REPORTS

Metabolites of a Tobacco-Specific Carcinogen in Urine From Newborns

Gerd M. Lackmann, Ulrich Salzberger, Uwe Töllner, Menglan Chen, Steven G. Carmella, Stephen S. Hecht

Affiliations of authors: G. M. Lackmann, Zentrum für Kinderheilkunde, Heinrich-Heine-Universität, Düsseldorf, Germany; U. Salzberger, U. Töllner, Klinik für Kinder- und Jugendmedizine, Stadisches Klinikum, Fulda, Germany; M. Chen, S. G. Carmella, S. S. Hecht, University of Minnesota Cancer Center, Minneapolis.

Correspondence to: Stephen S. Hecht, Ph.D., University of Minnesota Cancer Center, Box 806 Mayo, 420 Delaware St., S.E., Minneapolis, MN 55455 (e-mail: hecht002{at}gold.tc.umn.edu).


    ABSTRACT
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
BACKGROUND: Cigarette smoking during pregnancy can result in fetal exposure to carcinogens that are transferred from the mother via the placenta, but little information is available on fetal uptake of such compounds. We analyzed samples of the first urine from newborns whose mothers did or did not smoke cigarettes for the presence of metabolites of the potent tobacco-specific transplacental carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). METHODS: The urine was collected and analyzed for two metabolites of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc). Gas chromatography and nitrosamine-selective detection, with confirmation by mass spectrometry, were used in the analyses, which were performed without knowledge of the origin of the urine samples. RESULTS: NNAL-Gluc was detected in 22 (71%) of 31 urine samples from newborns of mothers who smoked; NNAL was detected in four of these 31 urine samples. Neither compound was detected in the 17 urine samples from newborns of mothers who did not smoke. The arithmetic mean level of NNAL plus NNAL-Gluc in the 27 newborns of smokers for which both analytes were quantified was 0.14 (95% confidence interval [CI] = 0.083-0.200) pmol/mL. The levels of NNAL plus NNAL-Gluc in the urine from these babies were statistically significantly higher than those in the urine from newborns of nonsmoking mothers (geometric means = 0.062 [95% CI = 0.035-0.110] and 0.010 [considered as not detected; no confidence interval], respectively; two-sided P<.001). NNAL plus NNAL-Gluc levels in the 18 positive urine samples in which both analytes were quantified ranged from 0.045 to 0.400 pmol/mL, with an arithmetic mean level of 0.20 (95% CI = 0.14-0.26) pmol/mL, about 5%-10% of the levels of these compounds detected in the urine from adult smokers. CONCLUSIONS: Two metabolites of the tobacco-specific transplacental carcinogen NNK can be detected in the urine from newborns of mothers who smoked cigarettes during pregnancy.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Smoking during pregnancy is associated with a variety of negative consequences, including low birth weight, spontaneous abortion, placenta previa, and abruptio placentae (1-3). In spite of this, only 39% of smokers quit while pregnant and, of those who quit, 70% resume smoking within 1 year of giving birth (4). In the United States, approximately one million infants are prenatally exposed to cigarette smoke during pregnancy each year (5).

One of the potential negative effects of smoking during pregnancy is exposure of the fetus to carcinogens. Previous studies have provided some evidence for such exposures. Detected in the fetal blood were 4-aminobiphenyl-hemoglobin adducts, and their levels were higher in fetuses of smokers than in those of nonsmokers (6-8). Hydrophobic DNA adducts and polycyclic aromatic hydrocarbon-DNA adducts have been detected in the placenta of mothers who smoked; some of these adducts were strongly related to smoking, but others were unrelated; moreover, not all studies have detected the putative smoking-related adducts (9-16). Such adducts detected in fetal tissues showed no relationship to smoking (17,18). DNA adducts of the carcinogen benzo[a]pyrene have been detected in placental DNA, but they were unrelated to smoking (19,20). Levels of the DNA adducts O6-methyldeoxyguanosine and 8-oxodeoxyguanosine in human placenta were also unrelated to smoking (14,21). Mutations in the hprt gene have been detected in newborns in some studies (22) but not in others (23). Overall, with the exception of 4-aminobiphenyl, the literature is unclear with respect to carcinogen uptake by fetuses of smoking mothers. Many of the studies cited above used nonspecific methods or detected compounds that would have origins other than cigarette smoke.

