Cyp2e1 Paracetamol

[Breogan Heflin]

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CYP2El, a cytochrome P-450 that is well conserved across mammalian species, metabolizes ethanol and many low molecular weight toxins and cancer suspect agents. The cyp2e1 gene was isolated, and a mouse line that lacks expression of CYP2E1 was generated by homologous recombination in embryonic stem cells. Animals deficient in expression of the enzyme were fertile, developed normally, and exhibited no obvious phenotypic abnormalities, thus indicating that CYP2E1 has no critical role in mammalian development and physiology in the absence of external stimuli. When cyp2el knockout mice were challenged with the common analgesic acetaminophen, they were found to be considerably less sensitive to its hepatotoxic effects than wild-type animals, indicating that this P-450 is the principal enzyme responsible for the metabolic conversion of the drug to its active hepatotoxic metabolite.

In a US birth cohort, we found detection of cord biomarkers of acetaminophen was associated with DNAm level of CYP2E1 in cord blood. Our findings suggest that DNA methylation of CYP2E1 may be an important regulator of acetaminophen levels in newborns.

Acetaminophen, also known as paracetamol, is the most widely used over-the-counter analgesic and antipyretic medication in pregnant women worldwide. In the USA, over 65% of pregnant women reported any use of acetaminophen during pregnancy, and 48% reported the use of acetaminophen in the third trimester [1]. Existing literature has shown transplacental passage of acetaminophen and its metabolites to the developing offspring after maternal use [2,3,4]. However, little is known about the biological processes that regulate acetaminophen levels in the offspring.

A total of 570 participants were included in the current study. Acetaminophen, acetaminophen glucuronide and 3-(N-acetyl-L-cysteine-S-yl)-acetaminophen were detected in 16.8%, 17.5% and 28.9% of the cord plasma samples, respectively. Distributions of the raw intensities from liquid chromatography-tandem mass spectrometry (LC-MS) and the corresponding background noise levels are shown in Additional file 1: Fig. 1. We observed high detection agreement among the three acetaminophen biomarkers measured. Among samples with detectable acetaminophen, 92% also had detectable acetaminophen glucuronide and 95% had detectable 3-(N-acetyl-L-cysteine-S-yl)-acetaminophen.

Table 1 shows the characteristics of study participants, by detection of acetaminophen. Overall, 66% of the participants had a non-Hispanic black mother, 28% had a Hispanic mother, and 6% had a non-Hispanic white mother. We observed higher proportions of Hispanic mother and non-Hispanic white mother and a lower proportion of non-Hispanic black mother among participants with detectable cord acetaminophen than the not detected group. In addition, participants with detectable acetaminophen in cord plasma had lower gestational age at birth, lower birthweight and were more likely to have primiparous parity, maternal chronic diabetes, maternal preeclampsia and intrauterine inflammation, compared to participants without detectable acetaminophen in cord plasma.

As a sensitivity analysis, we further adjusted for maternal race and ethnicity in the linear regression models to evaluate whether this variable may explain the associations found in the main analysis. The results did not change after the further adjustment, suggesting that the association between cord acetaminophen and methylation at CYP2E1 was unlikely driven by maternal race or ethnicity (Additional file 1: Table 3).

Notably, at all the CpG sites showing a significant difference in methylation variation, we also observed a lower average methylation level among neonates with an acetaminophen detection than neonates without. The %difference in methylation at these sites was comparable to those at differentially methylated CpG sites (Fig. 2). Linear regression assumes equal variance (i.e., the variance of dependent variable is constant at any level of an independent variable). However, at the CpGs we identified as variably methylated, the assumption of equal variance was not met. To address this concern, we performed an ad hoc analysis using weighted linear regression to test for differential methylation. The results remain largely unchanged.

