Abstract
Background
Methamphetamine (MA) use during pregnancy is a significant public health concern in the United States and affects long term brain and behavioral development in children. We hypothesized that prenatal MA exposure would be related to greater DNA methylation of HSD11B2 and postnatal environmental stress.
Methods
The Infant Development, Environment, and Lifestyle Study (IDEAL), a longitudinal study of Prenatal MA exposure enrolled mother-infant dyads in California, Hawaii, Iowa, and Oklahoma. Prenatal exposure was defined by maternal self-report and/or meconium toxicology screening. At ages 10–11 years, 100 children were assessed for drug exposure and DNA methylation of HSD11B2. Hierarchical linear models were used to determine the association between Prenatal MA exposure and methylation of HSD11B2 at 4 CpG sites.
Results
Prenatal MA exposure (1.4% vs 0.31%, P<0.01) and early childhood adversity (3.0 vs 2.0, P<0.01) were associated with greater DNA methylation of HSD11B2 at the CpG2 site. The statistically significant effects of early childhood adversity (B=0.11, P<0.01) and Prenatal MA exposure (B=0.32, P=0.03) on DNA methylation remained after adjusting for covariates.
Conclusions
Prenatal MA exposure is related to postnatal childhood adversity and epigenetic alterations in HSD11B2, an important gene along the stress response pathway suggesting prenatal and postnatal programming effects.
Introduction
Recent reports on the public health risks of drugs have shown a surge in the abuse of methamphetamine (MA) in the United States (US) and worldwide.(1, 2) Current reports suggest methamphetamine is second only to heroin in its threat to public health in the US.(2) MA use during pregnancy continues to be a significant public health concern in the United States (3, 4) and affects long term brain and behavioral development in children. Further, in utero methamphetamine exposure is a form of toxic stress.(5) Toxic stress can affect development in children through biological embedding, including epigenetic pathways and as cumulative damage over time or allostatic load. We investigated the effects of Prenatal MA exposure on epigenetic changes in 10–11-year-old children. Based on previous work,(6) and consistent with the notion of toxic stress, we reasoned that Prenatal MA exposure could act as an intrauterine stressor that affects gene expression particularly of stress related genes such as hydroxysteroid 11-Beta dehydrogenase type 2 (HSD11B2) that converts cortisol to its inert form, cortisone, thereby reducing levels of circulating cortisol.(7) We have previously demonstrated alterations in placental HSD11B2 methylation following adverse intrauterine exposures.(8) We hypothesized that Prenatal MA exposure would be related to greater DNA methylation of HSD11B2 in children exposed to methamphetamine antenatally, and that because of the developmental nature of the exposure that this increased methylation would be persistent, and be evident in middle childhood. We further hypothesized that greater DNA methylation of HSD11B2 would be related to postnatal environmental stress measured by an environmental adversity index and hair cortisol/cortisone.
METHODS
Subjects
Subjects included 10–11-year-old children; 55 with Prenatal MA exposure and a comparison group (COMP) of 45 children without Prenatal MA exposure from 2 of the 4 original recruitment sites (Hawaii and California) for the Infant Development Environment and Lifestyle (IDEAL) study. Detailed recruitment methods for the IDEAL study have been reported previously.(9) Mothers were recruited for the study between delivery and discharge. The study was approved by the institutional review boards at all participating sites including a Certificate of Confidentiality from the National Institute on Drug Abuse and all child participants in this study provided written informed assent in addition to parental consent. Prenatal MA exposure was determined by self-reported MA use during this pregnancy and/or a positive meconium screen with gas chromatography/mass spectroscopy confirmation. Children in the COMP group were born to mothers who denied methamphetamine use during pregnancy and their meconium screened negative for amphetamines and opiates. A matched case-control study design was used. Mothers in the control group were matched to those in the MA exposure group on maternal race, private versus public insurance, maternal education (completed high school versus not completed), and infant birth weight category (< 1500, 1500– 2500, >2500 g).