To our knowledge, there have been no reports on the analysis of carcinogens or their metabolites in the urine from newborns. In this study, we have analyzed the urine from newborns for metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen. Uptake of this carcinogen or its metabolites by the fetus could result only from exposure to tobacco products and would potentially provide a reliable indicator of fetal exposure to cigarette smoke carcinogens.

NNK is a potent transplacental carcinogen in the Syrian golden hamster (24-26). Treatment of pregnant hamsters with NNK causes tumors in their offspring in a variety of tissues, including the respiratory tract, pancreas, and adrenal glands. The pancreatic tumors are markedly enhanced by ethanol consumption by the mothers. Transplacental carcinogenicity in the hamster has been observed after low doses of NNK administered subcutaneously or intratracheally. NNK is also a transplacental tumorigen in the mouse; its activity in the mouse is weaker than in the hamster (27,28). NNK is a well-established pulmonary carcinogen in adult rats, mice, and hamsters and is likely to play an important role in lung cancer induction in smokers (29). A major metabolite of NNK in rodents and humans is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) that has carcinogenic activity similar to that of NNK (29). In this study, we analyzed first urine from newborns for NNAL and its glucuronide, NNAL-Gluc. These metabolites were chosen because previous studies (29,30) demonstrated their presence, but not the presence of NNK, in the urine from adult smokers.


    SUBJECTS AND METHODS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Pregnant women who agreed to participate in the study were patients at the Klinik für Kinder- und Jugendmedizin, Stadtisches Klinikum, Fulda, Germany. The investigation was approved by the Committee on Ethics in Medical Research of Phillips University, Marburg, Germany. Written informed consent was obtained from each study participant or their guardian before enrollment. Mothers of newborns answered questions about smoking during pregnancy. The smokers smoked an average of 12.4 (95% confidence interval [CI] = 9.7-15.1) cigarettes per day until the day of delivery. All newborns were full term. A sterile adhesive plastic bag was applied to the newborns' perineum immediately after birth, and the first urine void was collected. Cases in which this was not possible were excluded from the study. The urine samples (31 from newborns of smoking mothers and 17 from newborns of nonsmoking mothers) were frozen at -20 °C and then shipped to the University of Minnesota Cancer Center, Minneapolis, on dry ice. They were stored at -80 °C until analysis.

Analyses of NNAL, NNAL-Gluc, Nicotine, and Cotinine

All analyses were carried out without knowledge of whether the urine samples were from newborns of smoking or nonsmoking mothers.

NNAL and NNAL-Gluc. These analyses were performed essentially as previously described (30,31). Urine (4-9 mL) was thawed and added to a 50-mL centrifuge tube. The volume was adjusted to 10 mL with H2O. The pH was adjusted to 7. The mixture was extracted three times with ethyl acetate, and the extracts were combined in a 50-mL centrifuge tube. One nanogram of 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) was added as an internal standard, and the ethyl acetate extracts were dried with Na2SO4. The extracts, containing free NNAL, were then transferred to another 50-mL centrifuge tube and concentrated to dryness.

The aqueous layer from above was concentrated to 70% of its initial volume. Then, 10 000 U of ß-glucuronidase (type IX-A from Escherichia coli; Sigma Chemical Co., St. Louis, MO) and 1 ng iso-NNAL were added. The solution was incubated at 37 °C overnight with shaking. The solution was extracted three times with CH2Cl2, and the extracts were combined in a 50-mL centrifuge tube and dried (Na2SO4). The extracts were transferred to a clean 50-mL tube and concentrated to dryness.

The ethyl acetate and CH2Cl2 layers containing free NNAL and NNAL released from its glucuronide, respectively, were purified further by high-performance liquid chromatography as described previously (30). The collected fractions were concentrated to dryness, silylated, and analyzed by capillary gas chromatography with nitrosamine-selective detection, as described previously (31). Some samples were also analyzed by gas chromatography-tandem mass spectrometry to confirm the identity of the NNAL-trimethylsilyl ether (31).

