- Email: [email protected]
Mutagenic effect of amniotic fluid from smoking women at term
Mutation Research, 171 (1986) 71-77 Elsevier
71
MTR01081
Mutagenic effect of amniotic fluid from smoking w o m e n at term Gro Nylander Rivrud 1,2, Khre Berg 1, Diana Anderson 3, Stephen Blowers 3 and Knut Bjoro 2 t Institute of Medical Genetics, University of Oslo, Blindern, Oslo 3, 2 Department of Gynecology and Obstetrics, Rikshospitalet, Oslo I (Norway), and 3 British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS (Great Britain) (Received 18 October 1985) (Revision received 6 March 1986) (Accepted 12 March 1986)
Summary Concentrated term amniotic fluid samples from 44 women smokers and 44 controls were investigated with respect to mutagenic effect in the Salmonella/mammalian-microsome mutagenicity test, using tester strains TA98 and TA100. Tests with freeze-dried specimens of term amniotic fluid showed increases in the number of revertant colonies over background values, regardless of smoking status. However, samples from heavy smokers produced a higher number of revertants than did samples from nonsmokers in several experiments with tester strain TA98. The increase was statistically significant, using either total tar content or number o f cigarettes smoked to identify heavy smokers. Experimental series with tester strain TA100 also resulted in higher group means for heavy smokers than for nonsmokers, but the difference was not statistically significant with the concentrations used in this assay. We conclude that heavy smokers may expose their unborn children to mutagenic substances.
A broad spectrum of adverse effects on a foetus exposed to maternal smoking has been well documented. Most of these studies have concentrated on low birth weight or increased perinatal mortality, and the diagnostic term "foetal tobacco syndrome" was recently introduced (Nieburg et al., 1985). Well controlled follow-up studies have shown long-term effects on the growth and intellectual development in children of smokers, even independent of birth weight (Dunn et al., 1976; Naye and Peters, 1984). A serious consequence of smoking during pregCorrespondence to Dr. Rivrud, Institute of Medical Genetics, University of Oslo, Blindern, Oslo 3, Norway.
nancy could be damage to the genetic material of the unborn child, possibly causing cancer or unfavourable mutations that may be passed on to future generations. Several studies of childhood cancers have included information about maternal smoking. Some workers have found an increased relative risk in children of smoking mothers, while others have not (see review, Everson, 1980). To the best of our knowledge, no study has been published relating maternal smoking to adult-onset tumours in man. Transplacental carcinogenic effects of cigarette smoking have been reported in studies on experimental animals. These studies indicate that tumours resulting from prenatal exposure usually
0165-1218/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 do not develop until adulthood, and that their morphology and site may not differ from those of spontaneously occurring tumours (Everson, 1980). Tomatis (1979) found increased tumour frequencies in later hamster generations after one single transplacental exposure to mutagens, presumably caused by germ line mutations that made the animals cancer prone. Damage to DNA is probably a critical event in carcinogenesis. In the Salmonella/mammalian microsome mutagenicity test there is a 85-90% correlation between known carcinogenic effects of chemicals and induction of point mutations (Purchase et al., 1978). The presence of mutagens in body fluids indicates that the organism was exposed to DNA/protein-damaging compounds. Everson et al. (1985) tested small volumes of amniotic fluid from 16-week pregnancies and found no association between maternal smoking and mutagenicity. We have reported suggestive evidence of increased mutagenic activity of amniotic fluid from heavy smokers at term. No such increase was found when samples were obtained in connection with amniocentesis at 16 weeks of pregnancy (Nylander Rivrud et al., 1985). This study is an extension of previous work on the problem of possible mutagenicity of amniotic fluid from smokers. We now report confirmatory evidence of the mutagenic effect of term amniotic fluid from some very heavy smokers.
mental series the dry material was dissolved in DMSO, resulting in a 25-fold concentration. 2 samples were not sufficiently dissolved to permit testing. In the subsequent experimental series sterile distilled water was used, and a 33-fold concentration was achieved. Processed samples were checked for bacterial contamination. 10 amniotic fluid samples from smokers and nonsmokers were assessed for the amount of free amino acids in a Kontron automatic analyser. The mean value of histidine was 27.8 + 16.4 #moles/1.
Interviews All women were interviewed in the obstetrics department, usually shortly before or after delivery, when they could still remember the number of cigarettes smoked. They were asked about smoking habits during pregnancy, and in great detail about smoking during the 3 days preceding labour. Questions covered brand of cigarettes used, number of cigarettes smoked per day, inhalation habits, smoking habit of spouse and other possible sources of passive smoking. Tar content of cigarettes smoked was calculated from published figures for individual brands and multiplied by the number of cigarettes smoked. The women were also questioned about exposure to other possible mutagens such as alcoholic beverages, coffee, tea, chocolate (because of its theobromine content), certain hair dyes and smoked or grilled food (Stich et al., 1982).
Patients, samples and methods
Mutagenicity assays Sampfing techniques Amniotic fluid specimens were obtained after informed consent from 44 women who smoked and from the same number of nonsmokers, all at term. At caesarean section delivery, amniotic fluid was withdrawn before the uterus was incised. At vaginal delivery a sample was collected when the membranes ruptured, either spontaneously or by amniotomy. Blood-stained samples were discarded.