Measurements
DNA Collection.
Buccal derived DNA from saliva samples were collected from children using the Oragene-DNA saliva collection system. Children were asked to provide a saliva sample by expectorating into the provided collection tube up to a marked line. The collection tube was sealed, releasing a stabilizing solution into the collected sample to allow for processing of the sample at a later period. Sample collection tubes were bar-coded and labeled with ID numbers. DNA was isolated from the collection tubes following the Oragene methods, which reliably result in 50–150 μg of high-quality DNA for downstream analysis.
DNA Methylation Analysis.
A sodium bisulfite pyrosequencing approach was used to assess the methylation status of HSD11B2. Characteristics of the assay were assessed using dilution series of fully methylated referent DNA into fully unmethylated referent DNA. The PyroMark MD system (QIAGEN Inc., Germantown, MD) was used for all pyrosequencing assays. Quantitative assessment of the extent of DNA methylation at each of the 4 HSD11B2 CpG sites was performed by using the integrated Pyro-Q-CpG software (QIAGEN Inc.) to analyze data using previously published methods described further in Supplementary Material (online).(10) Genomic coordinates are GRch37/hg19: 67464389, 67464395, 67464399, 67464412.Methylation variables for the CpG sites were winsorized and natural log transformed and analyzed as a continuous measure based on percent of methylation at each CpG site.
Early Adversity.
Early adversity was coded using a single index score similar to those used in previous studies.(11,12) The early adversity index accounted for cumulative measures of adversity from birth through age 5 years by calculating the sum of a set of binary indicators (Table 1).
Table 1.
Early adversity index binary indicators
| Binary indicators of early adversity |
|---|
| any self-reported maternal substance use/abuse during the first 3 years of life |
| any extreme poverty experienced between birth and 5 years (as indicated by annual income <$ 10,000, approximately 50% of the U.S. Department of Health and Human Services poverty line for families with two to five members at the time of data collection) |
| any transition to new primary caregiver from birth through 5 years |
| any maternal subscale score on the Brief Symptom Inventory(28) greater than the clinical cut-off point (Derogatis, 1993) through 3 years |
| maternal depression at least one standard deviation above the mean from birth through 3 years indicated by the Beck Depression Inventory(29) (M=9.44, SD=7.03; Beck, Steer & Brown, 1996) |
| quality of the living environment at least one standard deviation below the mean at 2.5 years as indicated by the Home Inventory(30) (M=37.69, SD=4.39; Caldwell & Bradley, 2003) |
| community violence at least one standard deviation above the mean from birth through 3 years as indicated by the Neighborhood Problems section of the Lifestyle Interview (M=1.70, SD=1.84) |
| social position at least one standard deviation below the sample mean from birth through 5 years as indicated by the Index of Social Position which reflects a weighted average of parental occupational status and education level(31,32) (M=31.32, SD=8.92; see Hollingshead, 1975; LaGasse et al., 1999) |
Hair Collection.
A proximal crown head hair sample was obtained from the child at 10–11 years of age. Hair was collected from 87 of the 100 participants. There were 5 refusals, 1 hair specimen was too short, and the reason not collected was unknown for 7 children. The 3-cm hair segment was analyzed by the United States Drug Testing Laboratories (USDTL) using a new liquid chromatography-tandem mass spectrometry assay to measure cortisol and cortisone.(13) Specimens were analyzed according to a slightly modified version of a previously published procedure.(14) Of the 87 hair specimens, 63 had a value of “0” for cortisol indicating that cortisol was not detected. However of the 87 samples, 72 had positive cortisone values, therefore cortisone data was used in the analysis. Raw cortisone values (pg/mg) were right skewed and normalized using a natural log transformation. Outliers 3 SD or more from the mean were winsorized by replacing their value with the nearest value less than 3 SD from the mean. Cortisone is used as a biological marker of chronic stress in this study.
