Introduction

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Fetal effects of exposure to cigarette smoke include decreases in human fetal birth weight, intrauterine growth retardation, premature delivery, perinatal mortality, spontaneous abortion and fetal malformations including cleft palate (Khoury et al., 1987, 1989). Exposure of children to cigarette smoke is also associated with increased rates of sudden infant death syndrome, respiratory illness, asthma and middle ear effusion (Law and Hackshaw, 1996; American Academy of Pediatrics, 1997).

At least 43 of the 3800 chemicals found in cigarette smoke, including NNK, are carcinogenic in experimental animals (Hecht, 1996). NNK is the most potent carcinogen of at least seven identified tobacco-specific nitrosamines, and is present at high levels in commercial tobacco products (Hoffmann and Hecht, 1985).

Although NNK-initiated carcinogenesis is well documented, it is still unknown if in utero exposure to NNK is teratologically important. Studies have shown that NNK can cross the mouse and hamster placenta, and can initiate various tumors in fetuses born to NNK-treated dams (Anderson et al., 1989; Correa et al., 1990). In humans, there is an increase in DNA-carcinogen adducts in the placenta from women who smoked during their pregnancy compared to nonsmokers (Everson et al., 1988). Furthermore, maternal administration of radiolabeled NNK in mice results in covalent binding to gestational day-18 fetal tissues (Castonguay et al., 1984), and NNK initiates both micronucleus formation in fetal hamster liver (Alaoui-Jamali et al., 1989) and DNA oxidation in fetal mouse tissues (Sipowicz et al., 1997), indicating placental transfer and bioactivation of NNK by maternal and/or fetal tissues.

NNK, a derivative of the N-nitrosation of nicotine, can undergo reversible carbonyl reduction leading to the formation of two enantiomers of the N-nitroso alcohol, NNAL (fig. 1). P450-catalysed -hydroxylation of either NNK or NNAL at either the -methyl or the -methylene carbons can lead to the formation of reactive intermediates capable of methylating DNA (O⁶-methyldeoxyguanine, 7-methyldeoxyguanine, O⁴-methyldeoxythymidine) or pyridyloxobutylating DNA, respectively (Hecht, 1996).

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Postulated metabolic pathway mediating the toxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Both NNK and NNAL can be -hydroxylated by various cytochromes P450 (P450s) at either the -methyl or the -methylene carbons, producing reactive intermediates that can methylate or pyridyloxobutylate DNA. NNK can also initiate DNA oxidation, possibly via the formation of ROS, but the bioactivating enzymes have not been characterized.

Although NNK can form an electrophilic reactive intermediate, recent studies have shown that NNK also may initiate the formation of ROS. Radical "scavengers" reduced the amount of NNK-initiated DNA single strand breaks in cultured human lung cells, suggesting that at least part of the genotoxicity of NNK was ROS-mediated (Weitberg and Corvese, 1993). Similarly, Xu et al. (1992) showed that multiple dosing of NNK increased DNA oxidation in mouse lung, which was reduced with concomitant administration of green tea that contains an antioxidant [polyphenol, ()-epigallocatechin gallate]. Similarly, in rat skin fibroblasts, NNK-initiated micronucleus formation was inhibited by the antioxidative enzyme superoxide dismutase (Kim and Wells, 1996).

The results of the above studies support the hypothesis that, in addition to the toxicity initiated by electrophiles, alternative ROS-initiated DNA damage may contribute to NNK toxicity. It is unclear at this point whether P450s and/or other enzymes, such as peroxidases, bioactivate NNK to reactive intermediates capable of producing ROS. Peroxidases, such as PHS are known to bioactivate various xenobiotics to free radical intermediates, leading to ROS formation (Marnett, 1990).