Moreover, we observed a larger sample variance of methylation at the differential and/or variably methylated CpGs (located at CpG island or South Shore) compared to the surrounding CpGs (Table 3), suggesting that DNA methylation at these sites is likely under the influence of some upstream factors, either environmental or genetic, and their interactions. A previous study found that genetic polymorphism can affect the methylation level of CYP2E1 in newborns [13]. It is possible that the association between acetaminophen and CYP2E1 DNA methylation varies by genotype, particularly at CpG sites with different DNA methylation variance. Unfortunately, we were unable to directly assess this aspect as genetic data are not currently available for this analysis, which we recognize as a limitation. Further studies should incorporate genetic data to gain further insight into the complex interplay between genetics, acetaminophen exposure and DNA methylation in relation to CYP2E1 regulation.

Previous EWAS has scanned the genome to identify CpG sites showing differential methylation in cord blood [16, 17] and placenta [18] related to prenatal exposure to acetaminophen. However, they did not identify CpG sites in CYP2E1, after correcting for multiple comparisons. Several major differences between our study and the previous EWAS, including study design, characteristics of participants, exposure measurement method and timing, and biospecimen type could potentially explain the inconsistency. Additionally, previous EWAS studies are likely still underpowered to detect the methylation changes at CYP2E1 associated with acetaminophen exposure. In fact, at all the CpG sites of CYP2E1 we identified in the study, the directions of effect are consistent with the results from the placenta EWAS [18] in extremely low gestational age newborns.

A major strength of our study is the use of rich empiric molecular data obtained from BBC participants, which allowed us to evaluate the association between an objective measure of acetaminophen exposure, i.e., cord plasma metabolite measures, and CYP2E1 cord blood DNA methylation measures. The objectively measured acetaminophen biomarkers were less prone to measurement errors due to inaccurate self-report and were able to capture acetaminophen exposure during a specific short time window before delivery.

We observed significant differences in maternal pregnancy conditions and birth outcomes between individuals with detectable and undetectable acetaminophen metabolites in cord blood. Specifically, mothers of neonates with detectable acetaminophen showed a higher prevalence of intrauterine inflammation, characterized by common symptoms such as intrapartum maternal fever [20], and preeclampsia, indicated by symptoms like severe headache and epigastric pain [21]. These pregnancy conditions are known risk factors for preterm birth and low birth weight [22, 23], which may explain the significant differences in these birth outcomes between the acetaminophen groups. These observations underscore the importance of carefully considering potential confounding by indication in our analysis. To address this concern, we adjusted for relevant maternal clinical variables during pregnancy, aiming to minimize the potential impact of confounding. Furthermore, we conducted sensitivity analysis and observed similar changes in methylation among newborns whose mother did not have gestational diabetes. This suggested that the results were not driven by residual confounding due to gestational diabetes, a pregnancy condition that has been previously associated with CYP2E1 DNA methylation [11]. However, it is important to acknowledge that despite our best efforts, unmeasured confounding remains a potential limitation due to the inherent nature of observational data in this study.

In this study, we considered the measured acetaminophen metabolites as indicators of maternal acetaminophen exposure, because unlike NAPQI (the toxic metabolite), production of these metabolites is not directly related to CYP2E1 enzyme activities. We propose two conceptual models that could explain the observed association between acetaminophen metabolites and DNA methylation at the CYP2E1 locus (Fig. 4). DNA methylation of CYP2E1 may modify the effect of maternal acetaminophen exposure on production of NAPQI and its potential fetal effects (Fig. 4A). It is also possible that DNA methylation of CYP2E1 lies on the causal pathway from maternal acetaminophen exposure to NAPQI production and acts as a mediator (Fig. 4B). However, due to the study design and the absence of NAPQI measurements in the current study, we were unable to differentiate between these two models. Nevertheless, the findings from this study are significant as they provide crucial initial evidence in population-based cohort, demonstrating an association between acetaminophen metabolites and CYP2E1 DNA methylation level. This association underscores the need for further investigation into the specific biological mechanisms involved, whether they pertain to effect modification or mediation by DNA methylation at the CYP2E1 locus. Future studies employing longitudinal data and/or experimental designs, along with the inclusion of direct measurement of acetaminophen toxicity, are warranted to elucidate the effects of maternal acetaminophen exposure on fetal development and the precise roles of DNA methylation in regulating this process.

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