crease in revertant number with at least one concentration being at least two times the solvent control.
2.3. Sister-chromatid exchange (SCE) assays Chinese hamster ovary (CHO; ATCC-CCL61CHO-K1, proline-requiring) cells were used for this assay. This cell line has an average cycle time of 12 to 14 h with a modal chromosome number of 20. The cells were grown in an atmosphere of 5% CO 2 at 37°C in McCoy's 5a medium with 15% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin solution to maintain exponential growth. This medium was also used during exposure
Table 3 Sister-chromatid exchange induction by nicotine and metabolites without metabolic activation Treatment ( / x g / m l )
Time in BrdUrd (h)
Cell cycle stages (%)
1000
26.0 26.0 26.0 26.0
1.0 4.0 1.0 43.0
99.0 96.0 99.0 57.0
0 0 0 0
10.10 + 0.45 9.72 + 0.44 9.20 + 0.43 11.00-t-0.51
Cotinine 0 500 750 1000
26.0 26.0 26.0 26.0
1.0 5.0 2.0 7.0
99.0 95.0 98.0 93.0
0 0 0 0
10.10 8.50 9.46 10.54
+ + + _
0.45 0.41 0.43 0.46
26.0 26.0 26.0 26.0
1.0 1.0 1.0 5.0
99.0 99.0 99.0 95.0
0 0 0 0
10.10 9.88 9.40 9.82
+ + + +
0.45 0.44 0.43 0.44
26.0 26.0 26.0 26.0
1.0 5.0 0.0 3.0
99.0 95.0 100.0 97.0
0 0 0 0
10.10-t-0.45 10.14 _+ 0.45 9.34 + 0.43 9.86 + 0.44
26.0 26.0 26.0 26.0 26.0 26.0
2.0 0.0 5.0 3.0 5.0 13.0
98.0 100.0 95.0 97.0 95.0 87.0
0 0 0 0 0 0
12.06 9.10 9.88 10.38 9.68 9.14
26.0
0
100.0
0
30.34 + 0.78
MI
M2
SCE/cell ~ M3
Nicotine 0 500 750
Nicotine-N'-oxide 0 500 750 1000
Trans-3'-hydroxycotinine 0 500 750 1000
Cotinine-N-oxide 0 100 250 500 750 1000
+ 0.49 ___0.43 + 0.44 + 0.46 + 0.44 ___0.43
Positit'e control-rnitomycin C 0.01
Values represent the mean ± S.E.M., n = 50 cells. * Significant increase in the level of SCE compared to solvent control ( p < 0.05).
100
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
of the cells to the test article without metabolic activation. McCoy's 5a medium with 2.5% FBS (containing L-glutamine and penicillin-streptomycin in the above concentrations) was used during the exposure of cells to the test articles with metabolic activation. An Aroclor 1254-induced rat liver homogenate preparation ($9, obtained from Molecular Toxicology, Inc.), was used in the metabolic activation system. Liver enzymes were induced by injecting adult male Fischer-344 rats with Aroclor 1254(500 m g / k g ) five days before they were killed. The
metabolic activation consisted of one part $9 to nine parts McCoy's 5a medium containing 2.5% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin solution. Cofactors were added at concentrations of 24 mg of NADP and 45 mg of sodium isocitrate per milliliter of $9. The metabolic activation mixture was freshly prepared and kept on ice before use. The SCE experiments were conducted by using modifications of the protocol of Galloway et al. (1987). In the nonactivation assays, the cells were exposed to the test article continuously until approximately 2.5 h prior to harvest of the cells. In the
Table 4 Sister-chromatid exchange induction by nicotine and metabolites with metabolic activation Treatment ( ~ g / m l )
Nicotine 0 500 750
1000 Cotinine 0 500 750 1000
Time in BrdUrd (h)
Cell cycle stages (%)
26.0 26.0 26.0 26.0
2.0 2.0 2.0 6.0
98.0 98.0 98.0 94.0
0 0 0 0
11.78 11.24 11.88 11.86
_+ 0.49 _+ 0.47 _+ 0.49 + 0.49
26.0 26.0 26.0 26.0
2.0 1.0 0.0 2.0
98.0 99.0 100.0 98.0
0 0 0 0
11.78 10.88 11.40 11.02
_+ 0.49 + 0.47 + 0.48 _+ 0.47
2.0 0.0 2.0 4.0
98.0 100.0 98.0 96.0
0 0 0 0
11.78 11.38 12.88 11.84
+ 0.49 _+ 0.48 + 0.51 ___0.49
2.0 6.0 0.0 8.0
98.0 94.0 100.0 92.0
0 0 0 0
11.78 11.46 12.08 12.40
_+ 0.49 ___0.48 + 0.49 + 0.50
0.0 3.0 0.0 8.0 5.0 5.0
100.0 97.0 100.0 92.0 95.0 95.0
0 0 0 0 0 0
14.00 13.04 14.04 13.98 13.50 13.70
_ 0.53 + 0.51 _+ 0.53 _+ 0.53 _+ 0.52 _+ 0.52
2.0
98.0
Nicotine-N'-oxide 0 26.0 500 26.0 750 26.0 1000 26.0 Trans 3'-hydroxycotinine 0 26.0 500 26.0 750 26.0 1000 26.0 Cotinine-N-oxide 0 26.0 100 26.0 250 26.0 500 26.0 750 26.0 1000 26.0 Positiee control-cyclophosphamide 1.50 26.0
M1
M2
Values represent the mean + S.E.M., n = 50 cells. Significant increase in the level of SCE compared to solvent control ( p < 0.05).
SCE/cell a M3
35.32 _+ 0.84
D.J. Doolittle et al. / Mutation Research 344 (I 995) 95-102
metabolic activation assays, the CHO cells were exposed to the test article for 2 h in metabolic activation mix. Bromodeoxyuridine (BrdUrd) was added 2 h after the beginning of exposure to the test article. Cells used for SCE analysis were scored in their second metaphase after initiation of chemical exposure. SCE levels for individual treatments were compared to the solvent controls using a t-test employing the Bonferoni correction for multiple comparisons. Dose response was analyzed using a trend test (Margolin et al., 1986).
