Abstract
Background
Some epidemiologic studies suggest that maternal consumption of cured meat during pregnancy may increase risk of brain tumors in offspring. We explored whether this possible association was modified by fetal genetic polymorphisms in genes coding for glutathione S-transferases (GSTs) that may inactivate nitroso compounds.
Methods
We assessed six GST variants: GSTM1 null, GSTT1 null, GSTP1I105V (rs1695), GSTP1A114V (rs1138272), GSTM3*B (3 bp deletion), and GSTM3A-63C (rs1332018) within a population-based case-control study with data on maternal prenatal cured meat consumption (202 cases and 286 controls born in California or Washington, 1978-1990).
Results
Risk of childhood brain tumor increased with increasing cured meat intake by the mother during pregnancy among children without GSTT1 (odds ratio [OR]=1.29, 95% confidence interval [CI] 1.07-1.57 for each increase in the frequency of consumption per week) or with potentially reduced GSTM3 (any -63C allele, OR=1.14, 95% CI 1.03-1.26), whereas no increased risk was observed among those with GSTT1 or presumably normal GSTM3 levels (interaction p=0.01 for each).
Conclusions
Fetal ability to deactivate nitrosoureas may modify the association between childhood brain tumors and maternal prenatal consumption of cured meats.
Impact
These results support the hypothesis that maternal avoidance during pregnancy of sources of some nitroso compounds or their precursors may reduce risk of brain tumors in some children.
Keywords: brain neoplasms, child, glutathione transferase, meat, nitro compounds
Introduction
Childhood brain tumors (CBT) are the second most common pediatric cancer. Ionizing radiation is the only conclusively established non-genetic risk factor, but several epidemiologic studies suggest that maternal consumption of cured meats during pregnancy increases risk of CBT in offspring (1, 2). Although some studies have not observed this association (1, 3), the potential relationship remains compelling because cured meat is an important source of nitrite that can combine with other components of meat to form N-nitroso compounds (NOCs), including nitrosoureas (4). These are potent neurocarcinogens in non-human primates (5) and other animals, especially when exposure occurs in utero (6, 7).
Unlike some NOCs, nitrosoureas do not require enzymatic activation to act as carcinogens. Individual variation in a mother or child’s ability to detoxify (denitrosate) these chemicals is key to understanding their potential impact on cancer risk. Glutathione S-transferases (GSTs) are important in the detoxification of nitrosoureas (8-10). These include the alpha (GSTA), mu (GSTM), pi (GSTP) and theta (GSTT) subfamilies. The various GSTs are structurally similar with some overlap in substrate specificity, but their activity with respect to nitrosoureas differs. The GSTs’ relative expression levels in human brain, including during the fetal period, also differ. Therefore, some GSTs may play a more important role than others in protecting the fetal brain from nitrosourea compounds. Notably, GSTP1 is highly expressed in the fetal brain as early as 12 weeks gestation, including in astrocytes (11), the cell of origin for glial tumors, the tumor type most consistently associated with maternal cured meat consumption (2, 12). In addition, GSTP1 overexpression is associated with brain tumor resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine) in vitro (13), consistent with a role of GSTP1 in nitrosourea metabolism in the brain. GSTT1 and GSTM3 also are highly expressed in the brain (14), and are particularly efficient in the metabolism of BCNU in humans (9). GSTA isoforms are not expressed in fetal brain (11). We thus focused our explorations on genetic polymorphisms in GSTP1, GSTT1 and the GSTM4-GSTM2-GSTM1-GSTM5-GSTM3 gene cluster containing GSTM3.
Both GSTM1 in this cluster, and GSTT1, contain a common genetic polymorphism that results in the complete absence of the respective enzyme activity among homozygous carriers of the variant allele (null status). The functional GSTM1 *A allele is linked with a 3 bp deletion (*B) in GSTM3 that creates a Yin Yang 1 binding site (15). In the 5’ promoter resides another functional polymorphism, GSTM3A-63C; the C allele is associated with reduced GSTM3 expression (16). GSTP1 contains two frequently studied polymorphisms, GSTP1I105V and GSTP1A114V that result in amino acid changes near the enzyme’s catalytic center. These affect enzyme activity in a substrate-dependent manner, and are associated with survival among anaplastic glioma patients (17).
