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Nitrosatable drugs, such as secondary or tertiary amines and amides, form N-nitroso compounds in the presence of nitrite. Various N-nitroso compounds have been associated with neural tube defects in animal models. Using data from the National Birth Defects Prevention Study, the authors examined nitrosatable drug exposure 1 month before and 1 month after conception in 1,223 case mothers with neural tube defect-affected pregnancies and 6,807 control mothers who delivered babies without major congenital anomalies from 1997 to 2005. Nitrite intakes were estimated from mothers’ responses to a food frequency questionnaire. After adjustment for maternal race/ethnicity, educational level, and folic acid supplementation, case women were more likely than were control women to have taken tertiary amines (odds ratio = 1.60, 95% confidence interval (CI): 1.31, 1.95). This association was strongest with anencephalic births (odds ratio = 1.96, 95% CI: 1.40, 2.73); odds ratios associated with tertiary amines from the lowest tertile of nitrite intake to the highest tertile were 1.16 (95% CI: 0.59, 2.29), 2.19 (95% CI: 1.25, 3.86), and 2.51 (95% CI: 1.45, 4.37), respectively. Odds ratios for anencephaly with nitrosatable drug exposure were reduced among women who also took daily vitamin supplements that contained vitamin C. Prenatal exposure to nitrosatable drugs may increase the risk of neural tube defects, especially in conjunction with a mother’s higher dietary intake of nitrites, but vitamin C might modulate this association.
Various N-nitroso compounds have been associated with neural tube defects (NTDs) in animal models (1, 2), and DNA alkylation of embryonic cells has been suggested as one of the mechanisms for teratogenicity (1). Extensive experimental evidence indicates that N-nitroso compounds can be formed in vivo by nitrosatable amines or amides reacting with nitrosating agents, such as nitrite, in an acidic environment like that found in the stomach (3). Endogenous N-nitroso compound formation contributes 40%–75% of exposure to such compounds in humans (4), and a variety of drugs contribute nitrosatable amines or amides in the endogenous formation of N-nitrosamines and N-nitrosamides. In experiments with simulated gastric conditions (5–7), the combination of drugs containing secondary or tertiary amines or amides with nitrite have yielded a range of N-nitroso compounds, depending on the chemical structure of the drug.
Few epidemiologic studies have been conducted on the relation between nitrosatable drugs and NTDs. Olshan and Faustman (8) noted an association between exposure to nitrosatable drugs during the first 4 months of pregnancy and spina bifida, whereas Croen et al. (9) did not detect this association with NTDs in their study population. In a study that also took into account dietary nitrate and nitrite intakes, Brender et al. (10) noted a significant positive association between prenatal use of drugs with the potential for nitrosation in Mexican-American women and NTDs in their offspring, especially among offspring of women who also had higher prenatal dietary intakes of nitrites. This finding was consistent with results from an experimental study with mice in which the percentage of NTDs and other defects increased in conjunction with a nitrosatable compound as the dose of nitrite increased (11), indicating that the combined teratogenicity of these compounds might be due to the nitrosation products formed within the stomach.
Previous studies of maternal exposure to nitrosatable drugs and birth defects were based on an incomplete assessment of drugs with respect to their nitrosatability. In their recent review, Brambilla and Martelli (12) identified 182 drugs that had been tested, of which 173 (95%) were found to form N-nitroso compounds or other reactive species. Furthermore, none of the previous studies examined the effects of nitrosatable drugs by their molecular structure (secondary or tertiary amines vs. amides) or examined the potential modulating effect of vitamin C, a well-documented inhibitor of nitrosation (13). Many drugs that are tertiary amines form N-nitrosodimethylamine in the presence of nitrite (12). In animal studies, prenatal administration of acetoxymethyl-methylnitrosamine, which has the same active intermediate metabolite as N-nitrosodimethylamine, was associated with exencephaly and other defects at doses not associated with maternal toxicity (1). In the present study, we examined 1) the relation between prenatal exposure to nitrosatable drugs by their molecular structure (secondary amines, tertiary amines, and amides) in conjunction with dietary intake of nitrites and NTDs in offspring and 2) the effect of supplemental and dietary intake of vitamin C on these associations.