3. Results and discussion Nicotine and four of its major metabolites were evaluated for mutagenicity in Ames strains TA98, TAI00, TA1535, TA1537 and TA1538, both with and without metabolic activation with $9 at concentrations ranging from 0 to 1000/xg per plate. Neither nicotine nor any of the four metabolites induced an increase in revertant frequency in any strain under either condition of metabolic activation (Tables 1 and 2). The same five compounds were also evaluated for their potential to increase the frequency of SCEs in CHO cells. Nicotine, cotinine, nicotine-N'-oxide and trans-3'-hydroxycotinine were examined for the induction of SCEs on a single experimental day while cotinine-N-oxide was evaluated on a second experimental day. No increases in SCE frequency were observed when nicotine and four of its major metabolites were tested at concentrations ranging up to 1000 /xg per ml of culture medium (Tables 3 and 4). Cytotoxicity, as indicated by cell cycle delay, was observed only with the high dose of nicotine (1000 /xg/ml) under the condition of no metabolic activation. Very slight delays in the cell cycle were observed with cotinine and cotinine-N-oxide, again only under the condition of no metabolic activation. Trivedi et al. (1990) reported that nicotine, at concentrations similar to those used here, increased the frequency of chromosomal aberrations in CHO cells, and Munzner and Renner (1989) reported that high doses of nicotine (Barnes and Eltherington, 1973) increased the frequency of chromosome aberrations in the bone marrow cells of Chinese ham-
101
sters. However, the reported increase in the frequency of chromosomal aberrations observed in the presence of nicotine (Trivedi et al., 1990) was arrived at by incorporating chromosome and chromatid gaps into the primary statistical analysis. International guidelines (OECD, 1983) recommend that gaps or other achromatic lesions not be included when calculating chromosome aberration frequency, since they may result from staining artifacts. As Trivedi et al. indicate, if gaps are excluded, nicotine did not increase the frequency of chromosome aberrations following either 2 or 4 h of treatment, and only slightly increased the frequency of chromosome aberrations following 24 h of continuous treatment (Trivedi et al., 1990). Munzner and Renner (1989) did not provide statistical support for their conclusions. However, it is clear from their data that the slight increase referred to by these authors is due to increased numbers of gaps. If gaps are excluded, nicotine clearly had no significant effect on the frequency of chromosome aberrations in these animals, since the standard deviations from the control and all treatment groups overlap markedly. In conclusion, although cigarette smoke condensate is genotoxic as measured in a wide range of in vitro assays (Doolittle et al., 1990), the results of the present study indicate that the observed genotoxicity is due to constituents other than nicotine. This is consistent with published data demonstrating that the genotoxic potential of tobacco smoke is not related to the nicotine content of the cigarette (Mizusaki et al., 1977; Gairola, 1982), and data indicating that the concentrations of nicotine and cotinine in the urine of human smokers smoking cigarettes which yield nicotine in the absence of tobacco pyrolysis products are unrelated to the mutagenicity of the urine (Rahn et al., 1991). The concentrations evaluated in the present study (up to 1000 /xg/ml) represent massive exaggerations of the average concentrations found in the plasma or urine of human smokers. Taken together, the data indicate that nicotine and its major metabolites do not represent a genotoxic hazard.
Acknowledgements The authors thank Cindy Fulp for excellent technical assistance.
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References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s with the Salmonella~mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Barnes, C.D. and L.G. Eltherington (1973) Drug Dosages in Laboratory A n i m a l s - - A Handbook, Univ. of California Press, Berkeley, p. 171. Byrd, G.D., K.-M. Chang, J.M. Greene and J.D. deBethizy (1992) Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers, Drug Metab. Dispos., 20, 192-197. Crooks, P.A., B.S. Bhatti, A. Ravard, R.M. Riggs and W.S. Caldwell (1992) Synthesis of N-glucuronic acid conjugates of cotinine, cis-3-hydroxycotinine, and trans-3-hydroxycotinine, Med. Sci. Res., 20, 881-883. Curvall, M. and E. Kazemi-Vala (1993) Nicotine and metabolites: analysis and levels in body fluids, in: J.W. Gorrod and J. Wahren (Eds.), Nicotine and Related Alkaloids: Absorption, Distribution, Metabolism and Excretion, Chapman and Hall, London. Dagne, E. and N. Castagnoli (1972) Cotinine-N-oxide, a new metabolite of nicotine, J. Med. Chem., 15, 840-841. Davis, R.A., M.F. Stiles, J.D. deBethizy and J.H. Reynolds (1991) Dietary nicotine: a source of urinary cotinine, Food Chem. Toxicol., 29, 821-827. De Flora, S., P. Zanacchi, A. Camoirano, C. Bennicelli and G.S. Badolati (1984) Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test, Mutation Res., 133, 161-198. Domino, E.F., E. Hornbach and T. Demana (1993) The nicotine content of common vegetables, New Eng. J. Med., 329, 437. Doolittle, D.J., C.K. Lee, J.L. Ivett, J.C. Mirsalis, E. Riccio, C.J. Rudd, G.T. Burger and A.W. Hayes (1990) Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco, Environ. Mol. Mutagen., 15, 93-105. Doolittle, D.J., C.A. Rahn and C.K. Lee (1991) The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine, Mutation Res., 260, 9-18. Florin, I., L. Rutberg, M. Curvall and C.R. Enzell (1980) Screening of tobacco smoke constituents for mutagenicity using the Ames' test, Toxicology, 15, 219-232. Gairola, C. (1982) Genetic effects of fresh cigarette smoke in Saccharomyces cerevisiae, Mutation Res., 102, 123-136.
Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson and E. Zeiger (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals, Environ. Mol. Mutagen., 10 (Suppl. 10), 1-175. Leete, E. (1983) Biosynthesis and metabolism of the tobacco alkaloids, in: S.W. Pelletier (Eds.), Alkaloids Chemical and Biological Perspectives, Vol. 1, John Wiley and Sons, New York, NY, pp. 86-139. Margolin, B.H., M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom and E. Zeiger (1986) Statistical analyses for in vitro cytogenetic assays using Chinese hamster ovary cells, Environ. Mutagen., 8, 183-204. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mizusaki, S., H. Okamoto, A. Akiyama and Y. Fukuhara (1977) Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate, Mutation Res., 48, 319-325. Munzner, R. and H.W. Renner (1989) Genotoxic investigations of tobacco protein using microbial and mammalian test systems, Z. Ern~ihrungswiss., 28, 300-309. OECD Guideline for Testing of Chemicals (1983) Genetic toxicology: In vitro mammalian cytogenetic test, 473, 1-6. Phillipson, J.D. and S.S. Handa (1975) Nicotine N-oxides, Photochem., 14, 2683-2690. Rahn, C.A., G. Howard, E. Riccio and D.J. Doolittle (1991) Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets, Environ. Mol. Mutagen., 17, 244-252. Riebe, M., K. Westphal and P. Fortnagel (1982) Mutagenicity testing, in bacterial test systems, of some constituents of tobacco, Mutation Res., 101, 39-43. Riebe, M. and K. Westphal (1983) Studies on the induction of sister-chromatid exchanges in Chinese hamster ovary cells by various tobacco alkaloids, Mutation Res., 124, 281-286. Surgeon General (1988) The health consequences of smoking: nicotine addiction, U.S. Dept. of Health and Human Services. Trivedi, A.H., B.J. Dave and S.G. Adhvaryu (1990) Assessment of genotoxicity of nicotine employing in vitro mammalian test system, Cancer Lett., 54, 89-94. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.
ELSEVIER
Mutation Research 344 (1995) 95-102
The genotoxic potential of nicotine and its major metabolites David J. Doolittle a.*, Richard Winegar b, Chin K. Lee a, William S. Caldwell a, A. Wallace Hayes a,1, j. Donald deBethizy a a R.J. Rey'nolds Tobacco Company, Environmental and Molecular Toxicology, Research and Development, P.O. Box 1236, Winston-Salem, NC 27102, USA b SRI, Menlo Park, CA, USA
Received 7 March 1995; revised 30 May 1995; accepted 31 May 1995
Abstract Nicotine is a naturally occurring alkaloid found primarily in members of the solanaceous plant family, which includes tobacco. Nicotine is rapidly absorbed by humans and then metabolized, primarily by cytochrome P450's. Studies on the genotoxic potential of these metabolites are limited. Nicotine and four of its major metabolites: cotinine, nicotine-N'-oxide, cotinine-N-oxide, and t r a n s - Y - h y d r o x y c o t i n i n e were evaluated for genotoxic potential in the Salmonella mutagenicity assay (strains TA98, TA100, TA1535, TA1537, and TA1538) at concentrations ranging from 0 to 1000 /xg/plate and in the Chinese hamster ovary sister-chromatid exchange (SCE) assay at concentrations ranging from 0 to 1000 /xg/ml. All assays were conducted with and without $9 metabolic activation. None of the five compounds increased the frequency of mutations or the frequency of SCEs. These results indicate that nicotine and its major metabolites are not genotoxic in the assays conducted.
I. Introduction Nicotine is a naturally occurring alkaloid found primarily in members of the solanaceous plant family, but widely distributed in the plant kingdom through 12 families and 24 genera (Leete, 1983). Although nicotine exposure may come from eating common solanaceous vegetables such as potato, egg plant, green pepper and tomato (Davis et al., 1991; Domino et al., 1993), the principal source of human exposure is through the use of tobacco and nicotine replacement therapies such as the transdermal nico-
* Corresponding author. Present address: The Gillette Company, Boston, MA, USA.
tine patch and nicotine-containing gum (Surgeon General, 1988). Nicotine is rapidly absorbed via the respiratory tract following cigarette smoking, via the buccal cavity following oral tobacco and gum use, and via the skin following exposure to tobacco leaves during harvesting and when using the transdermal patch. Systemic exposure to nicotine absorbed via the gastrointestinal tract is low because of substantial hepatic metabolism and excretion. Daily intake of nicotine varies from individual to individual and among the different nicotine-containing products. The venous plasma concentration of nicotine in cigarette smokers ranges from about 10 to 70 n g / m l (Curvall and Kazemi-Vala, 1993). Recent advances in mea-
0165-1218/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0165- 1218(95)00037-2
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D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
suring total nicotine uptake have permitted more accurate estimates of daily exposure to nicotine. For example, Byrd et al. (1992) reported that smokers of full-flavor low-'tar' cigarettes absorb about 20 mg of nicotine per day and Curvall and Kazemi-Vala (1993)
reported that daily nicotine absorption in oral snuff users varied from 10 to 40 rag/day. Studies on the genotoxic potential of nicotine generally show negative results although a few isolated positive results have been reported. Nicotine is