To elucidate the CBT-cured meat association, we examined whether it is modified by these six functional GST polymorphisms. Using population-based case-control data in which maternal cured meat consumption was associated with CBT (18), we assessed these polymorphisms using DNA from dried blood spots (DBS) from newborn screening archives in California and Washington. We hypothesized that the previously-observed CBT-cured meat association would be greater among children whose genotype might result in decreased denitrosation of nitrosoureas (i.e. reduced GST levels or activity), than among children with greater denitrosation capabilities.
Materials and Methods
Methods for obtaining interview data and specimens have been described (18-20). Briefly, all children were ≤10 years old and living in Seattle-Puget Sound (Washington), San Francisco-Oakland (California), or Los Angeles County (California) at the time of either diagnosis with a primary tumor of the brain, cranial nerves, or cranial meninges (ICD-O codes 191.0–192.1) in 1984-1991 (N=202 cases) or recruitment via random digit dialing in 1989-1993 (N=286 controls). These are all of the participants from the earlier population-based case-control study of CBT (18) for whom a DBS was located in state newborn screening archives. Among those born in California or Washington in a year with specimens archived (1978-1990), we obtained a DBS for 94% of cases and 86% of controls from Seattle (19), 93% of cases and 75% of controls from San Francisco, and 92% of cases and 85% of controls from Los Angeles (20). This represents 93% of cases and 83% of controls born in California or Washington in archived years, and 37% of cases and 36% of controls from the original study.
We ascertained frequency of maternal prenatal consumption of cured meat (ham, bacon, hot dogs, sausage, luncheon meat, and “other cured meats”) by structured in-person interviews with mothers, on average 5.3 years after birth for cases and 6.4 years for controls. Institutional Review Board approvals were received from all relevant agencies prior to study initiation, informed consent was obtained prior to the interview, and DBS were anonymized prior to release from the archives. In Washington, specimens were labeled only with a randomly assigned identification number (19), and identifying information was removed from study data. Similar methods for assuring anonymity were used in California (20).
The Functional Genomics Laboratory at the University of Washington obtained DNA from DBS using the QIAamp DNA Mini Kit (QIAGEN, Valencia, CA), and conducted genotyping for 6 variants: GSTP1I105V (rs1695), GSTP1A114V (rs1138272), GSTM3*B (rs36120609-rs1799735-rs58210492), and GSTM3A-63C (rs1332018) using TaqMan assays (Applied Biosystems, Foster City, CA); and GSTM1 and GSTT1 null using one multiplex PCR-based assay (21). A portion of the β-globin gene was co-amplified to verify that double-null status was not an artifact of PCR failure. Complete genotyping data were available for 200 (99%) cases and 279 (98%) controls. Duplicate or quadruplicate specimens for 6% of cases and 6% of controls from Washington were analyzed, blind to initial results, with complete concordance. When stratified by race/ethnicity, state and case status, no genotype frequencies failed chi square tests for Hardy-Weinberg equilibrium, with the exception of Californian Hispanics for GSTM3*B. However, this was statistically significant only for cases, and we confirmed that as reported previously (15) this allele was less frequent among GSTM1 null individuals (Pearson chi square p<0.0005).
