Environmental Concentrations of the Type 2 Diabetes Medication Metformin and Its Transformation Product Guanylurea in Surface Water and Sediment in Ontario and Quebec, Canada.

Metformin, used to treat Type 2 diabetes, is the active ingredient of one of the most prescribed drugs in the world, with over 120 million yearly prescriptions globally. In wastewater-treatment plants (WWTPs), metformin can undergo microbial transformation to form the product guanylurea, which could have toxicological relevance in the environment. Surface water samples from 2018 to 2020 and sediment samples from 2020 were collected from six mixed-use watersheds in Quebec and Ontario, Canada, and analyzed to determine the metformin and guanylurea concentrations at each site. Metformin and guanylurea were present above their limits of quantification in 51.0% and 50.7% of all water samples and in 64% and 21% of all sediment samples, respectively. In surface water, guanylurea was often present at higher concentrations than metformin, while the inverse was true in sediment, with metformin frequently detected at higher concentrations than guanylurea. In addition, at all sites influenced solely by agriculture, concentrations of metformin and guanylurea were <1 µg/L in surface water, suggesting that agriculture is not a significant source of these compounds in the investigated watersheds. These data suggest that WWTPs and potentially septic system leaks are the most likely sources of the compounds in the environment. Guanylurea was detected at many of these sites above environmental concentrations of concern, where critical processes in fish may be affected. Due to the scarcity of available ecotoxicological data and the prominence of guanylurea across all sample sites, there is a need to perform more toxicological investigations of this transformation product and revisit regulations. The present study will help provide toxicologists with environmentally relevant concentration ranges in Canada. Environ Toxicol Chem 2023;42:1709-1720. © 2023 His Majesty the King in Right of Canada and The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada.

Long and Short Duration Exposures to the Selective Serotonin Reuptake Inhibitors (SSRIs) Fluoxetine, Paroxetine and Sertraline at Environmentally Relevant Concentrations Lead to Adverse Effects on Zebrafish Behaviour and Reproduction.

Selective serotonin reuptake inhibitors (SSRIs) are currently the most prescribed class of psychotropic medications. Their increased global manufacture and use have become growing concerns for aquatic toxicologists and environmental biologists, who assess both the direct and indirect effects of substances on the environment and on human health. In order to assess the potential impact of environmentally relevant levels of SSRIs on fish development, behaviour and reproduction, we exposed juvenile and adult zebrafish to a select group of SSRIs using two separate exposure paradigms. In the first paradigm, juvenile zebrafish were exposed to Fluoxetine (Prozac), Paroxetine (Paxil), Sertraline (Zoloft) or a mixture of the three beginning at environmentally relevant levels (10 µg/L) for 135 days (long-term exposure) beginning at 5 days post fertilization (dpf). In the second paradigm, adult zebrafish were exposed to matching concentrations of the same SSRIs for 35 days (short-term exposure). The long-term exposure paradigm proved to have little to no overt effect on growth or development at sub-lethal concentrations (10 and 100 µg/L). However, both the stress/anxiety response (novel tank tests) and reproduction (fecundity and fertility) were dramatically reduced. Importantly, the short-term exposure of reproductively mature fish led to similar adverse effects on both the stress response and reproduction. Following both the short and long duration exposure paradigms, a 2-week washout period led to a small reduction in the adverse effects. These findings highlight the potential for SSRIs to negatively impact population dynamics in zebrafish and may be of particular value should they be found in other fish species in the environment.

P-Glycoprotein (P-gp)/ABCB1 plays a functional role in extravillous trophoblast (EVT) invasion and is decreased in the pre-eclamptic placenta.

Dysregulation of trophoblast differentiation is implicated in the placental pathologies of intrauterine growth restriction and pre-eclampsia. P-glycoprotein (P-gp encoded by ABCB1) is an ATP-binding cassette transporter present in the syncytiotrophoblast layer of the placenta where it acts as a molecular sieve. In this study, we show that P-gp is also expressed in the proliferating cytotrophoblast (CT), the syncytiotrophoblast (ST) and the extravillous trophoblast (EVT), suggesting our hypothesis of a functional role for P-gp in placental development. Silencing of ABCB1, via siRNA duplex, results in dramatically reduced invasion and migration, and increased tube formation and fusion in the EVT-like HTR8/SVneo cell line. In both EVT and CT explant differentiation experiments, silencing of ABCB1 leads to induction of the fusion markers human hCG, ERVW-1 and GJA1 and terminal differentiation of both trophoblast subtypes. Moreover, P-gp protein levels are decreased in both the villous and the EVT of severe early-onset pre-eclamptic placentas. We conclude that, in addition to its role as a syncytial transporter, P-gp is a key factor in the maintenance of both CT and EVT lineages and that its decrease in severe pre-eclampsia may contribute to the syncytial and EVT placental pathologies associated with this disease.

Lymphoblastoid cell lines reveal associations of adult DNA methylation with childhood and current adversity that are distinct from whole blood associations.