Processing of amniotic fluid samples All samples were frozen at -20°C. Before use they were thawed and centrifuged at 2000 rpm for 10 rain. The supernatant fluid was freeze-dried and the powder crushed. In the first two experi-
All samples were coded and blind-tested in the plate incorporation assay according t o the standard revised methods for the Salmonella mutagenicity testing (Maron and Ames, 1983). This test system utilizes mutant Salmonella typhimurium bacteria that have lost their ability to synthesize histidine and therefore will not grow under the conditions employed. When exposed to certain mutagens these bacteria undergo reverse point mutations and some may regain the ability to grow without histidine. To mimic metabolic processes in the mammalian organisms a liver homogenate containing large amounts of microsomes and enzymes is added to the test system ($9 fraction). To each Vogel-Bonner plate of 20 ml was
73 TABLE 1 LABORATORY CONDITIONS AND EXPERIMENTAL VARIATIONS TESTING TERM AMNIOTIC FLUID WITH TESTER STRAIN TA98 IN THE AMES TEST Experiment
Solvent
Concentration of amniotic fluid
Amniotic fluid conc./plate (/~1)
Equivalent amount of amniotic fluid (ml)
1 2
DMSO
25 x
100
2.5
3 4
Distilled water
33 ×
100
3.3
added 0.5 ml of filter-sterilized $9 activation mix, 2 ml of biotin/histidine-containing top agar, 0.1 ml of a fresh bacterial culture containing > 2 × 108 b a c t e r i a / m l and 0.1 ml of the test sample. Table 1 presents the laboratory conditions for the 4 consecutive experiments conducted. Individual samples of amniotic fluid were tested in duplicate or triplicate with both bacterial strains. Appropriate controls were included (Table 2). Plates were incubated at 37°C for 48 h and the number of colonies assessed independently by 2 persons. The use of an automatic counting device was omitted to avoid problems with minute particles of undissolved amniotic fluid components.
Bacterial strains, chemicals and media Salmonella typhimurium tester strains TA98 and TA100 were generous gifts from Professor Bruce Ames, University of California. The bacteria were' tested before each experiment to establish the appropriate sensitivity to crystal violet and resistance to ampicillin in order to ensure that they were permeable to large molecules and contained the R-plasmid. The $9 mix for mammalian metabolic activation was prepared from homogenates of livers obtained from 200-g male Sprague-Dawley rats for the two first experimental series and from 180-g male Wistar rats for the later experimental series. The rats had been treated intraperitoneally with 500 m g / k g body weight Aroclor 1254 (Monsanto Chemical Co. Ltd.). Unless otherwise specified all procedures, chemicals and media were as described by Maron and Ames (1983).
Statistical analyses Differences between groups in mean number of revertant colonies were assessed by a t test. For division into groups of light and heavy smokers a cut-off point of 500 mg tar in cigarettes smoked in the last 48 h before delivery was used. Statistical assessment by a t test of smokers and nonsmokers in all 4 experimental series was performed after adjusting each person's mean revertant score to a multiple of the background value in the experiment where her sample was included. A multiple linear regression of individuals' adjusted mean plate counts on mg tar was performed using the Bio Medical D a t a Program 1R at the DEC 10 computer of the University of Oslo. Results 4 samples had to be excluded; 2 because of solution problems and 2 because of unreadable results after incubation of plates. Table 2 and Fig. 1 present the group mean results for women with different smoking history compared to solvent controls in the 4 experiments. Revertant counts are presented as the mean _ the standard error of the mean. Plates containing amniotic fluid generally showed an increase in number of revertant colonies with a minimum increase of 1.3 times background counts for any one group. In the 3 experiments where amniotic fluid from heavy smokers was included, group means for all smokers showed an increase in mean value over
74 TABLE 2 AMNIOTIC FLUID FROM SMOKING AND NONSMOKING WOMEN AT TERM TESTED IN THE AMES TEST WITH Salmonella typhimurium BACTERIAL TESTER STRAIN TA98 Mean number ( 5- S.E.) of revertant colonies for samples from individuals in each category Expt. No. la,b
2b
3c
4c
Solvent controls d
28.3+ 1.8
29.0+ 5.3
42.5+ 9.2
44.0+ 8.0
Nonsmokers Increase ¢ Number of subjects
45.5 + 14.6 1.60 6
38.1 + 6.9 1.31 15
64.6 + 9.9 1.52 12
57.5 + 13.9 1.30 10
All smokers Increase Number of subjects
52.4 + 16.9 1.85 11
40.5 + 6.0 1.40 13
65.9 + 10.6 1.55 8
106.0 + 70.0 2.40 9
Smokers < 500 mg tar f Increase Number of subjects
34.3 + 9.9 1.21 4
37.0 + 4.2 1.28 3
65.9 + 10.6 1.55 8
52.4 + 7.0 1.91 5
Smokers > 500 mg tar f Increase Number of subjects
62.5+ 9.9 * 2.20 7
41.55- 6.3 1.43 10
B[a]P controls h Increase
603.05- 5.5 21.3
267.05-22.8 9.20
None 0 0
173.55-45.9 ** 3.94 4
437.5 + 14.8 10.29
431.05-23.1 9.79
a b c d e r s h
Experiment performed at BIBRA, all others at University of Oslo. Bacteria from BIBRA, originally from Professor B.N. Ames. Bacteria received directly from Professor B.N. Ames. 100 /~1 with 500 #1 $9 mix (20/~1 $9). Increase = multiple of solvent controls. In cigarettes smoked within 48 h of delivery. 4 extreme smokers with > 65 cigarettes smoked within 48 h of delivery. Benzo[a]pyrene 5/zg/plate. * Difference nonsmokers/heavy smokers significant with P < 0.025. ** Difference nonsmokers/heavy smokers significant with P < 0.005.