We estimated odds ratios (ORs) and 95% confidence intervals (CIs) for the CBT-cured meat association using unconditional logistic regression, adjusted for study center, age, sex and race/ethnicity. We categorized the latter as African American/black (either parent African American/black), Hispanic (either parent Hispanic, neither parent African American), white (both parents non-Hispanic white), and Asian/other. We adjusted for age, sex and study center because they were frequency-matching variables, and for race/ethnicity because of previously reported associations with CBT, genotype and cured meat consumption. Adjustment for birth year or maternal education did not materially affect ORs or CIs further, and therefore were not included in final models. For ORs between CBT and cured meat, we categorized maternal cured meat consumption as previously (18): never; ≤1 time/week; >1 time/week but ≤3 times/week; >3 times/week but ≤7 times/week; >7 times/week. We also evaluated consumption as a continuous (frequency per week) variable. We then stratified by genotype; dichotomization was required for GSTT1 and GSTM1 because the assay does not separate heterozygous and homozygous non-null individuals, and for the other 4 polymorphisms because homozygous variants were uncommon. We assessed interaction between maternal cured meat consumption (continuous) and genotype on the multiplicative scale, while including exposure and genetic main effects terms in the model. To the extent sample size allowed, we explored the consistency of results between our two largest racial/ethnic groups (non-Hispanic whites, non-black Hispanics); and by CBT histological subtype (astroglial tumors [ICD-O histology codes 9380, 9382, 9400, 9401, 9420, 9421]; medulloblastoma/primitive neuroectodermal tumors [PNET, codes 9470, 9471, 9473]; and “other” tumors [all other codes]).
Results
Cases and controls for whom a DBS was located were similar to original study participants with regard to race/ethnicity and maternal education (Table 1). Those with DBS were born in more recent birth years (when archival samples were stored), and therefore were younger. The median age at diagnosis/reference for both cases and controls with DBS was 3 years (data not shown). Consistent with this relatively young age, proportionally fewer astroglial and proportionally more medulloblastoma/PNET cases were included than in the original study (Table 1). Only three (1%) cases and three (1%) controls had a personal or family history of Li-Fraumeni Syndrome, neurofibromatosis or tuberous sclerosis, or had a first-degree relative with a brain tumor (data not shown).
Table 1.
Demographic and clinical characteristics of children with and without brain tumors, overall and among those with a dried blood spot for genotyping, West Coast Childhood Brain Tumor Study, births in 1965-1990
| All participants | Participants with a dried blood spot for genotyping | |||
|---|---|---|---|---|
| Cases N=540 n (%) | Controls N=801 n (%) | Cases N=202 n (%) | Controls N=286 n (%) | |
| Birth year | ||||
| 1965-1977 | 194 (36) | 292 (36) | -- | -- |
| 1978-1984 | 232 (43) | 325 (41) | 99 (49) | 142 (50) |
| 1985-1990 | 114 (21) | 184 (23) | 103 (51) | 144 (50) |
| Age at diagnosis/reference (years) | ||||
| ≤4 | 188 (35) | 287 (36) | 168 (83) | 222 (78) |
| 5-9 | 158 (29) | 232 (29) | 34 (17) | 61 (21) |
| 10+ | 194 (36) | 282 (35) | 0 (0) | 3 (1) |
| Study center | ||||
| Los Angeles | 304 (56) | 315 (39) | 110 (54) | 99 (35) |
| San Francisco | 102 (19) | 205 (26) | 26 (13) | 50 (17) |
| Seattle | 134 (25) | 281 (35) | 66 (33) | 137 (48) |
| Male | 298 (55) | 448 (56) | 121 (60) | 168 (59) |
| Race/ethnicity* | ||||
| Non-Hispanic white | 313 (58) | 532 (67) | 105 (54) | 192 (68) |
| Hispanic | 147 (27) | 183 (23) | 62 (32) | 61 (22) |
| African American | 42 (8) | 41 (5) | 14 (7) | 13 (5) |
| Asian/other | 38 (7) | 44 (6) | 15 (8) | 17 (6) |
| Maternal education (college)* | ||||
| None | 270 (50) | 318 (40) | 103 (51) | 112 (39) |
| Some | 170 (32) | 267 (33) | 57 (28) | 88 (31) |
| Degree | 99 (18) | 215 (27) | 42 (21) | 85 (30) |
| Histological tumor type | ||||
| Astroglial | 308 (57) | -- | 96 (48) | -- |
| Medulloblastoma/PNET† | 107 (20) | -- | 55 (27) | -- |
| Other | 125 (23) | -- | 50 (25) | -- |
Proportions exclude those with missing data on maternal race/ethnicity, paternal race/ethnicity and/or maternal education
Primitive neuroectodermal tumor
The CBT-cured meat association did not markedly vary by whether an archival DBS was obtained, although among the relatively contemporary group with DBS (median birth year 1985), there was no indication of increased risk for the lowest category of exposure in slight contrast to those without a specimen (median birth year 1977) (Table 2). Similar to results reported for the full sample (18), the CBT-cured meat association was suggested among participants with DBS but remained statistically non-significant for each of the three histologic tumor type categories (ORs of 1.68, 1.40, and 1.89 for cured meat >7 times/week vs. never for astroglial tumors, medulloblastoma/PNET and “other” tumors, respectively, data not shown).