Nicotine and cotinine. These analyses were carried out by use of a method similar to that described previously (32,33). The analyses were performed in the Analytical Chemistry and Biomarkers Facility of the University of Minnesota Cancer Center. [CD3]Cotinine (500 ng) and [CD3]nicotine (50 ng) (Sigma Chemical Co.) were used as internal standards. They were added to 0.2 mL of urine, and the volume was adjusted to 1 mL. The resulting solution was mixed with 1 mL of 50% aqueous K2CO3 and then extracted once with 2 mL of CH2Cl2. The CH2Cl2 extract was separated and mixed with 0.2 mL of methanol. The solution was concentrated under a stream of N2 to a total volume of 0.1-0.2 mL of methanol and then analyzed by gas chromatography-mass spectrometry with selective ion monitoring.

Statistical Analyses

Birth weights were compared by use of the two-sided Student's t test. To compare NNAL plus NNAL-Gluc with cotinine or nicotine, we added the individual NNAL and NNAL-Gluc values. These values were treated as missing data if either was not quantified. For samples in which analytes were not detected, one half of the detection limit was used. Detection limits are given in Table 1, A,Go footnote {ddagger}. Since a substantial number of subjects had values below the detection limits, the distributions of the data were highly skewed to the left. Therefore, (a) CIs were formed on a log scale, then end points were exponentiated, and means in Table 1Go, B, are geometric; (b) comparisons were performed by use of the Wilcoxon test, a nonparametric procedure, and all reported P values are two-sided; and (c) correlations were investigated by use of Kendall's tau (coefficient of correlation is denoted by r, with P values being two-sided). In summary, all reported P values were derived from two-sided statistical tests.


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Table 1, A. Levels of NNAL-Gluc, NNAL, cotinine, and nicotine in the first urine from newborns* 1, B. Statistical comparisons between newborns of mothers who smoked and of mothers who did not smoke

 

    RESULTS
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Mean gestational age was 38.5 (95% CI = 38.0-39.0) weeks (range, 36-41 weeks) in the smoking group (n = 31) and 39.0 (95% CI = 38.3-39.7) weeks (range, 36-41 weeks) in the nonsmoking group (n = 17). Mean birth weights were 2838 (95% CI = 2633-3043) g (range, 1640-4100 g) in the smoking group and 3441 (95% CI = 3079-3803) g (range, 2050-5380 g) in the nonsmoking group. The difference in birth weights was statistically significant (P<.01).

The analysis of NNAL and NNAL-Gluc in the urine from newborns was carried out by gas chromatography with nitrosamine-selective detection. A representative chromatogram from the urine from a newborn of a smoking mother is illustrated in Fig. 1.Go The indicated peaks are the internal standard iso-NNAL-TMS and the analyte NNAL-TMS. NNAL-TMS is the trimethylsilyl ether of NNAL. NNAL was released from NNAL-Gluc by hydrolysis with ß-glucuronidase. Eight samples, five from newborns of smoking mothers, were also analyzed by gas chromatography-tandem mass spectrometry. Selected reaction monitoring was performed for mass-to-charge ratio (m/z) 282->132 and m/z 282->162. An example of this analysis is presented in Fig. 2,Go A and B. Fig. 2Go, A, shows the presence of internal standard and NNAL-TMS in urine extracts from a newborn of a smoking mother; Fig. 2Go, B, shows the same analysis of urine from the newborn of a nonsmoker. These results clearly demonstrate the presence of NNAL-TMS, released from NNAL-Gluc, only in the urine from the newborn of the smoking mother.



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Fig. 1. Analysis for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc) in the urine of newborns was carried out by gas chromatography with detection by a Thermal Energy Analyzer (TEA), a nitrosamine-selective detector. A representative chromatogram from the urine from a newborn of a smoking mother is illustrated. The indicated peaks are iso-NNAL-TMS, the trimethylsilyl ether of the internal standard iso-NNAL, and NNAL-TMS, the trimethylsilyl ether of NNAL. NNAL was released from NNAL-Gluc by hydrolysis with ß-glucuronidase. The chart speed was changed between 20 and 25 minutes.

 


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Fig. 2. Urine samples analyzed by gas chromatography-tandem mass spectrometry. Representative chromatograms of the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)-glucuronide fractions from the urine from newborns born of mothers who smoked (A) or did not smoke (B) are shown here. The indicated peaks are iso-NNAL-TMS, the trimethylsilyl ether of the internal standard iso-NNAL, and NNAL-TMS, the trimethylsilyl ether of NNAL. NNAL was released from NNAL-glucuronide by hydrolysis with glucuronidase. Upper panels of A and B show selected reaction monitoring of mass-to-charge ratio (m/z) 282->132. Lower panels of A and B show selected reaction monitoring of m/z 282->162. E+04 7.068 = 7.068 x 104, etc.