Data Analysis
One-way analysis of variance (ANOVA) was used for analysis of continuous measures and Chisquare for categorical measures. Hierarchical multiple regression using SPSS (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp.) examined the effects of Prenatal MA exposure, early adversity, and chronic stress on DNA methylation of HSD11B2 after controlling for covariates. The models were adjusted for the following covariates: gestational age at birth, quantity of self-reported use of tobacco, alcohol, and marijuana use. Covariates were selected based on maternal and child characteristics that significantly differed between mothers in the MA exposure and comparison groups (P<0.05) yet were not highly correlated with other covariates (r>0.70), trends in the literature, and conceptual reasons.
RESULTS
Included mothers reported greater prenatal alcohol use and differed by race with a higher number of white and Hispanic included in the study relative to those who were not included in this sample. There were no differences in newborn characteristics between children who were included in the study and those not included (Table 2). Children with prenatal MA exposure were exposed to more tobacco and marijuana, were more likely to be born to single mothers, had lower socioeconomic status (SES) and were less likely to have received any prenatal care relative to the COMP group (Table 3). The children exposed to methamphetamine weighed on average 267 g less, measured 2.9 cm shorter, had 0.7 cm smaller head circumference, and were 1.36 weeks younger than children in the COMP group.
Table 2.
Comparison of dyads included and not included in the analysis
| N (%) or mean (SD) | Included N=100 | Not included N=212 | P |
|---|---|---|---|
| Maternal/demographic characteristics | |||
| Race | <0.01 | ||
| White | 33 (33.0%) | 49 (23.1%) | |
| Hispanic | 35 (35.0%) | 51 (24.1%) | |
| Hawaii & Pacific Islander | 15 (15.0%) | 56 (26.4%) | |
| Asian | 12 (12.0%) | 45 (21.2%) | |
| Black | 3 (3.0%) | 11 (5.2%) | |
| American Indian | 1 (1.0%) | 0 (0.0%) | |
| Other | 1 (1.0%) | 0 (0.0%) | |
| Low SES | 27 (27.0%) | 59 (28.1%) | 0.84 |
| Partner at birth | 53 (53.0%) | 118 (55.7%) | 0.66 |
| Education <12 years | 48 (48.0%) | 95 (45.0%) | 0.62 |
| Prenatal care | 95 (95.0%) | 196 (92.5%) | 0.40 |
| Prenatal tobacco use | 50 (50.0%) | 115 (54.2%) | 0.48 |
| Average number of cigarettes/day across pregnancy | 3.20 (5.87) | 4.20 (7.40) | 0.24 |
| Prenatal alcohol use | 30 (30.0%) | 41 (19.3%) | 0.04 |
| Average oz. absolute alcohol/day across pregnancy | 0.05 (0.30) | 0.07 (0.43) | 0.71 |
| Prenatal marijuana use | 17 (17.0%) | 31 (14.6%) | 0.59 |
| Average number of joints/day across pregnancy | 0.03 (0.15) | 0.09 (1.03) | 0.53 |
| Prenatal methamphetamine (MA) use | 55 (55.0%) | 102 (48.1%) | 0.26 |
| Maternal age (yr) | 25.24 (6.21) | 25.38 (5.78) | 0.85 |
| Neonatal characteristics | |||
| Sex (Male) | 52 (52.0%) | 115 (54.2%) | 0.71 |
| Birth weight (g) | 3229 (642) | 3234 (580) | 0.95 |
| Length (cm) | 50.0 (3.9) | 51.0 (3.0) | 0.17 |
| Head Circumference (cm) | 33.8 (1.9) | 33.9 (1.8) | 0.77 |
| Gestational age (wks) | 38.4 (2.6) | 38.7 (1.8) | 0.25 |
| Small for gestational age | 10 (10.0%) | 30 (14.2%) | 0.31 |
Table 3.