Table 2.
Childhood brain tumor and maternal consumption of cured meat during pregnancy, by availability of a dried blood spot for genotyping, West Coast Childhood Brain Tumor Study, births in 1965-1990
| Frequency of maternal cured meat* consumption during pregnancy | Participants without a dried blood spot for genotyping | Participants with a dried blood spot for genotyping | ||
|---|---|---|---|---|
| Cases/Controls N=338/515† | OR (95% CI)†‡ | Cases/Controls N=202/286† | OR (95% CI)†‡ | |
| Never | 69/109 | 1.0 (reference) | 35/51 | 1.0 (reference) |
| >0 to ≤1 times/week | 66/97 | 1.26 (0.80-1.97) | 38/73 | 0.84 (0.46-1.53) |
| >1 to ≤3 times/week | 88/148 | 1.04 (0.69-1.58) | 52/80 | 1.05 (0.59-1.86) |
| >3 to ≤7 times/week | 76/112 | 1.29 (0.83-2.00) | 54/63 | 1.37 (0.76-2.49) |
| >7 times/week | 37/43 | 1.71 (0.91-3.05) | 23/17 | 1.97 (0.88-4.41) |
| Continuous (per week) | 1.04 (1.00-1.08) | 1.03 (0.98-1.09) | ||
Ham, bacon, hot dogs, sausage, luncheon meat or “other” cured meats combined
Tabulation excludes participants with missing data on maternal prenatal cured meat consumption (2 cases and 6 controls without a dried blood spot, and 2 controls with a dried blood spot)
Odds ratio and 95% confidence interval, adjusted for age, study center, sex and race/ethnicity (non-Hispanic white, Hispanic, African American, Asian/other)
When we examined whether the CBT-cured meat association was modified by any of the selected functional polymorphisms, there was no indication that the CBT-cured meat association depended on either GSTP1 polymorphism (Table 3). However, the association appeared modified by GSTT1 genotype, with the association specifically observed among GSTT1 null children (Tables 3-4, interaction p=0.01). We confirmed this interaction among the subset of non-Hispanic whites (interaction p=0.01), but this sub-analysis included only 12 GSTT1 null cases (data not shown). We also observed a statistically significant interaction with GSTM3A-63C: The CBT-cured meat association was only present among children with the -63C (reduced expression) allele (Tables 3 and 5, interaction p=0.01). When we explored whether this potential cured meat-GSTM3A-63C interaction varied by other polymorphisms in the same gene cluster, it remained, irrespective of GSTM3 *B (interaction p=0.04-0.06) or GSTM1 genotype (interaction p=0.03-0.13, Table 3). In contrast, possible interactions between cured meat and GSTM3 *B and between cured meat and GSTM1 disappeared when stratifying by GSTM3A-63C (also shown in Table 3).
Table 3.