Ras proteins are involved in signal transduction controlling cell growth and differentiation, and are expressed at relatively high levels throughout all stages of development where a high degree of cellular differentiation is occurring (organogenesis) (Slamon and Cline, 1984; Barbacid, 1987). Ras oncogenes have acquired specific point mutations that code for Ras proteins that are constitutively active, and they are the most prevalent oncogenes detected in human cancers (Barbacid, 1987; Bos, 1989). Mutations in codon 12 of the K-ras gene have been detected in NNK-initiated lung tumors in mice (Belinsky et al., 1989; Ronai et al., 1993). Because embryogenesis, like tumorigenesis, is a process whereby cells proliferate extensively, but in a tightly controlled, tissue-specific fashion, oncogenes such as ras may play a role in ROS-mediated chemical teratogenesis. Evidence supporting a role for Ras in teratogenesis includes studies showing developmental abnormalities both in Drosophilia expressing an activated form of ras (Bishop and Corces, 1988), and in transgenic mice expressing activated H-ras (Quaife et al., 1987). Also, transgenic mice homozygous deficient for K-Ras function die early in gestation (Johnson et al., 1997). Finally, we have shown that the anticonvulsant drug phenytoin causes an increase in the amount of embryonic active GTP-bound Ras, and that phenytoin-initiated embryotoxicity can be completely blocked by an inhibitor of Ras activity (Winn and Wells, 1997, 1998). Accordingly, given that NNK causes ras mutations, which are known to mediate carcinogenesis, and that the underlying mechanisms mediating carcinogenesis and teratogenesis may be similar, an evaluation of NNK-initiated ras mutations in embryos is of considerable mechanistic interest.

The objective of this study was to determine the teratologic and embryotoxic consequences of NNK exposure in CD-1 mice, using in vivo and embryo culture approaches to assess both maternal and embryonic contributions. The teratological role of P450-catalysed biotransformation was evaluated in vivo by maternal pretreatment with the P450 inducer phenobarbital. PCR and PIREMA techniques were used to evaluate whether mutations in embryonic ras may mediate the teratogenicity of NNK.

	Methods

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Animals

Virgin female CD-1 mice (Charles River Canada Inc., St. Constant, Quebec) weighing 20 to 25 g were housed in plastic cages with ground corn cob bedding (Beta Chip, Northeastern Products Corp., Warrensburg, NY). Animals were kept in a temperature-controlled room with a 12-hr light-dark cycle automatically maintained. Food (Purina Rodent Chow, Ren's Feed and Supply, Oakville, Ontario, Canada) and tap water were provided ad libitum. One male mouse was housed with three females overnight between 1700 and 0900 hr. Pregnancy was ascertained the next morning by the presence of a vaginal plug, and this time was designated as GD 1.

Chemicals

NNK was purchased from Chemsyn Science Laboratories (Lenexa, KS). Phenobarbital was purchased from British Drug Houses (Toronto, Ontario, Canada). Hanks' balanced salt solution, Waymouth's MB 752/1, fetal bovine serum, sodium bicarbonate solution, HEPES [4(2-hydroxyethyl)-1-piperazine ethane sulfonic acid], L-glutamine and penicillin-streptomycin came from Gibco BRL (Toronto, Ontario, Canada). Male rat serum was obtained from retired CD-1 male breeder rats (Charles River) as described elsewhere (Winn and Wells, 1995). Amplitaq, MgCl₂ and the PCR buffer were obtained from Perkin Elmer Canada Ltd. (Mississauga, Ontario).

BstN1 was purchased from New England BioLabs (Mississauga, Ontario, Canada). Primers were synthesized and obtained from the Hospital for Sick Children Biotechnology Servive Centre (Toronto, Ontario, Canada). All other reagents used were of analytical grade.

In Vivo Study

NNK dosing. NNK, dissolved in 0.9% saline, was administered to pregnant CD-1 dams as a single i.p. dose of 100 mg/kg in an injection volume of 0.01 ml/g on 2 consecutive days, either GDs 10 and 11, 11 and 12 or 12 and 13, to determine the period of maximal susceptibility. In the P450 induction studies, phenobarbital dissolved in saline (60 mg/kg, i.p.) was administered on GDs 8, 9 and 10 followed by treatment with NNK (100 mg/kg, i.p.) on GDs 11 and 12.

Teratological assessment. Dams were observed for at least 1 hr after NNK administration to assess any overt signs of toxicity. On gestational day 19, 1 day before spontaneous delivery, dams were killed by cervical dislocation. After laparotomy, the uterus was exteriorized and the number and location of fetuses and resorptions were noted. Fetuses were then weighed and monitored under a heat lamp for 2 hr to detect postpartum deaths and then fixed in Carnoy's solution. At least 2 days after fixing fetuses were examined for cleft palates, ectopic kidneys and hydrocephalus.