nonsmokers. When only heavy smokers were comp a r e d w i t h n o n s m o k e r s , this d i f f e r e n c e was sign i f i c a n t in t h e 2 e x p e r i m e n t s t h a t i n c l u d e d t h e h e a v i e s t s m o k e r s ( T a b l e 2). E s s e n t i a l l y t h e s a m e r e s u l t s w e r e o b t a i n e d w i t h n o n - p a r a m e t r i c tests. T h u s t h e results w e r e n o t d e p e n d e n t o n the statist i c a l a s s u m p t i o n s u n d e r l y i n g t tests. The regression coefficient for individuals' a d j u s t e d m e a n p l a t e c o u n t s o n m g tar in c i g a r e t t e s s m o k e d w i t h i n 48 h o f d e l i v e r y w a s 0.0475. A regression of revertants on number of cigarettes smoked gave a comparable regression coefficient. An estimate of the regression coefficient indicates
t h a t a n i n c r e a s e o f 200 m g t a r o r 10 c i g a r e t t e s i n c r e a s e s t h e n u m b e r o f r e v e r t a n t s b y 1 0 - 1 2 % in plates with samples from very heavy smokers. S o m e s m o k e r s w h o w e r e e x p o s e d to l e s s t h a n 500 m g tar w i t h i n the last 48 h o f d e l i v e r y h a d fewer revertant colonies than nonsmokers. The d i f f e r e n c e b e t w e e n m e a n s was n o t s i g n i f i c a n t at t h e 5% level ( p = 0.21). T e s t s c o n d u c t e d w i t h t h e T A 1 0 0 s t r a i n also r e s u l t e d in a h i g h e r m e a n for h e a v y s m o k e r s t h a n f o r n o n s m o k e r s , b u t this d i f f e r e n c e w a s s m a l l a n d n o t s t a t i s t i c a l l y significant.
75
INCREASE *
5X
/,X
3X-
2X
•
B o
• o
BB ° uoo
ggo =o gg8 aoo
1X ¸
o
o
•
o
•
•
~
o oO OO
o
°o
'
.
O
~Go
,o'oo
,5'oo '
MG TAR
Fig. 1. Term amniotic fluid from smokers and nonsmokers tested in the Salmonella/mammalian-microsome mutagenicity test system with tester strain TA98. Increase * ( = multiple of solvent control) in number of revertant colonies (means from all assays for each person) plotted against amount of tar in cigarettes smoked within 48 h of delivery. HI, nonsmokers; ©, smokers < 500 mg tar; O, smokers > 500 mg tar.
Discussion The increase in mean number of revertants with samples from heavy smokers over nonsmokers suggests a mutagenic effect of term amniotic fluid from at least some women who were heavy smokers. While developed for testing chemicals, the Salmonella/mammalian-microsome test has also been very useful in testing various body fluids. Urine from smokers has been reported to be mutagenic following a 200-fold concentration (Yamasaki and Ames, 1977; Kado et al., 1983). The collection of amniotic fluid and preparation of the samples was very time consuming, especially in view of the numerous failures encountered when it proved impossible to obtain the desired volume. For this reason the samples were
collected over a period of 15 months and tested in 4 consecutive experiments. So far we have at most been able to achieve a 33-fold concentration of amniotic fluid. Because of the limited volume it is not possible to test each fluid sample with m a n y tester strains or a wide range of concentrations as recommended by Ames and others. Also, although a doubling or more of the number of revertants and a dose-response relationship may be a reasonable requirement before a chemical is considered mutagenic, our resuits from several different experiments with term amniotic fluid require careful consideration even in those experiments where such doubling was not found. The mutagenic effect of urine from smokers is found mainly in the evening of a day of smoking, and not the next morning (Yamasaki and Ames, 1977). Because of this, and the fact that there seems to be a total exchange of at least the water component of amniotic fluid every 48 h, we focussed our study on women who had smoked heavily during the last days and even preferably the last hours before delivery. The heavy smokers whose amniotic fluid resuited in a high number of revertants had usually delivered their babies vaginally, and m a n y had been smoking at least during the early stages of labour. W o m e n delivering by elective caesarean section had not been smoking for at least 8 h, usually longer. A possible explanation for the higher number of revertants from smokers' samples obtained at vaginal delivery could thus be the fairly quick turnover of amniotic fluid. Another reason could be a contamination of amniotic fluid with mutagenic substances at vaginal delivery. As a rule no disinfectant was used intravaginally when the samples were obtained, but in order to rule out that earlier vaginal examinations with a disinfectant cream could influence the results, this cream (Hibitane) was included in one experiment. With minute amounts no effect was seen compared to solvent controls, while no Salmonella bacteria survived in the plates where this cream had been added in amounts comparable to that used for a clinical examination. It is not clear why revertant counts for all amniotic fluid samples showed increases over