The National Birth Defects Prevention Study (NBDPS), previously described by Yoon et al. (14), is an ongoing population-based case-control study of birth defects in the United States that began in 1997. Ten Centers for Birth Defects Research and Prevention (CBDRP) (in Arkansas, California, Georgia, Iowa, Massachusetts, New York, and Texas (1998 to present); New Jersey (1998 to 2002); and North Carolina and Utah (2003 to present)) have participated or are currently participating in this national study. Case infants are identified from livebirths (all centers), stillbirths (all centers except New Jersey and New York from 1997 to 1999), and elective pregnancy terminations (Arkansas, California, Georgia, Iowa, North Carolina, Texas, and Utah) (15). Case definitions are standardized across centers, and clinical information on potential cases is evaluated by a clinical geneticist at each center (16) and independently reviewed by 1 or more other clinical geneticists. Infants with single-gene or chromosome abnormalities are excluded from the NBDPS (16). For this study on nitrosatable drugs, we included cases with NTDs (anencephaly, craniorachischisis, spina bifida, and encephalocele) and controls who had estimated delivery dates (EDDs) from October 1, 1997, through December 31, 2005.
Controls (livebirths who had no major birth defects and whose mothers resided in the study area at delivery) with EDDs during the same period were randomly selected from live birth certificates (Iowa, Massachusetts, New Jersey, North Carolina, and Utah) or hospital records (California, New York, and Texas) (15). Centers in Arkansas and Georgia initially selected controls from hospital records but switched to live birth certificates exclusively in January of 2001. The institutional review boards in each state and the Centers for Disease Control and Prevention approved the study protocol, and the institutional review boards of Texas A&M University, the University of Iowa, and the Texas Department of State Health Services also approved this project on nitrosatable drugs and birth defects.
In the NBDPS, women are interviewed via telephone by trained interviewers who administer a standard questionnaire after informed consent is obtained. The interview takes approximately 1 hour to complete and covers topics regarding maternal health (including medications being taken), diet (vitamin, food supplement, and food and beverage consumption), home/work (residence and occupation), demographic characteristics, and water use (14). Interviews are targeted for completion within 6 months of delivery, with a maximum time from delivery/termination to interview of no more than 24 months. Time of interview is determined by the EDD, and no women are interviewed until 6 weeks after the EDD (or delivery of a full-term infant).
As part of the interview, women in the NBDPS are questioned about their use of prescription and nonprescription drugs from 3 months before the estimated date of conception to the date of birth of the index pregnancy. In addition to the name of the medication, information is collected regarding the dates that the drug(s) were taken and the frequency of use. Reported drugs are linked to their active ingredients by using the Slone Epidemiology Center Drug Dictionary system (17), which identifies individual ingredients in multiple-component products (18). Methods used to classify drugs with respect to nitrosatability, functional groups, and indications have been published previously (19). Briefly, all orally administered and orally inhaled prescription and nonprescription medications and their active ingredients reported by case and control women were identified. These drugs were cross-referenced against previously compiled lists of nitrosatable medicinal compounds (12, 20) and categorized on the basis of the presence of amine (secondary or tertiary) and amide functional groups. Within its functional group, each component was also categorized by its primary indication or therapeutic use. Complete data on nitrosatable drug use and covariates were available for 1,168 (95.5%) participating case women and 6,553 (96.3%) participating control women. The Web Appendix (available at http://aje.oxfordjournals.org/) contains a list of the drugs that were identified as nitrosatable and reported as taken by NBDPS subjects with EDDs during the period of 1997–2005. For the present study on NTDs, we focused on drugs that women reported taking from 1 month before conception to 1 month after conception.