Embryo Culture Study

Pregnant CD-1 dams were killed on GD 9.5 by cervical dislocation and embryos were explanted according to the method of New (1978). Explanted embryos were kept at 37°C in a holding bottle which contained pregassed (5% CO₂ in air, Cannox Canada) "holding medium" (50 ml Waymouth's MB 752/1, 14 mM NaHCO₃, 2.5 mM HEPES, 1.0 mM L-glutamine and 17 ml male rat serum) until all embryos from all dams were explanted. Embryos at a similar stage of development (four to six somite pairs) were pooled and cultured in 25-cm² sterile cell culture flasks (Corning Glasswork Inc., Corning, NY) that contained 10 ml of CO₂ saturated embryo culture media [50 ml holding-media, penicillin (50 U/ml) and streptomycin (50 mg/ml)]. Flasks were incubated at 37°C (Forma Scientific, Toronto, Ontario, Canada) on a platform rocker (Bellco Biotechnology, Vineland, NJ).

Embryotoxicity. Embryos were exposed to NNK (10 and 100 µM) or the DMSO vehicle for 24 hr. After the culture period, embryonic morphological and developmental parameters were observed using a dissecting microscope (Carl Zeiss, Oberkochen, Germany) as described elsewhere (Winn and Wells, 1995). Developmental parameters included dorsal-ventral flexure (turning), anterior neuropore closure and somite development. Morphological assessment included yolk sac diameter (mm) and crown-rump length (mm).

K-ras 12 Mutational Analysis. Embryonic DNA was isolated from CD-1 embryos, cultured in the presence or absence of NNK (100 µM) for 24 hr as described above, using a QIAamp tissue kit (Qiagen, CA). Embryonic DNA was then analyzed for any K-ras codon 12 mutations using the polymerase chain reaction-primer introduced restriction with enrichment for mutant alleles (PCR-PIREMA) method described by Mills et al. (1995). This is a highly sensitive assay which detects mutant alleles present at the level of 0.1%. Embryonic DNA was first amplified using PCR with fully matched primers flanking exon 1 of the K-ras gene (model GeneAmp PCR System 9600, Perkin Elmer). The primers used were as follows: 5'-ACT GAG TAT AAA CTT GTG GTG GTT GGA GCT-3' (sense) and 5'-CGG CGT TAC CTC TAT CGT AGG GTC-3' (antisense). The PCR reaction included: 1 µM of each primer, 10 µM of each nucleotide and 1.2 mM MgCl₂ in a 50 µl reaction volume, cycled 25 times at 94°C for 1 min, 55°C for 2 min and 74°C for 3 min. A 5 µl aliquot of this PCR product was then amplified in a second PCR step using a 5'-mismatched sense primer (5'-ACT GAG TAT AAA CTT GTG GTG GTT GGA CCT-3') which introduces a BstN1 restriction site into normal alleles. This PCR reaction contained 1 µM of each primer (5'-mismatched sense and 5'-matched antisense), 4 µM of each nucleotide and 0.6 mM MgCl₂ in a 50 µl reaction volume and was cycled 25 times at 94°C for 1 min, 40°C for 2 min and 74°C for 3 min. A 2.5-µl aliquot of this PCR step was then digested overnight with BstN1 in a final volume of 10 µl. The second PCR step was then repeated on a 5-µl aliquot of the digestion product followed by overnight digestion with BstN1. A final PCR reaction was then carried out using a 5-µl aliquot of the digestion product, the 5'-mismatch primer, 120 µM of each nucleotide and 1.25 mM MgCl₂ in a 50 µl reaction volume and was cycled 40 times at 94°C for 1 min, 55°C for 2 min and 74°C for 3 min. After overnight digestion with BstN1 the PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide.

Statistical Analysis

Statistical significance between treatment groups in each study was determined using a standard, computerized statistical program (Statsview, Abacus Concepts, Inc., Berkeley, CA). Groups were compared using a one factor analysis of variance. Binomial data were examined using the [Chi]² test or the Fisher's exact test. The minimum level of significance used throughout was P < .05.