Childhood brain tumor and maternal consumption of cured meat during pregnancy, by selected functional polymorphisms in genes coding for four glutathione S-transferase (GST) enzymes, overall and by GSTM3C-63A, West Coast Childhood Brain Tumor Study, births in 1978-1990
| Genotype | All participants with dried blood spots for genotyping | GSTM3 -63AA (Normal expression) | GSTM3 -63AC/CC (Reduced expression) | |||
|---|---|---|---|---|---|---|
| Cases/controls N=202/286† | OR (95% CI)* | Cases/controls N=85/113† | OR (95% CI)* | Cases/controls N=117/169† | OR (95% CI)* | |
| GSTP1I105V | ||||||
| VV/IV | 117/191 | 1.03 (0.96-1.10) | 45/79 | 0.95 (0.86-1.05) | 72/109 | 1.23 (1.08-1.41) |
| II | 85/94 | 1.04 (0.96-1.12) | 40/34 | 1.03 (0.90-1.18) | 45/59 | 1.03 (0.88-1.21) |
| GSTP1A114V | ||||||
| VV/AV | 23/53 | 1.07 (0.95-1.20) | 9/24 | 1.21 (0.93-1.56)‡ | 14/27 | 1.35 (0.93-1.96) |
| AA | 179/232 | 1.05 (0.98-1.11) | 76/89 | 0.99 (0.91-1.08) | 103/141 | 1.13 (1.01-1.26) |
| GSTT1 | ||||||
| Not null | 169/235 | 1.00 (0.96-1.05) | 72/97 | 0.95 (0.88-1.03) | 97/136 | 1.07 (0.96-1.19) |
| Null | 31/50 | 1.29 (1.07-1.57) | 12/16 | 1.10 (0.91-1.33) | 19/32 | 1.61 (1.17-2.22) |
| GSTM1 | ||||||
| Not null | 105/140 | 1.06 (0.98-1.14) | 41/54 | 1.00 (0.90-1.12) | 64/83 | 1.13 (0.99-1.30) |
| Null | 95/145 | 1.01 (0.93-1.08) | 43/59 | 0.96 (0.87-1.06) | 52/85 | 1.18 (1.01-1.38) |
| GSTM3 *B | ||||||
| Any *B | 68/94 | 1.00 (0.91-1.09) | 43/52 | 0.98 (0.89-1.08) | 25/39 | 1.24 (0.96-1.61) |
| No *B | 134/191 | 1.06 (1.00-1.13) | 42/61 | 0.98 (0.89-1.08) | 92/129 | 1.15 (1.03-1.27) |
| GSTM3A-63C | ||||||
| AA | 85/113 | 0.98 (0.91-1.05) | -- | -- | -- | -- |
| AC/CC | 117/169 | 1.14 (1.03-1.26) | -- | -- | -- | -- |
Odds ratio and 95% confidence interval per frequency of maternal prenatal consumption per week of cured meats (ham, bacon, hot dogs, sausage, luncheon meat or “other” cured meats combined), adjusted for age (continuous), study center, sex and race/ethnicity (non-Hispanic white, Hispanic, African American, Asian/other) unless noted, excludes 2 controls with missing cured meat data and ≤2 cases and ≤4 controls with missing genotyping data
Numbers may not add to total due to missing genotyping data
Restricted to non-Hispanic whites (excludes 6 Hispanic controls) to control for race/ethnicity
Table 4.
