Polycyclic aromatic hydrocarbons (PAHs) are lipophilic compounds formed during the incomplete burning of coal, tobacco, or other organic substances. Human exposure is common through inhalation of tobacco smoke and smoke from other sources of combustion, through ambient air, and through consumption of PAHs particularly in charbroiled foods(ATSDR, 1995). Although environmental sources contribute to the total exposure burden, some of the highest exposure levels are found in the workplace ( Brandt and Watson, 2003; Hansen et al., 2008). Occupations where exposure is likely to occur include those involving coke ovens and coal tar use, iron and steel works, carbon electrode and carbon black manufacture, and asphalt manufacture and use. Additionally, exposures can occur in more common occupational settings such as restaurants( Sjaastad and Svendsen, 2009).

Maternal exposure to PAHs during gestation has been shown in mice to cause oral clefts (Shum et al., 1979). To the authors’ knowledge, no human studies assessing this association have been published. However, a major source of PAH exposure for humans is cigarette smoking (active or secondhand), which has been reported to show a moderate association with clefts in most studies (e.g., Ericson et al., 1979; Khoury et al., 1989; Shaw et al., 1996; Chung et al., 2000; Little et al., 2004a; Zeiger et al., 2005; Honein et al., 2007; Shi et al., 2007; Leite and Koifman, 2009; Shaw et al., 2009, Hackshaw et al., 2011), but not all( e.g., Werler et al., 1990; Grewal et al., 2008). The magnitude of the excess risk may be related to the genotype of the mother and/or fetus (van Rooij et al., 2001; Lammer et al., 2005; Shaw et al., 2005; Shi et al., 2007).

Because of high and potentially common workplace exposures to PAHs and evidence suggesting an association between an important source of PAHs (cigarette smoking) and oral clefts, the objective of this study was to determine if women’s periconceptional occupational exposure to PAHs was associated with risk of oral cleftsin off spring.

Participation was 69% among control mothers and 76% among case mothers. For the resulting analysis there were 2989 controls, 805 cases of CL±P, and 439 CP.

Risk of CL±Pamong offspring was statistically significantly higher among mothers who had diabetes before the index pregnancy, a multiple-fetus pregnancy, male children, lower education or household income, or a family history of clefts(Table 1). Risk was also higher among mothers who smoked or were exposed to secondhand smoke at work. Risk of CP was higher among mothers who were non-Hispanic White(compared to non-Hispanic Black or Hispanic), who smoked or were exposed to secondhand smoke at home, or who had a family history of clefts. Risk of CP varied across study sites.

Prevalence of occupational PAH exposure in controls was higher(p < 0.05) among mothers who were Hispanic, overweight or obese, had a lower education or household income, or who smoked or were exposed to secondhand smoke(data not shown). Prevalence of exposure was lower among women who drank alcohol.

Overall, 3.5% of the controls were classified as occupationally exposed to PAHs, as were 5.8% of the CL±P cases and 4.6% of the CP cases (Table 2). The distribution of SOC major job groups was similar between the 106 control mothers, 47 CL±P mothers, and the 20 CP mothers occupationally exposed to PAHs. The largest category for each group was “food preparation and serving related” jobs, followed by “sales and related” jobs. Combining those two occupations accounted for 76% of exposed control mothers, 79% of exposed CL±P mothers, and 65% of exposed CP mothers. The occupational settings where most exposed case and control mothers worked were restaurants, particularly fast food chains; the most common job duty mentioned by exposed mothers was cooking or food preparation. Cooking or food preparation accounted for a large portion of jobs with high intensity exposure to PAHs, while managing, providing personal care, or being a cashier were the most common low PAH exposure jobs within the participants’ work histories(data not shown) .

The crude OR (95% CI) for occupational PAH exposure and CL±P was 1.69 (1.18, 2.40), and 1.47 (1.02, 2.12) after adjusting for maternal education(Table 3). The ORs for CP were slightly elevated but not statistically significant ; the cOR was 1.30 (0.80, 2.12) and the OR adjusted for maternal secondhand smoke exposure at home was 1.24 (0.76, 2.03).