76 background values. There was no correlation between mutagenicity and histidine levels of samples analysed for free amino acids. Increasing the amount of amniotic fluid concentrate per plate generally did not seem to increase the number of revertants above the level reached with smaller amounts. Thi_s is in agreement with our earlier results that experimental addition of increasing levels of histidine up to 5-fold that used in the top agar, did not greatly influence the number of colonies after 48 h (Nylander Rivrud et al., 1985). Green and Muriel (1977) have reported, however, that the addition of extra nutrients can increase the background mutation frequency. Miller and Stoltz (1978) found that urine from rats injected with isonicotinic acid hydrazid was mutagenic only after lyophilization or prolonged storage at room temperature. A possible effect of procedural artefacts or extra histidine, biotin and other nutrients in amniotic fluid can probably be disregarded in this study as it would presumably affect smokers and nonsmokers equally. The somewhat surprising result that the mean number of revertants for light smokers was lower than or equal to that for the nonsmokers throughout all 4 experiments was not statistically significant. If, however, this reflects a true difference, one possible explanation could be that the foetuses of mothers who smoked have an increased capacity for dealing with mutagens, as long as the mutagenic load is not too heavy (Nebert et al., 1969; Everson, 1980). Also, if the time factor is as important as it appears to be with smokers' urine, a light smoker is less likely to smoke during labour than a tobacco addict (PoUin, 1984). The amount of tar in cigarettes smoked during the last 2 days was calculated. The results were significant also when number of cigarettes smoked was used for comparison. Very often the women who smoked many cigarettes per day would also prefer a strong type of tobacco, and Wald (1984) has shown that most smokers will increase the extent of inhalation when smoking milder cigarettes. Thus, for practical purposes it may not be essential to measure tar content rather than number of cigarettes smoked. The amniotic fluid comprises the internal as well as the external environment of the foetus who not only floats in it, but starts drinking it very
early. At term the foetus drinks about 500 ml of amniotic fluid per day, and voids about the same volume of urine. Before the foetal skin becomes keratinized at about 20 weeks, the amniotic fluid passes it freely. The potential hazard of exposing the foetus to amniotic fluid containing mutagenic substances, would seem to be another reason why pregnant women should not smoke.
Acknowledgements This work was supported by The Norwegian Society for Fighting Cancer. We greatly appreciate the cooperation of the Obstetrics Departments at Baerum Hospital and Ulleval Oslo City Hospital. We thank Guri Nilsen for excellent assistance in the laboratory.
References Dunn, H.C., A.K. McBurney, S. Ingram et al. (1976) Maternal cigarette smoking during pregnancy and the childs subsequent development: Physicalgrowth to age 6.5 years, Can. J. Publ. Health, 67, 499-505. Everson, R.B. (1980) Individuals transplacentally exposed to maternal smoking may be at increased cancer risk in adult life, Lancet, July 19, 123-127. Everson, R.B., K.L. Milne, D. Warburton, H.D. McClamrock and P.D. Buchanan (1985) Mutagenicity assays of human amniotic fluid, Environ. Mutagen., 7, 171-184. Green, M.H.L., and W.J. Muriel (1977) Mutagen testing using trp+ reversion in E. coli, in: B.J. Kilbey, M. Legator and W. Nichols (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 65-94. Kado, N.Y., D. Langley and E. Eisenstadt (1983) A simple modification of the Salmonella liquid-incubation assay, Mutation Res., 121, 25-32. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miller, C.T., and D.R. Stoltz (1978) Mutagenicity induced by lyophilization or storage of urine from isoniazid-treated rats, Mutation Res., 56, 289-293. Naye, R.L., and E.C. Peters (1984) Mental development of children whose mothers smoked during pregnan,cy, Obstet. Gynecol., 64, 60-107. Nebert, D.W., J. Winker and H.V. Gelboin (1969) Aryl hydrocarbon hydroxylase activity in human placenta from cigarette smoking and nonsmoking women, Cancer Res., 29, 1763-1769. Nieburg, P., J.S. Marks, N.M. McLaren and P.L. Remington (1985) The fetal tobacco syndrome, J. Am. Med. Ass., 253, 298-299. Nylander Rivrud, G., K. Berg, D. Anderson, S. Blowersand K. Bjoro (1985) Study of the amniotic fluid from smokers and non-smokers in the Ames test, Mutation Res., in press.
77 PoUin, W. (1984) The role of the addictive process as a key step in causation of all tobacco-related diseases, J. Am. Med. Ass., 252, 2874. Stich, H.F., M.P. Rosin, C.H. Wu and W.D. Powrie (1982) The use of mutagenicity testing to evaluate food products, in: J.A. Heddle (Ed.), Mutagenicity - - New Horizons in Genetic Toxicology, Academic Press, New York, p. 117. Tomatis, L. (1979) Prenatal exposure to chemical carcinogens and its effect on subsequent generations, in: J.M. Rice
(Ed.), Perinatal Carcinogenesis, National Cancer Institute Monograph 51, p. 159. Wald, N.J. (1984) Relative intakes of tar, nicotine, and carbon monoxide from cigarettes of different yields, Thorax, 39, 361-364. Yamasald, E., and B.N. Ames (1977) Concentration of mutagens from urine by adsorption with nonpolar resin XAD-2: Cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555-3559.