 
When the code for the study was broken, the results showed that NNAL-Gluc and in some cases NNAL were detected in 22 (71%) of 31 of the urine samples from newborns of smoking mothers but not in any of the 17 samples from newborns of nonsmoking mothers. The results are summarized in Table 1GoGo, A and B. Three of the 22 samples that were positive for NNAL-Gluc could not be quantified because of poor chromatography of the internal standard; free NNAL could not be quantified in one sample because of an interfering peak (Table 1Go, A). The arithmetic mean of NNAL plus NNAL-Gluc levels in the 27 newborns of smokers in which both analytes were quantified was 0.14 (95% CI = 0.083-0.200) pmol/mL. Levels of NNAL plus NNAL-Gluc in the urine from these babies were statistically significantly greater than those in the urine from newborns of nonsmoking mothers (P<.001; Table 1Go, B). Levels of NNAL plus NNAL-Gluc in the 18 positive samples in which they were both quantified ranged from 0.045 to 0.40 pmol/mL, with an arithmetic mean of 0.20 (95% CI = 0.14-0.26) pmol/mL.

Cotinine was detected in 28 (90%) of 31 urine samples from newborns of smoking mothers. Its levels ranged from not detectable to 2.9 nmol/mL, with an arithmetic mean of 0.87 (95% CI = 0.60-1.1) nmol/mL (n = 31). Cotinine levels in the urine from newborns of nonsmokers ranged from not detectable to 0.19 nmol/mL, with an arithmetic mean of 0.049 (95% CI = 0.023-0.075) nmol/mL (n = 17). The difference between smoke-exposed and nonexposed newborns was statistically significant, P<.001 (Table 1Go, B). Nicotine was detected in 18 (58%) of 31 urine samples from newborns of smoking mothers. Its levels ranged from not detectable to 3.7 nmol/mL, with an arithmetic mean of 0.63 (95% CI = 0.30-0.96) nmol/mL (n = 31). Nicotine was not detected in the urine from newborns of nonsmokers. The difference between smoke-exposed and nonexposed newborns was statistically significant, P<.001 (Table 1Go, B).

Levels of NNAL plus NNAL-Gluc correlated with levels of cotinine (r = .56; P<.001) and nicotine (r = .68; P<.001) when all samples from newborns of smoking mothers in which these analytes were quantified were compared as well as with the number of cigarettes smoked per day in the smokers (r = .65; P<.001).


    DISCUSSION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Our results clearly demonstrate the presence of two NNK metabolites, NNAL and NNAL-Gluc, in the urine from newborns of mothers who smoked cigarettes. These metabolites were not detected in the urine from newborns of nonsmoking mothers. To our knowledge, this is the first report of fetal uptake of a transplacental cigarette smoke carcinogen and/or its metabolites, specifically in smokers. 4-Aminobiphenyl, the only other cigarette smoke carcinogen for which there is clear evidence of fetal uptake due to the mothers' smoking, is not known to be a transplacental carcinogen (34,35). Our results support the hypothesis that in utero exposure to tobacco smoke could be carcinogenic later in life.