Table 1 Induction of his + revertants in Sahnonella typhimurium without metabolic activation Metabolite
Nicotine 0.0/zg/plate 62.5/xg/plate 125.0/xg/plate 250.0/zg/plate 500.0/zg/plate 750.0/zg/plate 1000.0 /xg/plate Cot±nine 0.0/xg/plate 62.5 /xg/plate 125.0/zg/plate 250.0 /xg/plate 500.0 /xg/plate 750.0 /zg/plate 1000.0 #g/plate Cot±nine-N-oxide 0.0 p,g/plate 62.5 /.tg/plate 125.0 p~g/plate 250.0 /zg/plate 500.0/zg/plate 750.0/zg/plate 1000.0/xg/plate Nicotine-N'-oxide 0.0/zg/plate 62.5 ~g/plate 125.0/xg/plate 250.0/xg/plate 500.0/zg/plate 750.0 /.~g/plate 1000.0/xg/plate Trans-3'-hydroxycotinine 0.0/zg/plate 62.5 /.Lg/plate 125.0 /xg/plate 250.0 /xg/plate 500.0 /xg/plate 750.0 /~g/plate 1000.0/.tg/plate Positice controls 2-Nitrofluorene 4.0 /xg/plate
Revertants per plate (mean _+ S.D.) TA98
TA 100
19.7 ± 3.5 25.3 + 2.5 20.7 ± 4.9 23.7 ± 5.8 24.7 ± 2.5 25.7 _+ 1.2 23.7 ± 7.5
80.7 99.0 73.3 86.0 71.0 90.0 72.3
± ± ± ± ± + ±
24.3 ± 22.7 ± 18.3 ± 22.0 + 24.0 ± 25.3 ± 22.3 ±
5.9 6.5 5.9 5.2 5.3 2.5 2.1
73.3 91.0 87.3 79.3 85.0 81.7 82.3
± ± ± ± ± ± ±
26.3 22.0 22.0 23.7 21.7 24.3 25.0
± ± ± ± ± ± ±
4.2 5.6 3.0 5.0 7.2 3.8 6.0
80.7 79.7 78.7 84.7 70.7 73.0 74.0
29.0 25.3 22.3 26.7 27.7 26.3 23.0
_+ 6.0 ± 3. I ± 2.9 ± 3.8 ± 7.8 ± 3.2 ± 3.6
28.3 24.3 29.7 25.0 27.0 25.3 23.3
_+ 10.8 ± 5.9 + 6.0 ± 8.2 ± 7.5 ± 4.6 _+ 4.5
3215.0 ± 205.5 2874.7 ± 163.1 1752.3 ± 99.5
TA 1535
TA 1537
TA 1538
10.1 4.0 5.7 8.2 6.2 5.2 4.2
19.3 ± 3.2 16.0 ± 1.0 13.3 ± 3.8 18.3 ± 5.1 17.0 ± 6.1 16.0 ___2.0 14.7 ± 2.5
12.3 ± 10.7 ± 11.7 ± 12.3 ± 13.7 ± 10.0 + 11.7 ±
3.5 2.3 1.2 3.1 2.9 3.0 2.3
18.0 ± 3.6 16.0 ± 2.6 17.3 _+4.2 19.0 ___ h0 15.0 ± 2.6 16.7 _+ 5.0 14.3 ± 3.1
6.5 7.2 15.3 7.8 4.0 10.0 10.6
17.3 ± 2.1 17.0 ± 2.0 17.3 _+ 1.5 14.0 + 3.6 17.0 ± 4.4 17.0 ± 1.0 15.7 ± 3.1
14.3 ± 3.5 11.7 ± 0.6 11.3 ± 2.9 13.3 + 1.5 11.3 _+ 3.2 14.3 ± 2.1 12.7 ± 3.8
19.3 ___4.0 15.7 ± 3.5 16.3 ± 3.8 16.0 + 3.0 15.7 ± 2.1 21.0 ± 5.3 18.3 ± 1.5
± 3.1 ± 11.6 _ 14.6 ± 12.0 _+ 12.2 ± 9.6 ± 9.2
14.3 ± 1.2 18.7 ± 6.0 15.3 ± 1.5 13.7 ± 2.9 15.7 _+ 2.9 15.0 ± 4.6 17.7 ± 1.5
14.0 ± 11.7 ± 10.7 ± 12.0± 11.3 ± 12.3 ± 11.0 ±
4.4 4.5 3.5 1.7 2.1 3.8 4.4
23.5 ± 2.1 22.0 ± 7.0 19.7 ± 6.0 19.0_+ 6.2 19.3 ± 1.2 15.7 ± 3.1 17.7 ± 1.5
131.3 ± 9.1 133.3 ± 10.3 138.7 _+ 9.2 136.3 ± 8.1 149.0 ± 18.7 145.3 ± I 1.7 148.0 _+ 3.6
19.0 ± 6.1 19.3 + 4.2 19.7 _+ 3.8 16.3 ± 3.5 16.3 ± 3.1 16.3 ± 1.2 16.7 ± 4.0
16.3 ± 15.0 ± 18.3 ± 14.3 ± 13.0 ± 16.0 ± 17.0 ±
1.2 3.0 5.1 2.3 1.0 3.6 3.5
25.0 27.0 22.7 25.0 21.3 23.7 23.7
± 7.2 _+ 1.7 ± 5.0 ± 4.4 ± 4.0 ± 2.1 ± 2.1
151.7 ± 135.0 ± 129.0 ± 134.7 ± 137.7 ± 132.3 ± 135.0 ±
19.0 ± 18.3 ± 18.0 ± 15.0 ± 19.7 ± 17.3 ± 16.3 ±
13.7 _+ 3.1 14.7 ± 1.5 13.3 ± 2.5 I 1.7 ± 1.2 9.0 ± 1.0 14.7 ± 0.6 13.7 ± 4.5
27.3 28.7 29.7 28.0 28.3 27.3 32.3
± 2.3 ± 5.1 ± 4.0 _± 2.0 ± 4.5 ± 4.9 ± 5.9
6.5 10.4 8.9 10.8 3.1 7.4 3.6
6.0 4.2 2.0 2.0 5.5 2.5 1.5
2097.7 ± 35.2 2835.7 ± 83.2 1483.0 ± 195.4
D,J. Doolittle et al. / Mutation Research 344 (1995) 95-102
97
Table 1 (continued) Metabolite
Revertants per plate (mean + S.D.) TA98
Sodium azide 1,0/xg/plate
TA 100
TA 1535
371.3 +_ 43.7 656,0 +_ 28.8 492.7 _+ 35.9
380.3 _+ 32.9 469.0 _+ 55.0 251.0 _+ 12.0
9-Aminoacridine 100.0/xg/plate
not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA1538 (Florin et al., 1980; Riebe et al., 1982; De Flora et al., 1984) but has been reported to be either positive (Riebe et al., 1982) or negative (De Flora et al., 1984) in Escherichia coli assays for inducible DNA repair. Riebe et al. (1982) speculated that the positive results they observed in the E. coli test for inducible DNA repair may be due to "an effect in the pol A - / p o l A system which does not lead to mutagenic alterations" since the authors did not observe mutations in the Salmonella strains. High concentrations of nicotine have been reported to increase SCEs (Riebe and Westphal, 1983; Trivedi et al., 1990) and chromosome aberrations (Trivedi et al., 1990) in CHO cells in the absence of metabolic activation. The slight increase in SCEs observed in the presence of high concentrations of nicotine was eliminated when a metabolic activation system was added (Riebe and Westphal, 1983). Nicotine has been reported to slightly increase the mean frequency of chromosome aberrations in bone marrow cells following administration of a high dose (Barnes and Eltherington, 1973) of 5 m g / k g , p.o. to Chinese hamsters (Munzner and Renner, 1989), but the administration of the maximum tolerated dose (0.8 m g / k g , i.p.) of nicotine to rats did not result in mutagenic urine (Doolittle et al., 1991). Nicotine is metabolized by mammalian enzymes to a number of major and minor metabolites, including cotinine, nicotine-N'-oxide, cotinine-N-oxide, and trans-3'-hydroxycotinine. Studies on the genotoxic potential of these metabolites are limited, although Riebe et al. (1982) reported that cotinine and nicotine-N'-oxide were not mutagenic in the Ames' Salmonella test and did not induce DNA repair in E. coli. The objective of the present study was to
TA 1537
TA 1538
172.3 _+ 28.7 129.3 _+ 31.0 216.7 _+ 18.5
evaluate nicotine and these four major metabolites for genotoxic potential, as measured in the Ames' bacterial mutagenesis assay and the mammalian sister-chromatid exchange assay. The concentrations of each metabolite tested were orders of magnitude higher than the concentrations observed in the plasma of smokers.