71
MTR01081
Mutagenic effect of amniotic fluid from smoking w o m e n at term Gro Nylander Rivrud 1,2, Khre Berg 1, Diana Anderson 3, Stephen Blowers 3 and Knut Bjoro 2 t Institute of Medical Genetics, University of Oslo, Blindern, Oslo 3, 2 Department of Gynecology and Obstetrics, Rikshospitalet, Oslo I (Norway), and 3 British Industrial Biological Research Association, Woodmansterne Road, Carshalton, Surrey SM5 4DS (Great Britain) (Received 18 October 1985) (Revision received 6 March 1986) (Accepted 12 March 1986)
Summary Concentrated term amniotic fluid samples from 44 women smokers and 44 controls were investigated with respect to mutagenic effect in the Salmonella/mammalian-microsome mutagenicity test, using tester strains TA98 and TA100. Tests with freeze-dried specimens of term amniotic fluid showed increases in the number of revertant colonies over background values, regardless of smoking status. However, samples from heavy smokers produced a higher number of revertants than did samples from nonsmokers in several experiments with tester strain TA98. The increase was statistically significant, using either total tar content or number o f cigarettes smoked to identify heavy smokers. Experimental series with tester strain TA100 also resulted in higher group means for heavy smokers than for nonsmokers, but the difference was not statistically significant with the concentrations used in this assay. We conclude that heavy smokers may expose their unborn children to mutagenic substances.
A broad spectrum of adverse effects on a foetus exposed to maternal smoking has been well documented. Most of these studies have concentrated on low birth weight or increased perinatal mortality, and the diagnostic term "foetal tobacco syndrome" was recently introduced (Nieburg et al., 1985). Well controlled follow-up studies have shown long-term effects on the growth and intellectual development in children of smokers, even independent of birth weight (Dunn et al., 1976; Naye and Peters, 1984). A serious consequence of smoking during pregCorrespondence to Dr. Rivrud, Institute of Medical Genetics, University of Oslo, Blindern, Oslo 3, Norway.
nancy could be damage to the genetic material of the unborn child, possibly causing cancer or unfavourable mutations that may be passed on to future generations. Several studies of childhood cancers have included information about maternal smoking. Some workers have found an increased relative risk in children of smoking mothers, while others have not (see review, Everson, 1980). To the best of our knowledge, no study has been published relating maternal smoking to adult-onset tumours in man. Transplacental carcinogenic effects of cigarette smoking have been reported in studies on experimental animals. These studies indicate that tumours resulting from prenatal exposure usually
0165-1218/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
72 do not develop until adulthood, and that their morphology and site may not differ from those of spontaneously occurring tumours (Everson, 1980). Tomatis (1979) found increased tumour frequencies in later hamster generations after one single transplacental exposure to mutagens, presumably caused by germ line mutations that made the animals cancer prone. Damage to DNA is probably a critical event in carcinogenesis. In the Salmonella/mammalian microsome mutagenicity test there is a 85-90% correlation between known carcinogenic effects of chemicals and induction of point mutations (Purchase et al., 1978). The presence of mutagens in body fluids indicates that the organism was exposed to DNA/protein-damaging compounds. Everson et al. (1985) tested small volumes of amniotic fluid from 16-week pregnancies and found no association between maternal smoking and mutagenicity. We have reported suggestive evidence of increased mutagenic activity of amniotic fluid from heavy smokers at term. No such increase was found when samples were obtained in connection with amniocentesis at 16 weeks of pregnancy (Nylander Rivrud et al., 1985). This study is an extension of previous work on the problem of possible mutagenicity of amniotic fluid from smokers. We now report confirmatory evidence of the mutagenic effect of term amniotic fluid from some very heavy smokers.
mental series the dry material was dissolved in DMSO, resulting in a 25-fold concentration. 2 samples were not sufficiently dissolved to permit testing. In the subsequent experimental series sterile distilled water was used, and a 33-fold concentration was achieved. Processed samples were checked for bacterial contamination. 10 amniotic fluid samples from smokers and nonsmokers were assessed for the amount of free amino acids in a Kontron automatic analyser. The mean value of histidine was 27.8 + 16.4 #moles/1.
Interviews All women were interviewed in the obstetrics department, usually shortly before or after delivery, when they could still remember the number of cigarettes smoked. They were asked about smoking habits during pregnancy, and in great detail about smoking during the 3 days preceding labour. Questions covered brand of cigarettes used, number of cigarettes smoked per day, inhalation habits, smoking habit of spouse and other possible sources of passive smoking. Tar content of cigarettes smoked was calculated from published figures for individual brands and multiplied by the number of cigarettes smoked. The women were also questioned about exposure to other possible mutagens such as alcoholic beverages, coffee, tea, chocolate (because of its theobromine content), certain hair dyes and smoked or grilled food (Stich et al., 1982).
Patients, samples and methods
Mutagenicity assays Sampfing techniques Amniotic fluid specimens were obtained after informed consent from 44 women who smoked and from the same number of nonsmokers, all at term. At caesarean section delivery, amniotic fluid was withdrawn before the uterus was incised. At vaginal delivery a sample was collected when the membranes ruptured, either spontaneously or by amniotomy. Blood-stained samples were discarded.