	Results

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In vivo Study. To our knowledge, there are no previous studies that have investigated the teratological effects of NNK, therefore we examined fetuses for external and internal morphological anomalies, including skeletal defects. All pregnant CD-1 dams treated with 100 mg/kg of NNK with or without phenobarbital induction survived to GD 19 and showed no obvious signs of toxicity, nor did the body weights of pregnant dams differ between the groups (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   In vivo teratogenicity of NNK in CD-1 mice. NNK (100 mg/kg i.p.) was given to pregnant dams on 2 consecutive gestational days (GDs) at three different times during organogenesis: either GDs 10 and 11, 11 and 12 or 12 and 13. The number of fetuses or implantations is given in parentheses.

View larger version (38K): [in this window] [in a new window]   Fig. 3.   In vivo effect of phenobarbital pretreatment on NNK teratogenicity in CD-1 mice. Pregnant dams were pretreated with phenobarbital (60 mg/kg i.p.) on GDs 8, 9 and 10, followed by treatment with NNK (100 mg/kg i.p.) on GDs 11 and 12. The number of fetuses or implantations is given in parentheses. *Difference from phenobarbital alone control group (P < .05). Difference from NNK alone group (P < .05). +Difference from combined saline and phenobarbital alone controls (P = .0083), but not from the phenobarbital alone group (P = .091).

Embryo culture study. The low NNK concentration (10 µM) significantly reduced yolk sac diameter, crown rump length and somite development, but had no effect on anterior neuropore closure or turning (P < .05) (fig. 4). The higher NNK concentration (100 µM) significantly decreased anterior neuropore closure and crown-rump length but did not decrease turning, yolk sac diameter or somite development (P < .05) (fig. 4).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   In vitro embryotoxicity of NNK in embryo culture. Day 9.5 embryos were incubated for 24 hr at 37°C in the presence of NNK (10 or 100 µM) or its DMSO vehicle. The number of embryos is given in parentheses. *Difference from DMSO control embryos (P < .05).

K-ras mutational analysis. DNA from embryos cultured with NNK (100 µM) analyzed for K-ras 12 mutations using the PCR-PIREMA method did not show any mutations (fig. 5). This technique is based on the introduction of a new restriction site into normal alleles, therefore a nondigested band at 118 bp should indicate the presence of any mutation, whereas a digested band at 89 bp would indicate that there was no mutation present. The positive control sample with the known mutation produced one strong nondigested band (118 bp), whereas the normal tumor samples, the embryonic DMSO and the embryonic NNK samples all produced two bands (118 and 89) with similar intensity, indicating that NNK did not cause any K-ras codon 12 mutation. The lack of complete digestion in normals is due to the high misincorporation rate of the taq polymerase (Mills et al., 1995).

View larger version (77K): [in this window] [in a new window]   Fig. 5.   Mutational analysis of codon 12 of the K-ras gene using the PCR-PIREMA technique. Tumor controls include DNA isolated from murine lung with a normal sequence (lanes 2 and 3, negative controls) and a known TGT mutation (lane 4, positive control). DNA also was isolated from CD-1 embryos exposed to DMSO vehicle (controls) (lanes 2-5) and NNK (100 µM) (lanes 2-7). Lane 1 in each case shows the base-pair ladder.

	Discussion

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Although previous studies have shown that NNK can initiate transplacental carcinogenesis (Anderson et al., 1989; Correa et al., 1990), no studies evaluating the potential teratogenic or embryotoxic effects of NNK have been conducted. We evaluated the effect of maternal administration of NNK during embryonic organogenesis to determine whether NNK was teratogenic. In transplacental carcinogenesis studies, NNK was administered to pregnant dams late in gestation, after organogenesis, when there is substantially higher activity of most embryonic P450s, the enzyme superfamily that, at least in adults, is thought to be primarily responsible for NNK bioactivation (Hecht, 1996) (fig. 1).