As part of the NBDPS interview, women are questioned about their average consumption of foods and beverages during the year before becoming pregnant with the index pregnancy through the use of a 58-item food frequency questionnaire adapted from the short Willett food frequency questionnaire (21, 22). Separate, more detailed questions to assess consumption of breakfast cereals cover intake from 3 months before conception through the end of pregnancy. From these 2 sources of information, dietary intakes of nitrates and nitrites in milligrams per day were estimated using procedures described in detail elsewhere (23, 24). Briefly, 1) weighted means for nitrates and nitrites in milligrams per 100 grams were calculated for each food item based on the relevant literature; 2) the respective means were multiplied by the serving size (in grams) assigned to each food; 3) nitrates and nitrites in each serving size were multiplied by the number of servings per month; and 4) nitrate and nitrite levels across all food items were summed and then divided by 30 to obtain milligrams per day of dietary nitrate and nitrite intake. We calculated total dietary nitrite intake with the formula suggested by Choi (25) (total nitrite = dietary nitrite intake + (0.05 × dietary nitrate intake)). Dietary intakes of nitrites and total nitrites were categorized into tertiles based on the control women’s distributions, as was done in a previous study (10). We excluded from the stratified analyses of nitrosatable drugs and dietary nitrites women whose calculated daily kilocalorie intake was less than 500 or greater than 5,000. These lower and upper limits are consistent with other dietary studies (26) and with what has previously been used with the NBDPS population (27, 28). Complete data for any nitrosatable drug use stratified by total nitrite intake were available for 1,132 (92.6%) participating cases and 6,376 (93.7%) participating controls.
Covariate selection was based on factors associated with NTDs in other studies and maternal factors associated with nitrosatable drug exposure (19). Potential confounders that were assessed included maternal race/ethnicity, educational level, and age; study site; body mass index based on self-reported height and weight (in kg/m2); folic acid supplementation around the time of conception; and dietary folate intake (expressed as dietary folate equivalents in quartiles based on the control women’s distribution).
Logistic regression was used to estimate odds ratios and 95% confidence intervals for NTDs in relation to any nitrosatable drug use and for secondary amines, tertiary amines, and amides. Women who did not report taking any drugs classified as nitrosatable around conception served as the reference group in all analyses. Final models for the association between nitrosatable drugs and NTDs included maternal race/ethnicity, educational level, and folic acid use as covariates.
Nitrosatable drug exposure was stratified by tertiles of dietary nitrite and total nitrite intake, and odds ratios and 95% confidence intervals were estimated for NTDs for each stratum, with adjustment for maternal race/ethnicity, educational level, folic acid use, and total energy intake. Multiplicative interaction was assessed by including the product terms of secondary and tertiary amines with dietary nitrite and total nitrite in the logistic models. Additive interaction was examined using the methods discussed by Andersson et al. (29), in which the relative excess risk due to interaction and the attributable proportion due to interaction were calculated with their respective 95% confidence intervals. In the absence of additive interaction, both measures equaled zero.
Nitrosatable drug exposure was also stratified by vitamin C supplementation during the first month of pregnancy and by dietary vitamin C intake (categorized as <85 mg/day or ≥85 mg/day, cutpoints that were based on recommended dietary allowances for pregnant women >18 years of age (30) and that corresponded to the 41st percentile for study participants). The presence of multiplicative and additive interactions was assessed for secondary and tertiary amines with vitamin C supplementation and dietary intake with the same methods used for these drugs as with dietary nitrite/total nitrite.
A total of 1,223 eligible case women with NTD-affected pregnancies (352 with anencephaly or craniorachischisis, 730 with spina bifida, and 141 with encephalocele) and 6,807 control women with an EDD during 1997–2005 participated in the NBDPS. Participation rates for case women with NTD-affected pregnancies and control women were 68% and 66%, respectively, with median time from EDD to interview of 9 months for case women and 8 months for control women. Case women were more likely than were control women to be Hispanic, to be less educated, to have a body mass index of 30 or higher, and to live in California or Texas, but they were slightly less likely than controls to use a folic acid preparation around the time of conception (Table 1).