Processing of amniotic fluid samples All samples were frozen at -20°C. Before use they were thawed and centrifuged at 2000 rpm for 10 rain. The supernatant fluid was freeze-dried and the powder crushed. In the first two experi-
All samples were coded and blind-tested in the plate incorporation assay according t o the standard revised methods for the Salmonella mutagenicity testing (Maron and Ames, 1983). This test system utilizes mutant Salmonella typhimurium bacteria that have lost their ability to synthesize histidine and therefore will not grow under the conditions employed. When exposed to certain mutagens these bacteria undergo reverse point mutations and some may regain the ability to grow without histidine. To mimic metabolic processes in the mammalian organisms a liver homogenate containing large amounts of microsomes and enzymes is added to the test system ($9 fraction). To each Vogel-Bonner plate of 20 ml was
73 TABLE 1 LABORATORY CONDITIONS AND EXPERIMENTAL VARIATIONS TESTING TERM AMNIOTIC FLUID WITH TESTER STRAIN TA98 IN THE AMES TEST Experiment
Solvent
Concentration of amniotic fluid
Amniotic fluid conc./plate (/~1)
Equivalent amount of amniotic fluid (ml)
1 2
DMSO
25 x
100
2.5
3 4
Distilled water
33 ×
100
3.3
added 0.5 ml of filter-sterilized $9 activation mix, 2 ml of biotin/histidine-containing top agar, 0.1 ml of a fresh bacterial culture containing > 2 × 108 b a c t e r i a / m l and 0.1 ml of the test sample. Table 1 presents the laboratory conditions for the 4 consecutive experiments conducted. Individual samples of amniotic fluid were tested in duplicate or triplicate with both bacterial strains. Appropriate controls were included (Table 2). Plates were incubated at 37°C for 48 h and the number of colonies assessed independently by 2 persons. The use of an automatic counting device was omitted to avoid problems with minute particles of undissolved amniotic fluid components.
Bacterial strains, chemicals and media Salmonella typhimurium tester strains TA98 and TA100 were generous gifts from Professor Bruce Ames, University of California. The bacteria were' tested before each experiment to establish the appropriate sensitivity to crystal violet and resistance to ampicillin in order to ensure that they were permeable to large molecules and contained the R-plasmid. The $9 mix for mammalian metabolic activation was prepared from homogenates of livers obtained from 200-g male Sprague-Dawley rats for the two first experimental series and from 180-g male Wistar rats for the later experimental series. The rats had been treated intraperitoneally with 500 m g / k g body weight Aroclor 1254 (Monsanto Chemical Co. Ltd.). Unless otherwise specified all procedures, chemicals and media were as described by Maron and Ames (1983).
Statistical analyses Differences between groups in mean number of revertant colonies were assessed by a t test. For division into groups of light and heavy smokers a cut-off point of 500 mg tar in cigarettes smoked in the last 48 h before delivery was used. Statistical assessment by a t test of smokers and nonsmokers in all 4 experimental series was performed after adjusting each person's mean revertant score to a multiple of the background value in the experiment where her sample was included. A multiple linear regression of individuals' adjusted mean plate counts on mg tar was performed using the Bio Medical D a t a Program 1R at the DEC 10 computer of the University of Oslo. Results 4 samples had to be excluded; 2 because of solution problems and 2 because of unreadable results after incubation of plates. Table 2 and Fig. 1 present the group mean results for women with different smoking history compared to solvent controls in the 4 experiments. Revertant counts are presented as the mean _ the standard error of the mean. Plates containing amniotic fluid generally showed an increase in number of revertant colonies with a minimum increase of 1.3 times background counts for any one group. In the 3 experiments where amniotic fluid from heavy smokers was included, group means for all smokers showed an increase in mean value over
74 TABLE 2 AMNIOTIC FLUID FROM SMOKING AND NONSMOKING WOMEN AT TERM TESTED IN THE AMES TEST WITH Salmonella typhimurium BACTERIAL TESTER STRAIN TA98 Mean number ( 5- S.E.) of revertant colonies for samples from individuals in each category Expt. No. la,b
2b
3c
4c
Solvent controls d
28.3+ 1.8
29.0+ 5.3
42.5+ 9.2
44.0+ 8.0
Nonsmokers Increase ¢ Number of subjects
45.5 + 14.6 1.60 6
38.1 + 6.9 1.31 15
64.6 + 9.9 1.52 12
57.5 + 13.9 1.30 10
All smokers Increase Number of subjects
52.4 + 16.9 1.85 11
40.5 + 6.0 1.40 13
65.9 + 10.6 1.55 8
106.0 + 70.0 2.40 9
Smokers < 500 mg tar f Increase Number of subjects
34.3 + 9.9 1.21 4
37.0 + 4.2 1.28 3
65.9 + 10.6 1.55 8
52.4 + 7.0 1.91 5
Smokers > 500 mg tar f Increase Number of subjects
62.5+ 9.9 * 2.20 7
41.55- 6.3 1.43 10
B[a]P controls h Increase
603.05- 5.5 21.3
267.05-22.8 9.20
None 0 0
173.55-45.9 ** 3.94 4
437.5 + 14.8 10.29
431.05-23.1 9.79
a b c d e r s h
Experiment performed at BIBRA, all others at University of Oslo. Bacteria from BIBRA, originally from Professor B.N. Ames. Bacteria received directly from Professor B.N. Ames. 100 /~1 with 500 #1 $9 mix (20/~1 $9). Increase = multiple of solvent controls. In cigarettes smoked within 48 h of delivery. 4 extreme smokers with > 65 cigarettes smoked within 48 h of delivery. Benzo[a]pyrene 5/zg/plate. * Difference nonsmokers/heavy smokers significant with P < 0.025. ** Difference nonsmokers/heavy smokers significant with P < 0.005.