In the first subanalysis, the unexposed group and occupationally exposed group were limited to subjects who were not exposed to PAHs from any other source that could be determined using the NBDPS data(smoking or secondhand smoke at home or work ) (data not shown). That reduced the number of exposed CL±P cases from 47 to 17. The crude OR remained elevated but was no longer statistically significant (1.65; 0.93, 2.93), as was the adjusted OR (1.70; 0.94, 3.06). The cOR for the six exposed CP cases was close to unity(1.05; 0.44, 2.50); the aOR was not estimable.

In the second subanalysis, no statistically significant effect measure modification was observed in either CL±P or CP for pre-pregnancy BMI, maternal age, race/ethnicity, education, folic acid supplement use, active smoking, or any smoking exposure (smoking or secondhand smoke at home or work) (data not shown). This did not seem to be related to statistical power, since (a) numbers of exposed cases were acceptable for most strata except some variables among CP, and (b) the ORs for the strata tended to be similar in magnitude, for example, 1.21 to 1.88 for CL±P in each stratum with at least 10 exposed cases.

In the analysis of exposure-response relationships, crude risk of CL±P exhibited a statistically significant monotonic trend by the Cochrane-Armitage test, which remained after adjustment for maternal education (p=0.02, Table 4). Compared to the no PAH exposure group, the aOR for the low PAH exposure level was 1.36 (0.82, 2.27), and the highest PAH exposure level was 1.66 (1.02, 2.70). There was no statistically significant exposure-response relationship observed for CP.

There were 705 cases of CL±P with no other major malformations; this made up 88% of our total CL±P cases. The associations with occupational PAH exposure observed with these cases were similar to those using the total sample, though sometimes slightly attenuated and often no longer statistically significant. For example, when adjusted for maternal education, the OR for any occupational PAH exposure and CL±P was 1.35 (0.92, 2.00) (data not shown) vs. 1.47 (1.02, 2.12) in the total sample. Compared to no occupational PAH exposure in the isolated group, the aOR for low exposure was 1.10 (0.62, 1.97) and for high exposure, 1.61 (0.96, 2.70); p for trend was 0.08. There were 361 cases or 82% of total CP without other major malformations; among them, the magnitude of associations were similar to total cases, and the test for trend remained not statistically significant.

This study found a positive association between maternal occupational exposure to PAHs and risk of cleft lip with or without cleft palate in offspring. This was true of crude and adjusted analyses(the aOR was 1.47, 95% CI 1.02, 2.12). Odds ratios increased with increasing total cumulative exposure to occupational PAHs, and the statistical significance of the trend remained after adjustment. Similar patterns were observed among CL±P cases without other major malformations, though there was no longer statistical significance, probably due in part to fewer subjects. When restricted to subjects without exposure to PAHs from active or secondhand smoking, the association with maternal occupational PAHs was no longer statistically significant , but the magnitude of effect was similar. No effect measure modification was observed for CL±P; ORs across strata were similar in magnitude. No statistically significant association was observed with cleft palate alone.

These observations were consistent with Shum et al., (1979) who found that PAH exposure caused oral clefts in mice. While no published papers were found regarding PAH exposure in humans and clefts in offspring, the current study was consistent with the frequent reports of cigarette smoking moderately increasing risk of clefts, since smoking is one of the major sources of PAH exposure in human beings. Many of those papers reported an association of smoking with combined oral clefts (Ericson et al., 1979; Van den Eeden et al, 1990; Chung et al., 2000; Wyszynski et al., 2002; Van Rooij et al., 2002; Shi etal., 2007; Shaw et al., 2009 , Hackshaw et al., 2011), or found statistically significant associations for both CL±P and CP (Khoury et al., 1987; Khoury et al., 1989; Shaw et al., 1996; Kallen, 1997; Wyszynski et al., 1997; Little et al., 2004a, 2004b; Meyer et al., 2004; Zeiger et al., 2005). The current study found statistically significant associations only with CL±P, which is consistent with several studies that examined both phenotypes (Lorente et al., 2000; Honein et al., 2007; Lie et al, 2008; Liete and Koifman, 2009), though one study found effects only with CP (Romitti et al., 1999). Some studies reported no association of smoking with combined clefts or with CL±P or CP (Hemminki et al., 1983; Werler et al., 1990; Christensen et al., 1999; Van Rooij et al, 2001; Grewal et al., 2008).