A higher proportion of case women (19.5%) than of control women (16.9%) reported taking drugs classified as nitrosatable (adjusted odds ratio (AOR) = 1.31, 95% confidence interval (CI): 1.12, 1.55), especially drugs classified as tertiary amines (AOR = 1.60, 95% CI: 1.31, 1.95) (Table 2). Women with anencephalic births were approximately twice as likely as were controls (AOR = 1.96, 95% CI: 1.40, 2.73) to report taking drugs classified as nitrosatable tertiary amines. Use of tertiary amines was less strongly associated with spina bifida (AOR = 1.48, 95% CI: 1.15, 1.91) and encephalocele (AOR = 1.48, 95% CI: 0.83, 2.63). Restriction of analyses to data from study centers that included terminations in case findings did not materially change the adjusted odds ratios for NTDs (AOR = 1.59), anencephaly (AOR = 1.93), or spina bifida (AOR = 1.44) associated with tertiary amine drug exposure.
Overall, NTDs were associated with drugs classified as tertiary amines across a broad range of indications, such as analgesic opioids (odds ratio (OR) = 1.25), antidiabetic drugs (OR = 2.04), antiemetic phenothiazines (OR = 1.34), antiepileptics (OR = 3.48), antihistamines (OR = 1.46), antiinfective macrolides (OR = 2.90), cough medications (OR = 1.24), gastrointestinal histamine 2 blockers (OR = 1.66), and stimulants (OR = 1.17) (data not shown; associations reported are restricted to drugs with at least 5 exposed cases and 5 exposed controls). Exclusion of women who took antidiabetic, antiepileptic, and antibiotic drugs slightly reduced the adjusted odds ratios for NTDs (AOR = 1.45), anencephaly (AOR = 1.83), and spina bifida (AOR = 1.36), but the 95% confidence intervals for these odds ratios excluded 1.0. Antihistamines were the most commonly taken tertiary amine drugs by both case women (5.5%) and control women (3.9%) and were most strongly associated with anencephalic births (AOR = 2.39, 95% CI: 1.56, 3.67).
The strongest associations between anencephaly and nitrosatable secondary and tertiary amines were among women with nitrite and total nitrite levels (sum of dietary nitrite and 5% dietary nitrate intake) in the upper 2 tertiles of estimated intake (Table 3). Notably, the odds ratios were near or below 1.0 for nitrosatable drug use in the lowest tertiles of nitrite and total nitrite intake. Adjusted odds ratios for anencephaly associated with tertiary amines for the lowest tertile to the highest tertile of total nitrite were 1.16 (95% CI: 0.59, 2.29), 2.19 (95% CI: 1.25, 3.86), and 2.51 (95% CI: 1.45, 4.37), respectively (P = 0.053 for multiplicative interaction; relative excess risk due to interaction = 1.095 (95% CI: −0.163, 2.35); and attributable proportion due to interaction = 0.552 (95% CI: 0.105, 0.998)). Exposures to nitrosatable secondary and tertiary amines were also most strongly associated with encephalocele in the highest tertiles of dietary nitrite and total nitrite intake (data not shown). The pattern of increasing odds ratios associated with nitrosatable drug exposure across increasing levels of dietary nitrites was less apparent for spina bifida (Table 4), and no significant multiplicative or additive interaction was noted. Exposure to tertiary amines was most strongly associated with spina bifida among women with an estimated dietary nitrite intake greater than 1.91 mg/day (AOR = 1.72, 95% CI: 1.12, 2.66).
Daily supplementation with preparations containing vitamin C diminished the association between nitrosatable drugs and anencephaly but not between nitrosatable drugs and spina bifida (Table 5). Among women who took a daily supplement with vitamin C, the odds ratio for having an anencephalic birth in association with nitrosatable tertiary amine use was lower (AOR = 1.52, 95% CI: 0.86, 2.71) than the corresponding estimates among women who did not take these supplements on a daily basis (AOR = 2.77, 95% CI: 1.48, 5.17) and among those who did not take the supplements at all (AOR = 2.11, 95% CI: 1.25, 3.57) during the first month of pregnancy (P = 0.510 for multiplicative interaction; relative excess risk due to interaction = 0.750 (95% CI: −0.377, 1.876); and attributable proportion due to interaction = 0.340 (95% CI: −0.106, 0.785)).