nonsmokers. When only heavy smokers were comp a r e d w i t h n o n s m o k e r s , this d i f f e r e n c e was sign i f i c a n t in t h e 2 e x p e r i m e n t s t h a t i n c l u d e d t h e h e a v i e s t s m o k e r s ( T a b l e 2). E s s e n t i a l l y t h e s a m e r e s u l t s w e r e o b t a i n e d w i t h n o n - p a r a m e t r i c tests. T h u s t h e results w e r e n o t d e p e n d e n t o n the statist i c a l a s s u m p t i o n s u n d e r l y i n g t tests. The regression coefficient for individuals' a d j u s t e d m e a n p l a t e c o u n t s o n m g tar in c i g a r e t t e s s m o k e d w i t h i n 48 h o f d e l i v e r y w a s 0.0475. A regression of revertants on number of cigarettes smoked gave a comparable regression coefficient. An estimate of the regression coefficient indicates
t h a t a n i n c r e a s e o f 200 m g t a r o r 10 c i g a r e t t e s i n c r e a s e s t h e n u m b e r o f r e v e r t a n t s b y 1 0 - 1 2 % in plates with samples from very heavy smokers. S o m e s m o k e r s w h o w e r e e x p o s e d to l e s s t h a n 500 m g tar w i t h i n the last 48 h o f d e l i v e r y h a d fewer revertant colonies than nonsmokers. The d i f f e r e n c e b e t w e e n m e a n s was n o t s i g n i f i c a n t at t h e 5% level ( p = 0.21). T e s t s c o n d u c t e d w i t h t h e T A 1 0 0 s t r a i n also r e s u l t e d in a h i g h e r m e a n for h e a v y s m o k e r s t h a n f o r n o n s m o k e r s , b u t this d i f f e r e n c e w a s s m a l l a n d n o t s t a t i s t i c a l l y significant.
75
INCREASE *
5X
/,X
3X-
2X
•
B o
• o
BB ° uoo
ggo =o gg8 aoo
1X ¸
o
o
•
o
•
•
~
o oO OO
o
°o
'
.
O
~Go
,o'oo
,5'oo '
MG TAR
Fig. 1. Term amniotic fluid from smokers and nonsmokers tested in the Salmonella/mammalian-microsome mutagenicity test system with tester strain TA98. Increase * ( = multiple of solvent control) in number of revertant colonies (means from all assays for each person) plotted against amount of tar in cigarettes smoked within 48 h of delivery. HI, nonsmokers; ©, smokers < 500 mg tar; O, smokers > 500 mg tar.
Discussion The increase in mean number of revertants with samples from heavy smokers over nonsmokers suggests a mutagenic effect of term amniotic fluid from at least some women who were heavy smokers. While developed for testing chemicals, the Salmonella/mammalian-microsome test has also been very useful in testing various body fluids. Urine from smokers has been reported to be mutagenic following a 200-fold concentration (Yamasaki and Ames, 1977; Kado et al., 1983). The collection of amniotic fluid and preparation of the samples was very time consuming, especially in view of the numerous failures encountered when it proved impossible to obtain the desired volume. For this reason the samples were
collected over a period of 15 months and tested in 4 consecutive experiments. So far we have at most been able to achieve a 33-fold concentration of amniotic fluid. Because of the limited volume it is not possible to test each fluid sample with m a n y tester strains or a wide range of concentrations as recommended by Ames and others. Also, although a doubling or more of the number of revertants and a dose-response relationship may be a reasonable requirement before a chemical is considered mutagenic, our resuits from several different experiments with term amniotic fluid require careful consideration even in those experiments where such doubling was not found. The mutagenic effect of urine from smokers is found mainly in the evening of a day of smoking, and not the next morning (Yamasaki and Ames, 1977). Because of this, and the fact that there seems to be a total exchange of at least the water component of amniotic fluid every 48 h, we focussed our study on women who had smoked heavily during the last days and even preferably the last hours before delivery. The heavy smokers whose amniotic fluid resuited in a high number of revertants had usually delivered their babies vaginally, and m a n y had been smoking at least during the early stages of labour. W o m e n delivering by elective caesarean section had not been smoking for at least 8 h, usually longer. A possible explanation for the higher number of revertants from smokers' samples obtained at vaginal delivery could thus be the fairly quick turnover of amniotic fluid. Another reason could be a contamination of amniotic fluid with mutagenic substances at vaginal delivery. As a rule no disinfectant was used intravaginally when the samples were obtained, but in order to rule out that earlier vaginal examinations with a disinfectant cream could influence the results, this cream (Hibitane) was included in one experiment. With minute amounts no effect was seen compared to solvent controls, while no Salmonella bacteria survived in the plates where this cream had been added in amounts comparable to that used for a clinical examination. It is not clear why revertant counts for all amniotic fluid samples showed increases over