2. Materials and methods
2.1. Test compounds Nicotine was obtained from Eastman Chemical (Kingsport, TN) and distilled in vacuo over solid sodium hydroxide (bp = 89.0°-90.5°C at 2 - 3 Torr) and stored in amber vials under dry nitrogen at - 2 0 ° C until used. The purity of the nicotine used for this study was > 99.5% as determined by capillary GC-MS. (S)-(-)-Cotinine, was obtained from Aldrich Chemical (Milwaukee, WI) and was 98.7% pure as determined by capillary GC-MS. NicotineN'-oxide, cotinine-N-oxide, and trans-3'-hydroxycotinine were provided by Dr. Peter Crooks (University of Kentucky). Nicotine-N'-oxide was prepared by the method of Phillipson and Handa (1975) and was > 99.0% pure as determined by HPLC with UV detection at 230 nm. Cotinine-N-oxide was prepared by the method of Dagne and Castagnoli (1972) and was 97.7% pure as determined by HPLC with UV detection at 230 rim. Trans-3'-hydroxycotinine was prepared by the method of Crooks et al. (1992), and was 98.6% pure as determined by capillary GC-MS.
2.2. Ames' mutagenici~ testing Mutagenicity was assessed in the Salmonella/microsome assay (Maron and Ames, 1983) with the
98
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
p r e i n c u b a t i o n m o d i f i c a t i o n d e s c r i b e d b y Y a h a g i et
Sprague-Dawley
al. ( 1 9 7 5 ) . T h e l i v e r h o m o g e n a t e ( $ 9 ) w a s p r e p a r e d
mg/kg
according
c o n c e n t r a t i o n in t h e $ 9 m i x w a s 5 % ( v / v ) ,
to
Ames
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(1975)
from
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The
Table 2 Induction of his + revertants in Salmonella typhimurium with 5% metabolic activation Metabolite
Nicotine 0.0/zg/plate 62.5/zg/plate 125.0/zg/plate 250.0/.Lg/plate 500.0 p,g/plate 750.0/xg/plate 1000.0/xg/plate Cotinine 0.0/zg/plate 62.5 ,ttg/plate 125.0/zg/plate 250.0 ,u,g/plate 500.0/zg/plate 750.0/zg/plate 1000.0 p~g/plate Cotinine-N-oxide 0.0 /xg/plate 62.5 txg/plate 125.0 /.~g/plate 250.0/.tg/plate 500.0/zg/plate 750.0 /zg/plate 1000.0/~g/plate Nicotine-N'-oxide 0.0/xg/plate 62.5 /xg/plate 125.0/xg/plate 250.0/.zg/plate 500.0 /xg/plate 750.0 p,g/plate 1000.0/zg/plate Trans-3'-hydroxycotinine 0.0 txg/plate 62.5 /xg/plate 125.0 #g/plate 250.0/zg/plate 500.0/zg/plate 750.0 /xg/plate 1000.0 p,g/plate Positice controls 2-Aminoantbracene 0.5 /zg/plate
2-Aminoanthracene 1.0 /xg/plate
Revertants per plate (mean 5: S.D.) TA98
TA 100
TA 1535
TA 1537
TA 1538
24.0 5:8.9 31.7 5:5.1 24.7 5:0.6 29.0 5:9.2 36.0 5:6.2 36.7 __ 1.5 34.0 5:6.2
73.0 5:10.4 69.7 _+ 7.6 74.7 5:12.4 77.3 5:9.0 66.0 _+ 13.0 73.3 5:4.2 75.0 5:2.0
12.3 _+ 1.5 13.3 5:2.1 11.7 5:0.6 16.5 5:0.7 12.7 5:2.1 10.7 5:1.5 12.3 + 0.6
11.7 + 0.6 11.7 5:0.6 12.7 5:3.2 10.0 + 1.7 11.7 5:2.5 8.7 5:2.1 12.3 5:1.5
21.3 21.3 23.0 23.0 22.0 20.0 21.0
25.7 24.7 27.0 25.0 32.3 22.0 28.3
5:1.5 5:0.6 5:3.0 5:7.0 5:1.2 5:1.7 5:4.2
71.0 5:10.0 76.0 + 14.7 71.3 5:10.2 77.0 5:10.4 81.3 5:17.2 74.7 + 12.3 81.0 5:5.3
12.7 5:4.0 12.0 5:2.6 11.0 5:1.7 15.3 __. 3.1 12.3 5:1.5 8.3 _ 2.5 I 1.0 5:2.0
14.7 5:2.1 13.7 5:2.5 I 1.3 5:2.1 13.3 5:4.5 10.3 5:3.8 8.3 5:4.2 12.7 5:2.9
22.7 5:6.0 21.0 5:3.6 21.7 5:6.7 25.0 5:10.0 18.7 5:4.5 19.7 5:7.4 18.7 5:6.1
26.7 29.3 33.3 27.3 23.7 22.0 23.3
5:3.2 5:4.5 5:4.7 _+ 3. I _ 4.7 5:6.6 _+ 8.4
74.7 89.7 75.0 66.3 73.7 64.0 73.3
11.3 _+ 2.9 I 1.3 5:2.1 11.0 _+ 3.5 11.7 5:5.1 12.0 5:3.0 14.7 5:0.6 13.0 5:2.0
12.0 5:4.4 12.7 5:1.5 11.7 5:3.2 10.0 5:2.6 11.3 5:2.5 8.7 5:3.8 12.7 __+1.5
20.0 20.0 24.3 20.0 24.7 27.0 26.7
5:4.0 5:3.0 5:4.7 5:4.4 5:2.3 5:1.0 5:2.1
25.0 28.7 33.0 35.0 33.7 36.3 32.7
