Association of Glyphosate Exposure with Blood DNA Methylation in a Cross-Sectional Study of Postmenopausal Women

Abstract

Background: Glyphosate is the most commonly used herbicide in the world and is purported to have a variety of health effects, including endocrine disruption and an elevated risk of several types of cancer. Blood DNA methylation has been shown to be associated with many other environmental exposures, but to our knowledge, no studies to date have examined the association between blood DNA methylation and glyphosate exposure.

Objective: We conducted an epigenome-wide association study to identify DNA methylation loci associated with urinary glyphosate and its metabolite aminomethylphosphonic acid (AMPA) levels. Secondary goals were to determine the association of epigenetic age acceleration with glyphosate and AMPA and develop blood DNA methylation indices to predict urinary glyphosate and AMPA levels.

Methods: For 392 postmenopausal women, white blood cell DNA methylation was measured using the Illumina Infinium MethylationEPIC BeadChip array. Glyphosate and AMPA were measured in two urine samples per participant using liquid chromatography-tandem mass spectrometry. Methylation differences at the probe and regional level associated with glyphosate and AMPA levels were assessed using a resampling-based approach. Probes and regions that had an false discovery rate $q<0.1$ in $\ge 90\%$ of 1,000 subsamples of the study population were considered differentially methylated. Differentially methylated sites from the probe-specific analysis were combined into a methylation index. Epigenetic age acceleration from three epigenetic clocks and an epigenetic measure of pace of aging were examined for associations with glyphosate and AMPA.

Results: We identified 24 CpG sites whose methylation level was associated with urinary glyphosate concentration and two associated with AMPA. Four regions, within the promoters of the MSH4, KCNA6, ABAT, and NDUFAF2/ERCC8 genes, were associated with glyphosate levels, along with an association between ESR1 promoter hypomethylation and AMPA. The methylation index accurately predicted glyphosate levels in an internal validation cohort. AMPA, but not glyphosate, was associated with greater epigenetic age acceleration.

Discussion: Glyphosate and AMPA exposure were associated with DNA methylation differences that could promote the development of cancer and other diseases. Further studies are warranted to replicate our results, determine the functional impact of glyphosate- and AMPA-associated differential DNA methylation, and further explore whether DNA methylation could serve as a biomarker of glyphosate exposure. https://doi.org/10.1289/EHP10174.

Figure 1.

Results from probe-level differential methylation analysis for urinary glyphosate in 332 postmenopausal California women. The volcano plot shows delta-M (difference in methylation M value for a 1-unit increase in the natural log of glyphosate) on the horizontal axis and $-{\log }_{10}\left(\mathrm{p}\right)$ on the vertical axis. Linear models adjusted for urinary creatinine, age, race/ethnicity, BMI, smoking status, alcohol consumption, self-reported organic eating habits, diet quality (Healthy Eating Index), estimated WBC type proportions, batch, and position on chip, were fitted for all probes on the Illumina HumanMethylationEPIC array, which remained after quality filtering. Results were pooled from 1,000 random subsamples of the training set ($n=299$ individuals per subsample). Probes that were statistically significant with FDR $q<0.1$ in 90% or more subsamples are marked as hypermethylated or hypomethylated. Note: BMI, body mass index; FDR, false discovery rate; WBC, white blood cell.

Figure 2.

Enrichment analysis for genomic context of glyphosate-associated differentially methylated probes (DMPs) identified in 332 postmenopausal California women. The proportions of glyphosate-associated DMPs compared to other probes on the array in each genomic context were compared with Fisher’s exact test; significant ($<0.05$) $p$-values are labeled.

Figure 3.

Performance of methylation index using 24 CpG sites to predict the natural logarithm of urinary glyphosate concentration in the training set (A) and the validation set (B, C). Panel C shows the methylation index was significantly associated with glyphosate tertile [median (IQR) $-2.4$ ($-2.7$ to $-2.2$ for lowest tertile vs. $-2.0$ ($-2.3$ to $-1.6$) for highest tertile, ANOVA $p=0.009$] in the validation set. Panel D shows the classification performance of the methylation index in the validation set for classifying the highest vs. the lowest tertile of urinary glyphosate. The index was developed using elastic net regression on methylation $\mathrm{\beta }$ values of differentially methylated probes associated with glyphosate in the training set, a population of 332 postmenopausal California women. Note: ANOVA, analysis of variance; IQR, interquartile range.