In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Vitamin B12 is an essential component of the 1-carbon cycle and data from both human
and animal studies strongly support the concept that maternal and/or fetal vitamin
B12 availability has important influences on health outcomes, including fetal growth,
neurodevelopment, and longer term cardiometabolic disease risk 34]-36]. Herein, we show, for the first time, that maternal smoking, which is known to reduce
the concentrations of vitamin B12 in pregnant women and their infants 17],18], is associated with widespread effects on 1-carbon metabolism, including alterations
in levels of enzyme transcripts and co-factors. Importantly, we also show that these
effects are present during early development and in a key target tissue, the fetal
liver.

The precise role of in utero methyl donor availability and/or effects on the 1-carbon pathway in the programming
of later disease risk remains unknown; however, one important mechanism may be through
changes in DNA methylation 11]. In animal models, dietary availability of methyl donors during pregnancy has a profound
influence on both phenotype and DNA methylation in offspring 12],37]. In humans, maternal vitamin B12 concentrations correlate inversely with global DNA
methylation in umbilical cord blood 15], and an inverse correlation between cord plasma homocysteine concentrations and genome-wide
DNA methylation at repetitive sequences has been reported 38]. Additionally, genome-wide and candidate gene studies have identified alterations
in DNA methylation in association with maternal smoking in accessible tissues at birth,
including cord blood, buccal cells, and placenta 8]-10]. Our findings that maternal smoking is associated with effects on DNA methylation
strengthen and extend previous studies by showing that such changes are detectable
in the fetal liver, a major target organ for developmental programming effects 39],40], and that they are present during early development.

The imprinted gene IGF2 has a major role in mediating fetal growth 41] and altered DNA methylation at the IGF2 DMRs is known to be associated with human syndromic growth disorders, including Silver
Russell and Beckwith Wiedemann syndromes 42]. Recent data suggest that more subtle alterations in DNA methylation at IGF2 are associated with patterns of fetal growth within the normal range 43]-46] and some studies report that infants born small for gestational age have reduced
DNA methylation at IGF2 in cord blood 43]. Thus, reduced IGF2 methylation in fetal liver during early-mid gestation could be one mechanism for
the reduction in birthweight seen with prenatal smoke exposure. DNA methylation at
IGF2 appears to be particularly sensitive to the prenatal environment 9],47],48] and our data support the concept that methylation at IGF2 may be a useful marker of in utero exposures 9]. DNA methylation at GR may also be influenced by diverse environmental cues in early life and animal and
human studies suggest that downstream effects on the expression of GR may play a role in mediating the associations between the early life environment
and subsequent cardiometabolic and neuropsychiatric disorders 31],39],49],50]. The methylation changes we report at both IGF2 and GR in association with smoke exposure are relatively small; however, they resemble those
described in previous studies, including studies of prenatal smoke exposure 9],47],51]. Additionally, the observed alterations in DNA methylation were region- and CpG site-specific,
consistent with reports in both animal models and human studies 31],52]. In agreement with previously published studies in humans, DNA methylation levels
did not necessarily predict changes in transcript levels, notably at IGF2, suggesting that there are alternative and/or additional mechanisms driving changes
in transcript expression 53]. Rather, such changes in DNA methylation may result in genes being ‘poised’ for activation
in response to future events 53],54].

Sex differences were evident in DNA methylation, which was higher at IGF2 and lower at GR in females compared with males and the most marked changes in DNA methylation in
association with maternal smoking occurred at these loci. Sexually dimorphic changes
in DNA methylation have been reported as a consequence of early life nutritional challenges
and exposure to maternal smoking 9],51],55],56]. The proposed mechanisms to explain these observations include differences in the
timing and speed of development, the influence of sex steroids, especially androgens,
and/or sex chromosome complement 22],57]. Our results suggest that sex differences in the concentrations of vitamin B12 and
homocysteine and in the expression of genes important in 1-carbon metabolism and DNA
methylation could also be important in determining the sex-specific effects of exposure
to an adverse environment in early life. Consistent with our previous studies 21],22], the smoke-induced changes in cobalt, vitamin B12, and homocysteine, and in the expression
of enzymes involved in both the 1-carbon cycle and DNA methylation, resulted in profiles
which resembled those seen in the opposite sex (summarized in Figure 5). These clearly suggest that smoke exposure results in masculinization of the female
liver and feminization of the male liver (Figure 5).

Figure 5. Summary of effects of fetal sex and maternal smoking on the human fetal second trimester
liver 1-carbon metabolism pathway. Redrawn and modified from 10]. In 7 out of 8 genes or compounds showing sex differences, maternal smoking reduced
the magnitude of the difference. Together with sex differences and sex-specific effects
of maternal smoking on DNA methylation this demonstrates the sensitivity of the fetal
liver to maternal smoking.

Temporal changes in gene expression and DNA methylation indicate that there may be
specific windows of susceptibility during development and, importantly, that this
is likely to differ between the sexes. Allelic methylation patterns at the IGF2 DMRs arise early in embryogenesis and change progressively during development 58] and here we show that this is also the case for GR between 12 and 20 weeks of gestation. Thus, since a worryingly high proportion of
women continue to smoke throughout pregnancy, the observed effects on DNA methylation
may change or become amplified with ongoing exposure. Additionally, the temporal changes
in DNA methylation profiles across gestation could be one explanation for the differences
between studies with respect to the association of DNA methylation at IGF2 and fetal growth 9],59]. Whilst the data shown here represent a single, albeit unique, 10-week snap-shot
of the effects of maternal smoking, they are consistent with previous studies showing
that maternal smoking is associated with altered DNA methylation in blood, buccal
cells, and placenta in exposed offspring at birth 8]. Importantly, these changes may be persistent and/or evolve postnatally since DNA
methylation patterns in peripheral blood and buccal cells in young children and in
peripheral blood in adolescence and young adulthood also associate with in utero exposure to maternal smoking 7],10],60],61]. Furthermore, some studies report that methylation differences only become apparent
some considerable time after in utero environmental exposures 51].