In the current study, occupational PAH exposure showed statistically significant associations only with CL±P. This might have been due partly to small numbers of exposed cases of CP (20, compared with 47 CL±P), since CP also exhibited a positive, though weaker and not statistically significant, association (aOR = 1.24, 95% CI 0.76, 2.03). As such, the current study is consistent with the meta-analysis by Little et al (2004a) reporting weaker associations with maternal smoking for CP than CL±P. The difference between phenotype associations might also have been due partly to the fact that the developmental errors that lead to CL±P are different from those that lead to CP (Fogh-Anderson, 1942, Fraser, 1955).

PAHs cross the placenta (Gladen et al., 2000)and have been found in cord blood (Madhaven and Naidu, 1995). They have been shown to form bulky DNA adducts in mothers and offspring(Topinka et al., 2009). Workers exposed to PAHs have higher levels of PAH-DNA adducts compared to the general population (Perera et al., 1994; Brandt and Watson, 2003). If not repaired, these adducts can disrupt the cell’s microenvironment, leading to inhibition of important enzymes, cell death, and alteration of other cells(Agency for Toxic Substances and Disease Registry, 1995; Choi et al., 2008). Those in turn could affect the directed growth, fusion, and differentiation required for normal palate and lip development. Another possible mechanism is if PAH exposure leads to periods of fetal hypoxia through reduced placental blood flow (Rennie et al., 2011); hypoxic events increase the risk of oral clefts in certain strains of mice (Millicovsky and Johnston, 1981; Bronsky et al., 1986; Bailey et al., 1995).

A major l imitation of this study was the potential for exposure misclassification. Although the three industrial hygienists had several years of experience and working knowledge, inaccurate assignment of exposure was nevertheless possible since the occupational information reported during the CATI was limited and did not include potentially important exposure-modifying factors such as use of personal protective equipment and ventilation practices. However, the expert rater approach used in our study was superior to relying exclusively on maternal self-report of PAH exposure, which is likely to be limited and could introduce recall bias(Olsson et al., 2010). In our dichotomized exposure analyses (any/none), non-differential errors in exposure misclassification would be expected to bias the ORs toward the null (Rocheleau et al., 2011).

There was no information about sources of environmental PAH exposure (e.g., residential proximity to industrial combustion smoke) other than personal tobacco use and exposure to ETS at home or at work. However, smoking is an important source of PAHs (ATSDR, 1995), and it is therefore a strength of our study that we were able to account for PAH exposure from smoking. Further, since levels of occupational PAH exposure are generally higher than environmental sources (Brandt and Watson, 2003; Hansen et al., 2008), it is important to consider exposure in the workplace separately from other sources. Biomarkers of exposure to PAHs( e.g., Naufal et al., 2010; Ren et al., 2011)are generally superior to the interview-based data used in this study in terms of accurate measurement of cumulative exposure from all sources, occupational or otherwise. However, biomarkers collected at birth may not accurately reflect PAH exposures experienced during the periconceptional period, which is the critical window of fetal development for the oral cavity and surrounding structures, and thus may introduce undesirable misclassification in the timing of exposure .

One of the major strengths of this study was its use of NBDPS data , which provided an extensive occupational exposure database. The occupational exposure assessment improved accuracy compared to the exclusive use of job title or self-reported exposure, and the cumulative exposure estimation allowed examination of exposure-response relationships. Its case-control study design was more practical than occupational cohort studies for very rare outcomes such as birth defects. It yielded data on potentially important confounding factors such as active and passive smoking. The extensive case classification by NBDPS clinical geneticists produced accurate and fairly homogeneous case groups for analysis, reducing outcome heterogeneity and allowing for the examination of specific subgroups of oral clefts.

In summary, this study found a positive association between maternal occupational exposure to PAHs and risk of cleft lip with or without cleft palate in offspring. Risk showed an exposure-response relationship which also persisted after adjustment. No statistically significant association was observed with cleft palate alone. This may be the first study of PAH exposure and orofacial clefts conducted in humans, and it is generally consistent with many previous studies of cigarette smoking and clefts. Future investigations of PAHs and clefts may benefit from additional measures of exposure such as biomarker data, as well as gathering information on maternal and fetal genotypes related to PAH metabolism(Shimada, 2006; Wassenberg et al., 2005)or genotypes suggested in previous studies of gene-smoking interactions and oral clefts (Shaw et al., 1996; Romitti et al., 1999; Van Rooij et al., 2001; Zeiger et al, 2005; Lammer et al., 2005; Shaw et al., 2005; Shi et al., 2007).