Childhood brain tumor and maternal consumption of cured meat during pregnancy, by fetal GSTT1 genotype, West Coast Childhood Brain Tumor Study, births in 1978-1990
| Cured meat* consumption during pregnancy | GSTT1 non-null (Some GSTT1) | GSTT1 null (No GSTT1) | ||
|---|---|---|---|---|
| ca/co N=169/235† | OR (95% CI)‡ | ca/co N=31/50 | OR (95% CI)§ | |
| Never | 33/41 | 1.27 (0.66-2.46) | 2/10 | 0.51 (0.04-3.53) |
| >0 to ≤1/week | 31/55 | 1.0 (reference) | 7/18 | 1.0 (reference) |
| >1 to ≤3/week | 46/70 | 1.21 (0.67-2.19) | 5/10 | 1.29 (0.25-6.23) |
| >3 to ≤7/week | 44/51 | 1.46 (0.79-2.71) | 9/11 | 3.64 (1.02-13.55) |
| >7 times/week | 15/16 | 1.48 (0.61-3.58) | 8/1 | |
Frequency of consumption of ham, bacon, hot dogs, sausage, luncheon meat or “other” cured meats combined
Tabulation excludes 2 controls with missing data on maternal cured meat consumption
Odds ratio and 95% confidence interval, adjusted for race/ethnicity, study center, age and sex
Exact unadjusted odds ratio and 95% confidence interval
Table 5.
Childhood brain tumor and maternal consumption of cured meat during pregnancy, by fetal GSTM3A-63C genotype, West Coast Childhood Brain Tumor Study, births in 1978-1990
| Cured meat* consumption during pregnancy | GSTM3 -63AA (Normal expression) | GSTM3 -63AC/CC (Reduced expression) | ||
|---|---|---|---|---|
| ca/co N=85/113 | OR (95% CI)† | ca/co N=117/169‡ | OR (95% CI)† | |
| Never | 16/24 | 1.0 (reference) | 19/27 | 1.0 (reference) |
| >0 to ≤1/week | 22/24 | 1.52 (0.62-3.73) | 16/46 | 0.55 (0.23-1.31) |
| >1 to ≤3/week | 18/32 | 0.86 (0.35-2.09) | 34/48 | 1.22 (0.55-2.69) |
| >3 to ≤7/week | 22/21 | 1.86 (0.74-4.71) | 32/41 | 1.20 (0.53-2.69) |
| >7 times/week | 7/12 | 0.73 (0.22-2.38) | 16/5 | 5.66 (1.62-19.78) |
Frequency of consumption of ham, bacon, hot dogs, sausage, luncheon meat or “other” cured meats combined
Odds ratio and 95% confidence interval, adjusted for race/ethnicity, study center, age and sex
Tabulation excludes 2 controls with missing data on maternal cured meat consumption
We observed the GSTT1-cured meat interaction regardless of GSTM3A-63C genotype, and vice versa, although these interactions were not always statistically significant. The CBT-cured meat association was stronger among children with absent/reduced levels of both GSTT1 and GSTM3 (OR=1.61, 95% CI 1.17-2.22 for each increase per week in the frequency of consumption), than among those without GSTT1 but with normal GSTM3 expression (OR=1.10, 95% CI 0.91-1.33), or those with reduced GSTM3 expression but some GSTT1 (OR=1.07, 95% CI 0.96-1.19, Table 3). Risk of CBT did not increase with increasing exposure among children with both GSTT1 and normal GSTM3 expression (OR=0.95, 95% CI 0.88-1.03). Although based on very sparse data, both the GSTT1 and GSTM3 interactions were suggested when we focused on astroglial tumors, on medulloblastoma/PNET, or on all other CBTs combined (all interaction p-values ≤0.11, data not shown).
Discussion
To our knowledge, this is the first study to examine whether the previously observed CBT-cured meat association may be modified by the child’s ability to metabolize potentially relevant carcinogens, as indicated by fetal GSTT1, GSTP1, GSTM1 and GSTM3 genotype. For two of six polymorphisms examined, any increase in CBT risk from prenatal cured meat was confined to children who presumably denitrosate (inactivate) NOCs more slowly, specifically those without GSTT1 (8), and carriers of the GSTM3 -63C allele that is associated with reduced gene expression (16). These similar yet independent interactions between maternal prenatal cured meat intake and functional GST polymorphisms are biologically plausible. GSTT1 and GSTM3 are among the GSTs most highly expressed in the placenta and adult brain (14). In both organs, expression of GSTT1 and GSTM3 are at least an order of magnitude greater than GSTM1. Although GSTP1 is highly expressed in both placenta (14) and fetal brain (11), the well-studied GSTP1 polymorphisms included here are amino acid changes that may not capture enzyme activity as well as a promoter region polymorphism such as GSTM3A-63C, or the GSTT1 null polymorphism resulting in a complete absence of enzyme activity. In addition, of the GSTs considered here, GSTT1 and GSTM3 may be the most efficient in inactivating nitrosoureas (9). Together, these results suggest that the possible association between cured meat consumption during pregnancy and CBT risk in offspring may be modified by the fetus’ ability to metabolize compounds potentially associated with the consumption of cured meats, such as nitrosoureas (4).