Maternal and neonatal characteristics by MA exposure
| N (%) or mean (SD) | PME N=55 | Comparison N=45 | P |
|---|---|---|---|
| Maternal/demographic characteristics | |||
| Race | 0.72 | ||
| White | 18 (32.7%) | 15 (33.3%) | |
| Hispanic | 18 (32.7%) | 17 (37.8%) | |
| Hawaii & Pacific Islander | 10 (18.2%) | 5 (11.1%) | |
| Asian | 7 (12.7%) | 5(11.1%) | |
| Black | 1 (1.8%) | 2 (4.4%) | |
| American Indian | 1 (1.8%) | 0 (0.0%) | |
| Other | 0 (0.0%) | 1 (2.2%) | |
| Low SES | 23 (41.8%) | 4 (8.9%) | <0.01 |
| Partner at birth | 24 (43.6%) | 29 (64.4%) | 0.04 |
| Education <12 years | 31 (56.4%) | 17 (37.8%) | 0.06 |
| Prenatal care | 50 (90.9%) | 45 (100.0%) | 0.04 |
| Prenatal tobacco use | 40 (72.7%) | 10 (22.2%) | <0.01 |
| Average number of cigarettes/day across pregnancy | 5.50 (7.06) | 0.44 (1.50) | <0.01 |
| Prenatal alcohol use | 18 (32.7%) | 12 (26.7%) | 0.51 |
| Average oz. absolute alcohol/day across pregnancy | 0.09 (0.40) | 0.01 (0.03) | 0.18 |
| Prenatal marijuana use | 15 (27.3%) | 2 (4.4%) | <0.01 |
| Average number of joints/day across pregnancy | 0.03 (0.14) | 0.02 (0.15) | 0.75 |
| Maternal age (yr) | 25.11 (5.79) | 25.40 (6.75) | 0.82 |
| GA at 1st prenatal visit | 13.6 (8.04) | 8.2 (5.77) | <0.01 |
| Neonatal characteristics | |||
| Sex (Male) | 31 (56.4%) | 21 (46.7%) | 0.33 |
| Birth weight (g) | 3109 (687) | 3376 (554) | 0.04 |
| Length (cm) | 48.7 (4.1) | 51.6 (2.8) | <0.01 |
| Head Circumference (cm) | 33.5 (2.0) | 34.2 (1.6) | 0.05 |
| Gestational age (wks) | 37.75 (3.12) | 39.11 (1.60) | <0.01 |
| Small for gestational age | 6 (10.9%) | 4 (8.9%) | 0.74 |
| DNA methylation ofHSBUB2 | |||
| CpG site 1 | 1.3 (1.7) | 1.6 (1.6) | 0.52 |
| CpG site 2 | 1.4 (1.7) | 0.3 (0.6) | <0.01 |
| CpG site 3 | 0.5 (0.7) | 0.6 (0.8) | 0.80 |
| CpG site 4 | 3.8 (2.2) | 3.6 (2.0) | 0.69 |
To determine if Prenatal MA exposure use, early adversity, and chronic stress predicts greater DNA methylation, hierarchical linear regression was conducted with DNA methylation as the dependent variable. In an initial model, Prenatal MA exposure children had higher levels of DNA methylation at the HSD11B2 CpG site 2 than children in the COMP group (B=0.34, 95% CI:0.11, 0.57, P=<0.01) (Table 4, Model 1). In Model 2, the early adversity index was added to the model. Higher scores on the early adversity index related to higher levels of DNA methylation at CpG site 2 (B=0.11, 95% CI:0.03, 0.19, P<0.05), however Prenatal MA exposure was no longer statistically significant. However, while the addition of cortisone was not related to amount of DNA methylation at CpG2 (B=−0.13, 95% CI: −0.36, 0.09, P=0.23) the inclusion of cortisone resulted in significant effects for early adversity (B=0.11, 95% CI: 0.03, 0.19, P<0.01) and Prenatal MA exposure (B=0.24, 95% CI:0.002, 0.48, P<0.05). In the final model (Model 4), covariates were added, including: gestational age at birth, and count variables reflecting the quantity of tobacco, alcohol, and marijuana consumed prenatally. None of the covariates were related to DNA methylation at the HSD11B2 CpG2 site, but the statistically significant effects of early adversity (B=0.11, 95% CI: 0.02, 0.19, P<0.05) and Prenatal MA exposure (B=0.32, 95% CI: 0.04, 0.60, P=0.03) remained. There was no significant interaction between Prenatal MA exposure and early adversity.