There are two ways in which NNAL and NNAL-Gluc could appear in the urine of newborns: 1) uptake of NNK or NNAL by the fetus followed by fetal metabolism of NNK to NNAL and NNAL to NNAL-Gluc or 2) metabolism of NNK to NNAL and of NNAL to NNAL-Gluc by the mother followed by transfer of these metabolites to the fetus. The available data favor the first pathway. Animal experiments (24-28,36,37) demonstrate that NNK and/or NNAL cross the placenta and are taken up by the fetus. When pregnant Syrian golden hamsters are treated with NNK, only small amounts of NNK are detected in fetal lung and placenta; concentrations of NNAL are about 20 times higher than those of NNK in these tissues (37). This observation suggests that the transplacental carcinogenicity of NNK in the hamster is due to NNAL, which would be consistent with studies in adult animals in which NNK and NNAL show similar tumorigenic activity (29). To our knowledge, there is no published information on the fetal metabolism of NNAL to NNAL-Gluc, but some human uridinediphosphoglucuronosyl transferases are present in fetal liver (38). It is not clear at present which form glucuronidates NNAL. With respect to the second pathway, metabolism of NNK to both NNAL and NNAL-Gluc occurs in the mother, on the basis of published studies in which they have both been quantified in smokers' urine (30,39,40). The mean ratio ± standard deviation of NNAL-Gluc to NNAL is 3.7 ± 2.2 in the urine of adult smokers (39). Placental transfer of NNAL-Gluc is less likely than that of NNAL, by analogy to studies on retinoic acid (41,42). If the sources of NNAL and NNAL-Gluc in the newborns were exclusively transfer from the mother, then we would have expected lower ratios of NNAL-Gluc to NNAL than were observed, since transfer of NNAL-Gluc would be less likely than transfer of NNAL. In fact, most urine samples from newborns of smoking mothers contained detectable levels of NNAL-Gluc only and, in the samples that contained both NNAL-Gluc and NNAL, the mean ratio ± standard deviation of NNAL-Gluc to NNAL was 3.5 ± 1.3, similar to that observed in adults. Collectively, the available data favor the view that the origin of NNAL and NNAL-Gluc in the urine from newborns is fetal uptake of NNK and/or NNAL followed by fetal metabolism of NNAL to NNAL-Gluc.

Previous studies (43,44) have quantified cotinine levels in the urine from newborns but not by the specific gas chromatography-mass spectrometry method used here. Our data for cotinine, mean 540 ng cotinine/mg creatinine (n = 31), were comparable to those reported by Etzel et al. (43), 1233 ng cotinine/mg creatinine, when one considers that their study was performed by radioimmunoassay and therefore would detect cotinine and some of its metabolites. Although earlier studies (45-47) have examined transplacental nicotine exposure, there do not seem to have been any previous measurements of nicotine in the urine from newborns. Levels of cotinine and nicotine measured here demonstrate and confirm relatively large fetal exposure to these compounds. Comparative levels of NNAL plus NNAL-Gluc, cotinine, and nicotine in the urine from smokers, nonsmokers exposed to environmental tobacco smoke, and newborns are summarized in Table 2.Go Levels of these compounds in the urine from newborns are 5%-10% of those observed in the urine from smokers and are greater than those detected in the urine from people exposed to environmental tobacco smoke. Exposure of the developing fetus to such substantial quantities of these compounds could have toxicologic consequences.


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Table 2. Comparative levels of NNAL plus NNAL-Gluc, cotinine, and nicotine in urine*

 
Levels of NNAL plus NNAL-Gluc correlated with cotinine levels in the urine from newborns. These results agree with those of several previous studies of urine from smokers, snuff users, and nonsmokers exposed to environmental tobacco smoke (30,31,39,40,49). Collectively, the results are consistent with the view that urinary NNAL plus NNAL-Gluc is a good biomarker for NNK uptake, just as urinary cotinine is a good biomarker for nicotine uptake (29).

It has been hypothesized that individuals transplacentally exposed to maternal smoking may be at increased risk of developing cancer in adult life (50). However, epidemiologic studies of transplacental exposure to cigarette smoke and cancer during childhood or later in life (51,52) have produced mixed results and are generally inconclusive. Difficulties in the conduct of such studies, including problems of distinguishing between preconceptional, prenatal, and postnatal effects of tobacco smoke exposure, have been discussed (51). Despite these difficulties, the available evidence, including the evidence from our study, indicates that transplacental induction of cancer by cigarette smoke is biologically plausible (51). There may be differences in the abilities of individual fetuses to detoxify NNAL by glucuronidation or to metabolically activate this carcinogen; these factors could affect risk for cancer development, but they have not been taken into account in previous studies.

The results reported here demonstrate that uptake of NNK or its metabolites by nonsmokers begins before birth. Most women who smoke during pregnancy will continue smoking after the birth of their child, and additional exposures to tobacco smoke carcinogens will occur (53). The clinical consequences to the newborn from transplacental exposure to these carcinogens are unknown, but the exposure raises some concern.


    NOTES
 
Supported by Public Health Service grant CA44377 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services.

We thank Sharon E. Murphy and Andrew Miller for the nicotine and cotinine analyses and Chap Le, head of the Biostatistics Core facility, University of Minnesota Cancer Center, for statistical consultation.


    REFERENCES
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received August 14, 1998; revised December 14, 1998; accepted December 31, 1998.


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