The results from our embryo culture studies provide the first evidence that NNK is embryotoxic, and suggests that the embryo itself can bioactivate NNK to a reactive intermediate. Although NNK toxicity in adult mice is known to be mediated by P450-catalysed bioactivation to electrophilic reactive intermediates capable of methylating or pyridyloxobutylating DNA (fig. 1) (Hecht, 1996), the enzymes involved in embryonic bioactivation of NNK remain unknown. Expression of most P450s in rodent embryonic tissue during organogenesis is thought to be low to negligible (Juchau et al., 1992; Raucy and Carpenter, 1993), and whether or not this activity is involved in embryonic bioactivation remains to be determined. However, there are some P450 isozymes, including CYP1B1, which are expressed at high levels in rodent embryonic tissues (Savas et al., 1994; Walker et al., 1995), although it is not known whether NNK or its metabolites are substrates for these P450 isozymes.

Peroxidase-catalysed bioactivation of NNK leading to the formation of ROS also may be involved in the embryonic bioactivation and embryotoxicity of NNK. NNK-initiated micronucleus formation in rat skin fibroblasts is blocked by the dual P450/peroxidase inhibitor 1-aminobenzotriazole, and by the dual PHS/lipoxygenase inhibitor eicosatetraynoic acid (Kim and Wells, 1996). Given the low P450 activity in such cultured cells, the protection against NNK genotoxicity afforded by these peroxidase inhibitors suggests an important role for peroxidases such as PHS in NNK bioactivation. It has recently been postulated that NNK can initiate the formation of ROS (Xu et al., 1992; Weitberg and Corvese, 1993; Kim and Wells, 1996), and it has been shown that the antioxidative enzyme SOD can prevent NNK-initiated micronucleus formation (Kim and Wells, 1996). ROS can damage essential cellular macromolecules including DNA, which may be a critical determinant in chemically initiated teratogenesis since mice deficient in DNA repair are more susceptible to the teratogenicity of both benzo[a]pyrene and phenytoin (Nicol et al., 1995; Laposa and Wells, 1995).

Several studies have shown that NNK-initiated lung tumors in mice contain K-ras 12 mutations (Belinsky et al., 1989; Ronai et al., 1993), which are thought to occur from the direct methylation of DNA resulting in a G to A transition mutation. In humans, 16% of lung tumors and 24% of adenocarcinomas have been shown to have mutated K-ras genes (Rodenhuis and Slebos, 1992). We did not see any mutations in the K-ras 12 codon in any of the NNK-treated embryos, which suggests that the mechanism of NNK-initiated embryotoxicity may be different from that for NNK-initiated carcinogenesis.

Although our embryo culture studies showed that NNK can initiate embryotoxicity, our in vivo results using NNK alone suggest that NNK is not a potent teratogen, at least with respect to structural defects. NNK can produce tumors in mice and rats at doses as low as 5 mg/kg (Hecht et al., 1988; Prokopczyk et al., 1991). When administered late in gestation, a 100-mg/kg dose of NNK has been shown to be a weak transplacental carcinogen in pregnant mice (Anderson et al., 1989) and a more potent transplacental carcinogen in hamsters at doses as low as 1 mg/kg (Correa et al., 1990). In our studies, NNK alone at a dose of 100 mg/kg was not significantly teratogenic, although it did cause one cleft palate and three open eye defects, neither of which were observed in any controls. In our experience, open eye and cleft palate are rare in the CD-1 mouse, therefore we suspect that given a larger control group, these anomalies likely would prove to be statistically associated with NNK, which is consistent with the two additional fetuses with cleft palate in the group treated with both phenobarbital and NNK, and the statistically significant association in humans of cleft palate with smoking (Khoury et al., 1989). The observation that NNK was embryotoxic in embryo culture, but not significantly teratogenic in vivo, suggests that in vivo, maternal elimination of NNK and its metabolites via glucuronidation may protect the fetus from exposure to high levels of NNK and its metabolite. This may be particularly relevant in humans, where it is known that 2 to 12% of the population have deficiencies in UGTs (Monaghan et al., 1996) and, unlike in rodents, the production and subsequent glucuronidation of NNAL (carbonyl reduced form of NNK) is extensive (Morse et al., 1990; Carmella et al., 1993) (fig. 1). In rat skin fibroblasts, NNK-initiated micronucleus formation is enhanced in UGT-deficient cells (Kim and Wells, 1996).