5:1.0 _+ 2.5 5:4.4 5:3.5 + 9.3 5:6.0 5:3.8
132.7 5:10.1 114.0 5:10.4 114.3 5:19.9 129.0 5:1.7 108.0 5:9.5 137.0 _+ 10.4 131.3 _+_8.4
14.0 _+ 1.0 9.0 5:2.0 11.7 + 0.6 13.0 5:1.0 12.0 5:2.6 13.0 5:1.0 16.7 5:1.5
17.0 5:5.6 16.3 5:3.2 20.0 5:3.6 18.3 _ 0.6 13.3 5:1.5 13.0 5:1.7 13.3 5:1.5
21.7 21.0 24.7 29.0 26.0 23.0 29.0
5:4.7 5:2.6 5:1.5 5:5.6 5:3.6 5:5.2 5:3.5
30.7 27.7 24.0 32.0 33.0 28.3 31.3
5:9.0 + 7.2 5:1.0 ___6.2 5:3.6 5:3.8 5:5.1
115.7 + 6.7 119.0 5:13.9 132.0 _+ 4.0 115.7 5:16.8 105.0 5:1.7 131.7 5:4.9 131.3 5:5.0
16.7 5:1.5 14.0 _+ 4.4 15.0 5:3.5 12.3 5:0.6 11.7 5:3.2 13.3 _+ 2.5 14.0 5:2.0
14.0 ___ 1.7 15.0 5:1.7 13.3 _+ 0.6 14.3 5:0.6 15.0 _+ 2.0 14.7 5:0.6 13.3 + 3.2
22.7 25.0 23.7 28.0 23.7 22.3 22.0
5:4.0 +_ 3.6 5:3.2 5:3.6 5:4.9 5:2.1 5:6.2
381.3 5:11.4 598.3 5:25.7 463.3 5:48.2
_+ 12.5 5:10.1 5:8.0 5:6.7 5:6.1 ___4.0 5:9.3
330.7 5:15.3 772.3 5:24.7 739.0 5:4.4
_+ 5.0 5:4.9 5:1.0 5:8.5 5:2.6 5:2.6 5:5.3
370.0 + 14.4 437.3 5:13.6 401.7 5:27.2 98.0 5:3.6 134.3 + 16.2 114.3 + 17.9
99.0 + 9.5 97.7 _ 4.0 93.7 5: 8. I
$9
a n d 0.5
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
ml of the $9 mix was added per plate. Components of the assay were added to a 15 × 85 mm test tube in the following order: the test sample, the $9 mix or 0.2 M phosphate buffer, and the test bacteria. The mixture was shaken and allowed to incubate for 20 min at 37°C prior to the addition of 2 ml molten top agar containing 0.5 mM histidine-biotin. The contents of the tube were then poured onto minimal glucose agar and incubated at 37°C for 48 h. Concurrent negative and positive controls were performed with all experiments (see Tables 1 and 2). All testing was done by using triplicate plates at each concentration. A sample was considered to be mutagenic if it induced a concentration-dependent in-
99
crease in revertant number with at least one concentration being at least two times the solvent control.
2.3. Sister-chromatid exchange (SCE) assays Chinese hamster ovary (CHO; ATCC-CCL61CHO-K1, proline-requiring) cells were used for this assay. This cell line has an average cycle time of 12 to 14 h with a modal chromosome number of 20. The cells were grown in an atmosphere of 5% CO 2 at 37°C in McCoy's 5a medium with 15% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin-streptomycin solution to maintain exponential growth. This medium was also used during exposure
Table 3 Sister-chromatid exchange induction by nicotine and metabolites without metabolic activation Treatment ( / x g / m l )
Time in BrdUrd (h)
Cell cycle stages (%)
1000
26.0 26.0 26.0 26.0
1.0 4.0 1.0 43.0
99.0 96.0 99.0 57.0
0 0 0 0
10.10 + 0.45 9.72 + 0.44 9.20 + 0.43 11.00-t-0.51
Cotinine 0 500 750 1000
26.0 26.0 26.0 26.0
1.0 5.0 2.0 7.0
99.0 95.0 98.0 93.0
0 0 0 0
10.10 8.50 9.46 10.54
+ + + _
0.45 0.41 0.43 0.46
26.0 26.0 26.0 26.0
1.0 1.0 1.0 5.0
99.0 99.0 99.0 95.0
0 0 0 0
10.10 9.88 9.40 9.82
+ + + +
0.45 0.44 0.43 0.44
26.0 26.0 26.0 26.0
1.0 5.0 0.0 3.0
99.0 95.0 100.0 97.0
0 0 0 0
10.10-t-0.45 10.14 _+ 0.45 9.34 + 0.43 9.86 + 0.44
26.0 26.0 26.0 26.0 26.0 26.0
2.0 0.0 5.0 3.0 5.0 13.0
98.0 100.0 95.0 97.0 95.0 87.0
0 0 0 0 0 0
12.06 9.10 9.88 10.38 9.68 9.14
26.0
0
100.0
0
30.34 + 0.78
MI
M2
SCE/cell ~ M3
Nicotine 0 500 750
Nicotine-N'-oxide 0 500 750 1000
Trans-3'-hydroxycotinine 0 500 750 1000
Cotinine-N-oxide 0 100 250 500 750 1000
+ 0.49 ___0.43 + 0.44 + 0.46 + 0.44 ___0.43
Positit'e control-rnitomycin C 0.01
Values represent the mean ± S.E.M., n = 50 cells. * Significant increase in the level of SCE compared to solvent control ( p < 0.05).