Among women whose dietary intake of vitamin C was estimated to be 85 mg/day or more, the adjusted odds ratio for spina bifida in conjunction with tertiary amine drug exposure was 1.15 (95% CI: 0.78, 1.69) compared with 1.81 (95% CI: 1.28, 2.56) among women with a lower dietary intake of this vitamin (data not shown) (P = 0.130 for multiplicative interaction; relative excess risk due to interaction = 0.659 (95% CI: −0.112, 1.430); and attributable proportion due to interaction = 0.337 (95% CI: 0.017, 0.656)). In contrast, tertiary amine drug exposure was more strongly associated with anencephaly in women with dietary vitamin C intakes estimated to be 85 mg/day or more (AOR = 2.69, 95% CI: 1.76, 4.12) than in women with intakes less than 85 mg/day (AOR = 1.15, 95% CI: 0.66, 2.02).
In the present study, a large, population-based case-control study of nitrosatable drugs and birth defects, women who had NTD-affected pregnancies were more likely than were control women to have taken drugs classified as nitrosatable during the period 1 month before conception to 1 month after conception. To our knowledge, this birth defects study is the first in which nitrosatable drugs have been examined specifically by molecular structure. We found that tertiary amines were more strongly associated with NTDs, particularly anencephaly, than were secondary amines or amides. Furthermore, the strongest associations between nitrosatable drug exposure and NTDs were noted among women with higher estimated intakes of dietary nitrites. In contrast, nitrosatable drug exposures showed little to no association with NTDs among women in the lowest tertile of dietary nitrite intake. The association between tertiary amine drug exposure and anencephalic births was reduced among women who took a daily supplement with vitamin C compared with women who took these supplements less than daily or not at all.
The findings of the present study show a pattern similar to that seen in a study of nitrosatable drug use among Mexican-American women in Texas and NTDs in their offspring (10). In the Texas study, odds ratios for NTDs associated with nitrosatable drug use for the lowest tertile to the highest tertile of total dietary nitrite intake were 0.9 (95% CI: 0.3, 3.4), 2.7 (95% CI: 0.8, 11), and 7.5 (95% CI: 1.8, 45), respectively. However, the overall odds ratio for any NTD in relation to nitrosatable drug use was higher (AOR = 2.7, 95% CI: 1.4, 5.3) than the odds ratio noted in the present study, including that found for Hispanic women in NBDPS (AOR = 1.26, 95% CI: 0.84, 1.89). Anencephaly and spina bifida were not examined separately by nitrite intake in the Texas study because of insufficient numbers by phenotype.
In 2 other studies, the relation between nitrosatable drug use and NTDs was examined. Utilizing data from the Collaborative Perinatal Project, Olshan and Faustman (8) found that nitrosatable drug use during the first 4 months of pregnancy was associated with spina bifida (OR = 4.55, 95% CI: 0.66, 31.62) but not with anencephaly (0 of 6 case mothers exposed). They identified 13 nitrosatable drugs taken by the cohort. Using the list of nitrosatable drugs developed by Olshan and Faustman (8), Croen et al. (9) noted minimal association between nitrosatable drug use and NTDs among births to California residents (OR = 1.2, 95% CI: 0.77, 1.8); odds ratios for specific phenotypes were not reported.
A number of compounds have been found to inhibit nitrosation, with vitamin C being the most studied (13). In experimental studies in human volunteers, concomitant administration of ascorbic acid with nitrate and nitrosatable precursors such as proline (31) or a fish meal rich in amines (32) significantly reduced the excretion of N-nitroso compounds in comparison with these combined exposures without ascorbic acid. It is noteworthy that Brambilla and Martelli (12) concluded in their extensive review of nitrosatable drugs that the nitrosatable drug-nitrite interaction could be substantially reduced by the administration of vitamin C. In the present study, the associations between drugs classified as secondary or tertiary amines and anencephaly were reduced in women who took a daily vitamin supplement with vitamin C during the periconceptional period. The associations between these drugs and spina bifida were reduced among women who had an estimated dietary intake of vitamin C of 85 mg/day or more. However, we have no explanation for why we did not observe reduced associations between anencephaly and higher dietary vitamin C intake or reduced associations between spina bifida and vitamin C supplementation.