Table 4.
Summary of multivariable analysis for predicting increased methylation at CpG2 of HSD11B2
| Model 1 | Model 2 | Model 3 | Model 4 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Variable | Estimate* | 95% CI | P | Estimate | 95% CI | P | Estimate | 95% CI | P | Estimate | 95% CI | P |
| Step 1 PME Exposure | 0.34 | 0.11, 0.57 | <0.01 | 0.23 | −0.01, 0.46 | 0.06 | 0.24 | 0.002, 0.48 | <0.05 | 0.32 | 0.04, 0.60 | 0.03 |
| Step 2 Early adversity | 0.11 | 0.03, 0.19 | <0.05 | 0.11 | 0.03, 0.19 | <0.01 | 0.11 | 0.02, 0.19 | <0.05 | |||
| Step 3 Cortisone | −0.13 | −0.36, 0.09 | 0.23 | −0.13 | −0.36, 0.09 | 0.24 | ||||||
| Step 4 | ||||||||||||
| Average number of cigarettes/day | −0.01 | −0.03, 0.02 | 0.57 | |||||||||
| Average absolute alcohol oz./day | −0.21 | −0.60, 0.19 | 0.30 | |||||||||
| Average number of joints/day | 0.39 | −0.41, 1.2 | 0.34 | |||||||||
| Gestational age | 0.02 | 0.02 | 0.38 | |||||||||
Estimate is unstandardized regression coefficient for percent change in DNA methylation at CpG site 2
DISCUSSION
This is the first study to measure epigenetic effects of Prenatal MA exposure on a stress-related gene. It is also the first study to measure the effects of postnatal early adversity on a stress-related gene in Prenatal MA exposure children aged 10–11 years. We found that Prenatal MA exposure and the postnatal early adversity index was associated with greater DNA methylation of HSD11B2 with adjustment for a biological marker of chronic stress (hair cortisone). Critically, these epigenetic effects represent 2 distinct pathways, a biological pathway due to in utero drug exposure and a pathway related to adversity in the postnatal environment. These findings provide evidence for the biological embedding of early experiences during sensitive periods that could have long term physical and mental health implications.(5)
We measured DNA methylation in the HSD11B2 promoter region because this region regulates the neuroendocrine system, including the HPA axis and stress response. The specific epigenetic effects that we observed involved increases in DNA methylation of HSD11B2. Increased methylation at CpG sites in the promoter and first exon of HSD11B2 inhibits the activity of HSD11B2 and thereby reduces the capacity to inactivate cortisol.(15) The resulting increased levels of circulating cortisol can be harmful as stress hormones are widely implicated in the development of mental and physical disorders, especially when related to chronic, toxic stress.(5) Toxic stress disrupts brain circuitry and normal organ and metabolic systems during sensitive developmental periods. These disruptions can produce physiological changes leading to learning and behavior impairments, as well as increased risks for developing physical and mental illness.(16) Toxic stress and early adversity are potential drivers of health disparities.(5) The Adverse Childhood Experiences Study found that parental substance abuse is a stressor that can yield toxic stress in children.(17) We have documented toxic stress both at the biological (Prenatal MA exposure) and environmental (early adversity) levels.