100
D.J. Doolittle et al. / Mutation Research 344 (1995) 95-102
of the cells to the test article without metabolic activation. McCoy's 5a medium with 2.5% FBS (containing L-glutamine and penicillin-streptomycin in the above concentrations) was used during the exposure of cells to the test articles with metabolic activation. An Aroclor 1254-induced rat liver homogenate preparation ($9, obtained from Molecular Toxicology, Inc.), was used in the metabolic activation system. Liver enzymes were induced by injecting adult male Fischer-344 rats with Aroclor 1254(500 m g / k g ) five days before they were killed. The
metabolic activation consisted of one part $9 to nine parts McCoy's 5a medium containing 2.5% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin solution. Cofactors were added at concentrations of 24 mg of NADP and 45 mg of sodium isocitrate per milliliter of $9. The metabolic activation mixture was freshly prepared and kept on ice before use. The SCE experiments were conducted by using modifications of the protocol of Galloway et al. (1987). In the nonactivation assays, the cells were exposed to the test article continuously until approximately 2.5 h prior to harvest of the cells. In the
Table 4 Sister-chromatid exchange induction by nicotine and metabolites with metabolic activation Treatment ( ~ g / m l )
Nicotine 0 500 750
1000 Cotinine 0 500 750 1000
Time in BrdUrd (h)
Cell cycle stages (%)
26.0 26.0 26.0 26.0
2.0 2.0 2.0 6.0
98.0 98.0 98.0 94.0
0 0 0 0
11.78 11.24 11.88 11.86
_+ 0.49 _+ 0.47 _+ 0.49 + 0.49
26.0 26.0 26.0 26.0
2.0 1.0 0.0 2.0
98.0 99.0 100.0 98.0
0 0 0 0
11.78 10.88 11.40 11.02
_+ 0.49 + 0.47 + 0.48 _+ 0.47
2.0 0.0 2.0 4.0
98.0 100.0 98.0 96.0
0 0 0 0
11.78 11.38 12.88 11.84
+ 0.49 _+ 0.48 + 0.51 ___0.49
2.0 6.0 0.0 8.0
98.0 94.0 100.0 92.0
0 0 0 0
11.78 11.46 12.08 12.40
_+ 0.49 ___0.48 + 0.49 + 0.50
0.0 3.0 0.0 8.0 5.0 5.0
100.0 97.0 100.0 92.0 95.0 95.0
0 0 0 0 0 0
14.00 13.04 14.04 13.98 13.50 13.70
_ 0.53 + 0.51 _+ 0.53 _+ 0.53 _+ 0.52 _+ 0.52
2.0
98.0
Nicotine-N'-oxide 0 26.0 500 26.0 750 26.0 1000 26.0 Trans 3'-hydroxycotinine 0 26.0 500 26.0 750 26.0 1000 26.0 Cotinine-N-oxide 0 26.0 100 26.0 250 26.0 500 26.0 750 26.0 1000 26.0 Positiee control-cyclophosphamide 1.50 26.0
M1
M2
Values represent the mean + S.E.M., n = 50 cells. Significant increase in the level of SCE compared to solvent control ( p < 0.05).
SCE/cell a M3
35.32 _+ 0.84
D.J. Doolittle et al. / Mutation Research 344 (I 995) 95-102
metabolic activation assays, the CHO cells were exposed to the test article for 2 h in metabolic activation mix. Bromodeoxyuridine (BrdUrd) was added 2 h after the beginning of exposure to the test article. Cells used for SCE analysis were scored in their second metaphase after initiation of chemical exposure. SCE levels for individual treatments were compared to the solvent controls using a t-test employing the Bonferoni correction for multiple comparisons. Dose response was analyzed using a trend test (Margolin et al., 1986).
3. Results and discussion Nicotine and four of its major metabolites were evaluated for mutagenicity in Ames strains TA98, TAI00, TA1535, TA1537 and TA1538, both with and without metabolic activation with $9 at concentrations ranging from 0 to 1000/xg per plate. Neither nicotine nor any of the four metabolites induced an increase in revertant frequency in any strain under either condition of metabolic activation (Tables 1 and 2). The same five compounds were also evaluated for their potential to increase the frequency of SCEs in CHO cells. Nicotine, cotinine, nicotine-N'-oxide and trans-3'-hydroxycotinine were examined for the induction of SCEs on a single experimental day while cotinine-N-oxide was evaluated on a second experimental day. No increases in SCE frequency were observed when nicotine and four of its major metabolites were tested at concentrations ranging up to 1000 /xg per ml of culture medium (Tables 3 and 4). Cytotoxicity, as indicated by cell cycle delay, was observed only with the high dose of nicotine (1000 /xg/ml) under the condition of no metabolic activation. Very slight delays in the cell cycle were observed with cotinine and cotinine-N-oxide, again only under the condition of no metabolic activation. Trivedi et al. (1990) reported that nicotine, at concentrations similar to those used here, increased the frequency of chromosomal aberrations in CHO cells, and Munzner and Renner (1989) reported that high doses of nicotine (Barnes and Eltherington, 1973) increased the frequency of chromosome aberrations in the bone marrow cells of Chinese ham-
101
sters. However, the reported increase in the frequency of chromosomal aberrations observed in the presence of nicotine (Trivedi et al., 1990) was arrived at by incorporating chromosome and chromatid gaps into the primary statistical analysis. International guidelines (OECD, 1983) recommend that gaps or other achromatic lesions not be included when calculating chromosome aberration frequency, since they may result from staining artifacts. As Trivedi et al. indicate, if gaps are excluded, nicotine did not increase the frequency of chromosome aberrations following either 2 or 4 h of treatment, and only slightly increased the frequency of chromosome aberrations following 24 h of continuous treatment (Trivedi et al., 1990). Munzner and Renner (1989) did not provide statistical support for their conclusions. However, it is clear from their data that the slight increase referred to by these authors is due to increased numbers of gaps. If gaps are excluded, nicotine clearly had no significant effect on the frequency of chromosome aberrations in these animals, since the standard deviations from the control and all treatment groups overlap markedly. In conclusion, although cigarette smoke condensate is genotoxic as measured in a wide range of in vitro assays (Doolittle et al., 1990), the results of the present study indicate that the observed genotoxicity is due to constituents other than nicotine. This is consistent with published data demonstrating that the genotoxic potential of tobacco smoke is not related to the nicotine content of the cigarette (Mizusaki et al., 1977; Gairola, 1982), and data indicating that the concentrations of nicotine and cotinine in the urine of human smokers smoking cigarettes which yield nicotine in the absence of tobacco pyrolysis products are unrelated to the mutagenicity of the urine (Rahn et al., 1991). The concentrations evaluated in the present study (up to 1000 /xg/ml) represent massive exaggerations of the average concentrations found in the plasma or urine of human smokers. Taken together, the data indicate that nicotine and its major metabolites do not represent a genotoxic hazard.