Because our in vivo study found that NNK alone was not significantly teratogenic, we evaluated whether or not pretreatment with phenobarbital could enhance the teratogenicity of NNK. Many of the other compounds present in tobacco smoke, or exposure to other drugs/xenobiotics such as alcohol, which often is concomitant, could potentially modulate enzymes implicated in NNK bioactivation and/or detoxification in the placenta (Manchester and Jacoby, 1982), or possibly the embryo. In mice, CYP2B1 and CYP2B2 have been shown to catalyze the -hydroylation of NNK, and are the two major P450 isozymes inducible by phenobarbital pretreatment (Thomas et al., 1983; Hecht, 1996). Although phenobarbital pretreatment has been shown to induce fetal rat P450s (Cresteil et al., 1986), CYP2B in mouse fetal liver was not induced (Chianale et al., 1988). Phenobarbital pretreatment in pregnant mice decreased the teratogenicity of both phenytoin (Harbison and Becker, 1970) and cyclophosphamide (Gibson and Becker, 1968), and conversely increased that of carbamazepine (Finnell et al., 1995). However, at least the inhibitory effects may have been due in part to induction of maternal metabolism that reduced the amount of drug reaching the fetus, rather than alterations in fetal metabolism (Wells and Winn, 1996).

We found that pretreatment of dams with phenobarbital caused a significant increase in NNK-initiated postpartum lethality, although mean fetal weight or fetal resorptions were unaffected (fig. 3). An increase in postpartum lethality may be potentially relevant in humans given that exposure to tobacco smoke has been associated with an increased incidence of sudden infant death syndrome (Blair et al., 1996; MacDorman et al., 1997).

When compared to the combined controls treated with phenobarbital alone and with saline, phenobarbital pretreatment also enhanced the incidence of fetal anomalies initiated by NNK, including cleft palate, excencephaly and kinky tail. This not only is consistent with the interpretation above that NNK alone is teratogenic, but also suggests that embryonic P450-catalysed bioactivation may contribute to the teratologic mechanism. An additional possibility includes enhanced maternal P450-catalysed formation of a stable metabolite that can cross the placenta and undergo embryonic bioactivation. Because the apparent increase in anomalies in the group treated with both phenobarbital and NNK was not statistically different when compared only to the phenobarbital alone controls, it is possible that the association was fortuitous; however, for the reasons given above in the discussion of studies with NNK alone, we believe that this teratologic enhancement is biologically significant.

It is important to note that the dose of NNK used in our in vivo study was very high (100 mg/kg), even considering that pregnant women would be exposing their fetus for many months to irreversible macromolecular damage initiated by tobacco-related xenobiotics. If a pregnant woman smoked 20 nonfiltered average cigarettes (425 ng of NNK/cigarette) (Adams et al., 1987) a day for 9 mo and weighed in the range of 50 to 70 kg, she would be exposing herself and her fetus to about 0.04 mg/kg of NNK, which is more than three orders of magnitude lower than the dose used. Even considering that, to provide an equivalent plasma concentration, the dose for a mouse may need to be roughly 10 times that for a human, the dose in our study was about 250 times higher. However, although our results appear to represent the extreme teratological potential for NNK, there can be any number of complicating factors when attempting to extrapolate these results in embryo culture and in vivo in mice to humans.

In summary, although NNK is a highly potent animal carcinogen, our results indicate that, compared to dual carcinogen/teratogens such as BP, NNK is a relatively weak structural teratogen in our mouse models, although this effect was enhanced in vivo by pretreatment with the P450 inducer phenobarbital. This suggests that human fetal anomalies initiated by cigarette smoking may be caused by teratogenic tobacco constituents more potent than NNK, and possibly by NNK with concomitant exposure to drugs and environmental chemicals that enhance NNK teratogenicity. However, complicating factors such as potential synergistic effects of other constituents of tobacco smoke, and substantial differences in rodent and human embryonic bioactivating activities, preclude direct extrapolation of these results. NNK did not cause mutations in codon 12 of the K-ras gene, suggesting teratological mechanisms different from that postulated for carcinogenesis. For a comprehensive understanding of the teratological potential of NNK, further studies are warranted to determine the functional fetal consequences of NNK exposure during the latter half of pregnancy, when brain development is predominant and the activities of P450 isozymes are increasing.