Epigenetic effects are highly tissue specific. We have previously measured changes in DNA methylation of HSD11B2 in placenta in several settings. We have shown that prenatal socioeconomic adversity affects placental HSD11B2 methylation.(18) There was a sex related difference. We have compared placental HSD11B2 methylation to measures of fetal growth and neural behavioral assessment.(19) We showed a higher level of methylation was greatest in infants with low birth weight. This increase in HSD11B2 methylation was associated with reduced neurodevelopmental scores on quality of movement. These results suggest that the programming effects of in utero exposures are among the biological pathways may be mediated by alterations in the epigenetic alterations in this important gene. We note that these are small magnitude effects. Small-magnitude epigenetic effect sizes are common findings in environmental epigenetic studies resulting from such exposures. A small difference in methylation means that a small fraction of the cells exhibits this difference at a particular CpG site. Depending on the identity and nature of that cell, small differences could substantially affect functioning of that cell and the functioning of that cell’s progeny because of mitotic heritability of DNA methylation. (20) Future work examining the functional implications of these small differences would be warranted.
The absence of cortisol in the hair was an unexpected finding. Unlike plasma in which concentrations of cortisol are significantly higher than that of cortisone, in human hair cortisone concentrations are higher than cortisol.(14) Higher concentrations of cortisone than cortisol in hair may be due to increased activity of HSD11B2 in the hair bulb.(21) Thus hair cortisone is thought to provide a more reliable measure of HPA axis activity than cortisol.(22) Alternatively, the lack of measured cortisol in the hair samples may be secondary to allostatic load which would be consistent with previous work showing cortisol blunting in children with prenatal methamphetamine(23) or cocaine exposure. (24)
This study is limited by its small sample size. However, this novel study can make a significant contribution to the field of epigenetics despite the small sample size because the findings provide an impetus for further study with additional subjects. Another possible limitation of this study is that DNA samples were collected from peripheral tissue. Although it is unlikely that patterns of DNA methylation in buccal cells will exactly match those of neural tissue, buccal cells and neural cells are both derived from the same germ layer, the ectoderm, during fetal development, and recent reports suggest that there are similarities in early epigenetic patterning as well as in susceptibility to epigenetic change during the developmental period.(25),(26) We have also demonstrated relationships between buccal cell derived DNA methylation and infant neurobehavioral outcomes.(27) We also acknowledge that due to the cell type specificity of DNA methylation, what we have observed as differential methylation may represent changes in the underlying cell type proportions, although this is still a biologically interesting phenomenon and is worthy of further examination. Finally, although our findings clearly have implications for the development of mental health and physical disorders we do not have measures of these outcomes.
CONCLUSION
Our findings provide a new insight into the study of prenatal and postnatal programming models in children with prenatal substance exposure which is somewhat surprising given the known relationship between maternal drug use during pregnancy and toxic stress. Intrauterine substances could act as stressors, or challenges that disrupt fetal-placental homeostasis, through DNA methylation, thus prompting the fetus to make compensatory adjustments. These homeostatic adjustments could, in turn, trigger the reprogramming, or recalibration, of physiological systems. Early adversity in the postnatal environment could produce a second order reprogramming of physiological systems that can be expected to have long term developmental and physical consequences.
Supplementary Material
Impact Statement:
Prenatal methamphetamine exposure has been associated with developmental issues in newborns, yet little is known about the stress pathophysiology of methamphetamine on neurobehavior.
This is the first evidence that prenatal methamphetamine exposure acts as a stressor, confirming the third pathophysiology of methamphetamine exposure.
Financial Support:
Supported by National Institute on Drug Abuse (NIDA) Grant 1RO1DA014948 and in part by the National Center on Research Resources Grant 1UL1-TR000124 and 5P20 RR11091.
Footnotes
Disclosure: The authors declare no conflict of interest
Statement of Consent: The study was approved by Western Institutional Review Board, Puyallup, WA (HI) and the John F. Wolf Human Subjects Committee, Los Angeles, CA. All child participants in this study provided written informed assent in addition to the parental consent.
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Associated Data
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