Acknowledgements The authors thank Cindy Fulp for excellent technical assistance.
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References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s with the Salmonella~mammalian microsome mutagenicity test, Mutation Res., 31,347-364. Barnes, C.D. and L.G. Eltherington (1973) Drug Dosages in Laboratory A n i m a l s - - A Handbook, Univ. of California Press, Berkeley, p. 171. Byrd, G.D., K.-M. Chang, J.M. Greene and J.D. deBethizy (1992) Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3'-hydroxycotinine in smokers, Drug Metab. Dispos., 20, 192-197. Crooks, P.A., B.S. Bhatti, A. Ravard, R.M. Riggs and W.S. Caldwell (1992) Synthesis of N-glucuronic acid conjugates of cotinine, cis-3-hydroxycotinine, and trans-3-hydroxycotinine, Med. Sci. Res., 20, 881-883. Curvall, M. and E. Kazemi-Vala (1993) Nicotine and metabolites: analysis and levels in body fluids, in: J.W. Gorrod and J. Wahren (Eds.), Nicotine and Related Alkaloids: Absorption, Distribution, Metabolism and Excretion, Chapman and Hall, London. Dagne, E. and N. Castagnoli (1972) Cotinine-N-oxide, a new metabolite of nicotine, J. Med. Chem., 15, 840-841. Davis, R.A., M.F. Stiles, J.D. deBethizy and J.H. Reynolds (1991) Dietary nicotine: a source of urinary cotinine, Food Chem. Toxicol., 29, 821-827. De Flora, S., P. Zanacchi, A. Camoirano, C. Bennicelli and G.S. Badolati (1984) Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test, Mutation Res., 133, 161-198. Domino, E.F., E. Hornbach and T. Demana (1993) The nicotine content of common vegetables, New Eng. J. Med., 329, 437. Doolittle, D.J., C.K. Lee, J.L. Ivett, J.C. Mirsalis, E. Riccio, C.J. Rudd, G.T. Burger and A.W. Hayes (1990) Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco, Environ. Mol. Mutagen., 15, 93-105. Doolittle, D.J., C.A. Rahn and C.K. Lee (1991) The effect of exposure to nicotine, carbon monoxide, cigarette smoke or cigarette smoke condensate on the mutagenicity of rat urine, Mutation Res., 260, 9-18. Florin, I., L. Rutberg, M. Curvall and C.R. Enzell (1980) Screening of tobacco smoke constituents for mutagenicity using the Ames' test, Toxicology, 15, 219-232. Gairola, C. (1982) Genetic effects of fresh cigarette smoke in Saccharomyces cerevisiae, Mutation Res., 102, 123-136.
Galloway, S.M., M.J. Armstrong, C. Reuben, S. Colman, B. Brown, C. Cannon, A.D. Bloom, F. Nakamura, M. Ahmed, S. Duk, J. Rimpo, B.H. Margolin, M.A. Resnick, B. Anderson and E. Zeiger (1987) Chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals, Environ. Mol. Mutagen., 10 (Suppl. 10), 1-175. Leete, E. (1983) Biosynthesis and metabolism of the tobacco alkaloids, in: S.W. Pelletier (Eds.), Alkaloids Chemical and Biological Perspectives, Vol. 1, John Wiley and Sons, New York, NY, pp. 86-139. Margolin, B.H., M.A. Resnick, J.Y. Rimpo, P. Archer, S.M. Galloway, A.D. Bloom and E. Zeiger (1986) Statistical analyses for in vitro cytogenetic assays using Chinese hamster ovary cells, Environ. Mutagen., 8, 183-204. Maron, D.M. and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mizusaki, S., H. Okamoto, A. Akiyama and Y. Fukuhara (1977) Relation between chemical constituents of tobacco and mutagenic activity of cigarette smoke condensate, Mutation Res., 48, 319-325. Munzner, R. and H.W. Renner (1989) Genotoxic investigations of tobacco protein using microbial and mammalian test systems, Z. Ern~ihrungswiss., 28, 300-309. OECD Guideline for Testing of Chemicals (1983) Genetic toxicology: In vitro mammalian cytogenetic test, 473, 1-6. Phillipson, J.D. and S.S. Handa (1975) Nicotine N-oxides, Photochem., 14, 2683-2690. Rahn, C.A., G. Howard, E. Riccio and D.J. Doolittle (1991) Correlations between urinary nicotine or cotinine and urinary mutagenicity in smokers on controlled diets, Environ. Mol. Mutagen., 17, 244-252. Riebe, M., K. Westphal and P. Fortnagel (1982) Mutagenicity testing, in bacterial test systems, of some constituents of tobacco, Mutation Res., 101, 39-43. Riebe, M. and K. Westphal (1983) Studies on the induction of sister-chromatid exchanges in Chinese hamster ovary cells by various tobacco alkaloids, Mutation Res., 124, 281-286. Surgeon General (1988) The health consequences of smoking: nicotine addiction, U.S. Dept. of Health and Human Services. Trivedi, A.H., B.J. Dave and S.G. Adhvaryu (1990) Assessment of genotoxicity of nicotine employing in vitro mammalian test system, Cancer Lett., 54, 89-94. Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Y. Hashimoto (1975) Mutagenicity of carcinogenic azo dyes and their derivatives, Cancer Lett., 1, 91-96.