Care must be taken in interpreting these results. First, our sample size was modest, which increased the probability of false positives (22). Second, the interactions were present in each histologic group, including the highly heterogeneous “other” tumors. This was unexpected because most epidemiologic studies suggest that the CBT-maternal cured meat association may be specific to astroglial tumors (2-3, 12, 23), as may be any association with nitrate or nitrite in tap water (24). However, in animal studies nitrosoureas induce a variety of brain tumor types (25). Also, the lack of tumor-specific associations does not suggest selection or information bias, because generally neither inflates interactions (26-27). Finally, much remains to be learned about the content of specific NOCs and nitrosatable alkylureas in cured meat or their in vivo formation (4); their detoxification by individual GSTs; and the expression of individual GSTs in fetal brain and placenta over the course of pregnancy. Animal models suggest species-specific periods of susceptibility. They also indicate that nitrosation-inhibitors such as vitamin C prevent neurogenic tumors in offspring of rodents simultaneously exposed to nitrite and nitrosatable ureas during pregnancy (28). Therefore, it is a limitation that our modest sample size combined with a nearly universal use of vitamin supplements precluded examination of the observed interactions by supplement use. Despite these limitations, this work builds on earlier studies focused either on cured meat (1-3, 12, 23) or GST genetic (29-31) main effects. Our results underscore the importance of considering genotype when assessing CBT-exposure associations. They also may suggest the need to assess multiple GSTM functional polymorphisms in studies of CBT and perhaps other outcomes relevant to substrates better metabolized by GSTM3 than GSTM1. These genes both reside in the GSTM4-GSTM2-GSTM1-GSTM5-GSTM3 gene cluster, and until stratifying by GSTM3A-63C, it unexpectedly appeared that the CBT-cured meat association was present among children with GSTM1 but not among GSTM1 null children. In addition, given some overlap in function, it may also be important to consider the joint effects of polymorphisms in different GST subfamilies, including GSTM3, GSTT1 and GSTP1. Our ability to do this in the context of estimating CBT-cured meat ORs was limited, and the corresponding results can only be viewed as exploratory.
The present work supports the premise that some NOCs and NOC precursors may play a role in initiation of brain tumors during human fetal development. Future studies will benefit from assessment of maternal cured meat intake by trimester of pregnancy, larger sample sizes, and the inclusion of children conceived in a wider range of birth years in order to examine the effect of decreasing levels (4) of nitrite in cured meats over time. It also may be informative to genotype both mothers and children, so that the effect of GST enzymes in mothers’ livers can be considered as well.
Acknowledgments
We thank the Washington State Department of Health Newborn Screening Program, Michael Glass and Michael Ginder; California Department of Public Health Genetic Disease Screening Program, Steve Graham, Marty Kharrazi, and Fred Lorey; and the Sequoia Foundation for obtaining specimens; and the Functional Genomics Core Laboratory, Center for Ecogenetics and Environmental Health, University of Washington, Jesse Tsai and Hannah-Malia A. Viernes for genotyping.
Financial support: This research was supported by grants R01 CA116724, R03 CA106011, NIEHS P30ES007033, NIEHS 5P30ES07048 and NIEHS T32ES07262, National Institutes of Health; contract N01-CN-05230 from the National Cancer Institute; and Fred Hutchinson Cancer Research Center.
Footnotes
Conflicts of interest: None
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