The present study had several other limitations. Approximately one-third of the eligible case and control women did not participate in the study, although participation rates were similar between cases and controls. Cogswell et al. (15) examined how well the NBDPS control women with EDDs from 1997 to 2003 represented the base population from which the case women were also identified. Control participants were similar to their base populations with respect to maternal age, smoking, and prevalence of diabetes mellitus but differed slightly by maternal race/ethnicity and educational level. Assessment of dietary nitrate and nitrite intakes was restricted to a food frequency questionnaire that might have not captured all dietary sources of these food contaminants or accurately reflected the actual amounts consumed (24). The potential for differential recall between case and control women regarding medications taken during early pregnancy is also of concern. However, women were not specifically asked about nitrosatable drug exposure, and it is unlikely that recall of medications taken would differ between cases and controls by type of nitrosatable drugs (e.g., secondary or tertiary amines) or by dietary intake of nitrites. Finally, although the present study utilized extensive reviews (12, 20) of nitrosatable drugs that were not available when previous studies of nitrosatable drugs and birth defects were conducted, exposures to some nitrosatable drugs might have been missed because the components have not been tested for nitrosatability or because results of such tests have not been published.
In conclusion, results of the present study suggest that early prenatal exposure to nitrosatable drugs, especially tertiary amines, in conjunction with nitrite intake might increase risk of NTDs. To our knowledge, this is the first study indicating that vitamin C might modulate the association between prenatal exposure to nitrosatable drugs and NTDs in offspring.
Author affiliations: Department of Epidemiology and Biostatistics, School of Rural Public Health, Texas A&M Health Science Center, College Station, Texas (Jean D. Brender, Ann M. Vuong, Mayura U. Shinde, Qi Zheng, John C. Huber, Jr.); Slone Epidemiology Center, Boston University, Boston, Massachusetts (Martha M. Werler, Katherine E. Kelley); Program for Research in Nutrition and Health Disparities, School of Rural Public Health, Texas A&M Health Science Center, College Station, Texas (Joseph R. Sharkey); Navy and Marine Corps Public Health Center, Portsmouth, Virginia (John S. Griesenbeck); Department of Epidemiology and Center for Education and Research on Therapeutics, College of Public Health, University of Iowa, Iowa City, Iowa (Paul A. Romitti); Birth Defects Epidemiology and Surveillance Branch, Texas Department of State Health Services, Austin, Texas (Peter H. Langlois, Mark A. Canfield); and Environmental Epidemiology and Disease Registries Section, Texas Department of State Health Services, Austin, Texas (Lucina Suarez).
This work was supported by the National Institute of Environmental Health Sciences at the National Institutes of Health (grants 5R01ES015634 and 3R01ES015634-03S1) and the Centers for Disease Control and Prevention/Texas Center for Birth Defects Research and Prevention (cooperative agreement U50/CCU613232).
Coding of drug information in the National Birth Defects Prevention Study used the Slone Drug Dictionary under license from the Slone Epidemiology Center of Boston University.
The authors thank the participating staff and scientists from all sites in the National Birth Defects Prevention Study. They also thank Dr. Roberta McKean-Cowdin of Norris Comprehensive Cancer Center/Department of Preventive Medicine, University of Southern California, for sharing the list of nitrosatable drugs developed by the late Dr. William Lijinsky for the childhood cancer studies. In addition, the authors thank Michelle Steck, Texas A&M Health Science Center, for her assistance in developing the nitrate and nitrite estimates for the food frequency questionnaire and Amy Hanson, University of Texas Health Science Center at Houston, for her assistance in the development of the nitrosatable drug files.
This work was presented at the 24th Annual Meeting of the Society for Pediatric and Perinatal Epidemiologic Research in Montreal, Canada, on June 21, 2011, and at the 3rd North American Congress of Epidemiology in Montreal, Canada, on June 23, 2011.
The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Environmental Health Sciences or the National Institutes of Health.
Dr. Martha Werler has provided consultation for Amgen, Bristol-Meyers Squibb, and Abbott regarding their pregnancy registries for rheumatoid arthritis drugs. These companies may manufacture drugs that were evaluated in this study.