76 background values. There was no correlation between mutagenicity and histidine levels of samples analysed for free amino acids. Increasing the amount of amniotic fluid concentrate per plate generally did not seem to increase the number of revertants above the level reached with smaller amounts. Thi_s is in agreement with our earlier results that experimental addition of increasing levels of histidine up to 5-fold that used in the top agar, did not greatly influence the number of colonies after 48 h (Nylander Rivrud et al., 1985). Green and Muriel (1977) have reported, however, that the addition of extra nutrients can increase the background mutation frequency. Miller and Stoltz (1978) found that urine from rats injected with isonicotinic acid hydrazid was mutagenic only after lyophilization or prolonged storage at room temperature. A possible effect of procedural artefacts or extra histidine, biotin and other nutrients in amniotic fluid can probably be disregarded in this study as it would presumably affect smokers and nonsmokers equally. The somewhat surprising result that the mean number of revertants for light smokers was lower than or equal to that for the nonsmokers throughout all 4 experiments was not statistically significant. If, however, this reflects a true difference, one possible explanation could be that the foetuses of mothers who smoked have an increased capacity for dealing with mutagens, as long as the mutagenic load is not too heavy (Nebert et al., 1969; Everson, 1980). Also, if the time factor is as important as it appears to be with smokers' urine, a light smoker is less likely to smoke during labour than a tobacco addict (PoUin, 1984). The amount of tar in cigarettes smoked during the last 2 days was calculated. The results were significant also when number of cigarettes smoked was used for comparison. Very often the women who smoked many cigarettes per day would also prefer a strong type of tobacco, and Wald (1984) has shown that most smokers will increase the extent of inhalation when smoking milder cigarettes. Thus, for practical purposes it may not be essential to measure tar content rather than number of cigarettes smoked. The amniotic fluid comprises the internal as well as the external environment of the foetus who not only floats in it, but starts drinking it very
early. At term the foetus drinks about 500 ml of amniotic fluid per day, and voids about the same volume of urine. Before the foetal skin becomes keratinized at about 20 weeks, the amniotic fluid passes it freely. The potential hazard of exposing the foetus to amniotic fluid containing mutagenic substances, would seem to be another reason why pregnant women should not smoke.
Acknowledgements This work was supported by The Norwegian Society for Fighting Cancer. We greatly appreciate the cooperation of the Obstetrics Departments at Baerum Hospital and Ulleval Oslo City Hospital. We thank Guri Nilsen for excellent assistance in the laboratory.
References Dunn, H.C., A.K. McBurney, S. Ingram et al. (1976) Maternal cigarette smoking during pregnancy and the childs subsequent development: Physicalgrowth to age 6.5 years, Can. J. Publ. Health, 67, 499-505. Everson, R.B. (1980) Individuals transplacentally exposed to maternal smoking may be at increased cancer risk in adult life, Lancet, July 19, 123-127. Everson, R.B., K.L. Milne, D. Warburton, H.D. McClamrock and P.D. Buchanan (1985) Mutagenicity assays of human amniotic fluid, Environ. Mutagen., 7, 171-184. Green, M.H.L., and W.J. Muriel (1977) Mutagen testing using trp+ reversion in E. coli, in: B.J. Kilbey, M. Legator and W. Nichols (Eds.), Handbook of Mutagenicity Test Procedures, Elsevier, Amsterdam, pp. 65-94. Kado, N.Y., D. Langley and E. Eisenstadt (1983) A simple modification of the Salmonella liquid-incubation assay, Mutation Res., 121, 25-32. Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miller, C.T., and D.R. Stoltz (1978) Mutagenicity induced by lyophilization or storage of urine from isoniazid-treated rats, Mutation Res., 56, 289-293. Naye, R.L., and E.C. Peters (1984) Mental development of children whose mothers smoked during pregnan,cy, Obstet. Gynecol., 64, 60-107. Nebert, D.W., J. Winker and H.V. Gelboin (1969) Aryl hydrocarbon hydroxylase activity in human placenta from cigarette smoking and nonsmoking women, Cancer Res., 29, 1763-1769. Nieburg, P., J.S. Marks, N.M. McLaren and P.L. Remington (1985) The fetal tobacco syndrome, J. Am. Med. Ass., 253, 298-299. Nylander Rivrud, G., K. Berg, D. Anderson, S. Blowersand K. Bjoro (1985) Study of the amniotic fluid from smokers and non-smokers in the Ames test, Mutation Res., in press.
77 PoUin, W. (1984) The role of the addictive process as a key step in causation of all tobacco-related diseases, J. Am. Med. Ass., 252, 2874. Stich, H.F., M.P. Rosin, C.H. Wu and W.D. Powrie (1982) The use of mutagenicity testing to evaluate food products, in: J.A. Heddle (Ed.), Mutagenicity - - New Horizons in Genetic Toxicology, Academic Press, New York, p. 117. Tomatis, L. (1979) Prenatal exposure to chemical carcinogens and its effect on subsequent generations, in: J.M. Rice
(Ed.), Perinatal Carcinogenesis, National Cancer Institute Monograph 51, p. 159. Wald, N.J. (1984) Relative intakes of tar, nicotine, and carbon monoxide from cigarettes of different yields, Thorax, 39, 361-364. Yamasald, E., and B.N. Ames (1977) Concentration of mutagens from urine by adsorption with nonpolar resin XAD-2: Cigarette smokers have mutagenic urine, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3555-3559.