BACKGROUND: Some cohort studies bank lymphoblastoid cell lines (LCLs) as a renewable source of participant DNA. However, although LCL DNA has proved valuable for genetic studies, its utility in epigenetic epidemiology research is unknown. METHODS: To assess whether LCL DNA can be used for life-course environmental epigenomics, we carried out a pilot methylomic study (using the Illumina Infinium Human Methylation 450 BeadChip) of nil-passage, Epstein-Barr virus (EBV)-transformed LCLs (n = 42) and 28 matched whole-blood (WB) samples. These were from adult male participants of the British 1958 birth cohort, selected for extremes of social economic position (SEP) in childhood and adulthood, with additional information available on childhood abuse and prenatal tobacco exposure. RESULTS: We identified a small number of weak associations between these exposures and methylation levels of both individual CpG sites and genomic regions in WB and LCLs. However, only one of the regional, and none of the individual CpG site associations were common to both sample types. The lack of overlap between the associations detected in LCL compared with those found in WB could either be due to the EBV-transformation process, or to the fact that, unlike WB, LCLs are essentially a single (CD19+) cell type. We provide evidence that the latter is the more potent explanation, by showing that CpG sites known to be differentially methylated between different types of blood cell have significantly lower correlations (R = 0.11) than average (R = 0.2) between WB and LCLs in our datasets, whereas sites known to be affected by EBV-transformation have significantly higher correlations (R = 0.3). CONCLUSIONS: This small pilot study suggests that the DNA methylation profile of LCLs is more closely related to that of B cells than WB and, additionally, that LCLs may nevertheless be useful for life-course environmental epigenomics.

The multidrug resistance 1 gene Abcb1 in brain and placenta: comparative analysis in human and guinea pig.

The Multidrug Resistance 1 (MDR1; alternatively ABCB1) gene product P-glycoprotein (P-gp), an ATP binding cassette transporter, extrudes multiple endogenous and exogenous substrates from the cell, playing an important role in normal physiology and xenobiotic distribution and bioavailability. To date, the predominant animal models used to investigate the role of P-gp have been the mouse and rat, which have two distinct genes, Abcb1a and Abcb1b. In contrast, the human has a single gene, ABCB1, for which only a single isoform has been validated. We and others have previously shown important differences between Abcb1a and Abcb1b, limiting the extrapolation from rodent findings to the human. Since the guinea pig has a relatively long gestation, hemomonochorial placentation and neuroanatomically mature offspring, it is more similar to the human, and may provide a more comparable model for investigating the regulation of P-gp in the brain and placenta, however, to date, the Abcb1 gene in the guinea pig remains to be characterized. The placenta and fetal brain are barrier sites that express P-gp and that play a critical role of protection of the fetus and the fetal brain from maternally administered drugs and other xenobiotics. Using RNA sequencing (RNA-seq), reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (QPCR) to sequence the expressed isoforms of guinea pig Abcb1, we demonstrate that like the human, the guinea pig genome contains one gene for Abcb1 but that it is expressed as at least three different isoforms via alternative splicing and alternate exon usage. Further, we demonstrate that these isoforms are more closely related to human than to rat or mouse isoforms. This striking, overall similarity and evolutionary relatedness between guinea pig Abcb1 and human ABCB1 indicate that the guinea pig represents a relevant animal model for investigating the function and regulation of P-gp in the placenta and brain.

Human leukocyte telomere length is associated with DNA methylation levels in multiple subtelomeric and imprinted loci.

In humans, leukocyte telomere length (LTL) is positively correlated with lifespan, and shorter LTL is associated with increased risk of age-related disease. In this study we tested for association between telomere length and methylated cytosine levels. Measurements of mean telomere length and DNA methylation at >450,000 CpG sites were obtained for both blood (N = 24) and EBV-transformed cell-line (N = 36) DNA samples from men aged 44-45 years. We identified 65 gene promoters enriched for CpG sites at which methylation levels are associated with leukocyte telomere length, and 36 gene promoters enriched for CpG sites at which methylation levels are associated with telomere length in DNA from EBV-transformed cell-lines. We observed significant enrichment of positively associated methylated CpG sites in subtelomeric loci (within 4 Mb of the telomere) (P < 0.01), and also at loci in imprinted regions (P < 0.001). Our results pave the way for further investigations to help elucidate the relationships between telomere length, DNA methylation and gene expression in health and disease.

Childhood abuse is associated with methylation of multiple loci in adult DNA.

BACKGROUND: Childhood abuse is associated with increased adult disease risk, suggesting that processes acting over the long-term, such as epigenetic regulation of gene activity, may be involved. DNA methylation is a critical mechanism in epigenetic regulation. We aimed to establish whether childhood abuse was associated with adult DNA methylation profiles. METHODS: In 40 males from the 1958 British Birth Cohort we compared genome-wide promoter DNA methylation in blood taken at 45y for those with, versus those without, childhood abuse (n = 12 vs 28). We analysed the promoter methylation of over 20,000 genes and 489 microRNAs, using MeDIP (methylated DNA immunoprecipitation) in triplicate. RESULTS: We found 997 differentially methylated gene promoters (311 hypermethylated and 686 hypomethylated) in association with childhood abuse and these promoters were enriched for genes involved in key cell signaling pathways related to transcriptional regulation and development. Using bisulfite-pyrosequencing, abuse-associated methylation (MeDIP) at the metalloproteinase gene, PM20D1, was validated and then replicated in an additional 27 males. Abuse-associated methylation was observed in 39 microRNAs; in 6 of these, the hypermethylated state was consistent with the hypomethylation of their downstream gene targets. Although distributed across the genome, the differentially methylated promoters associated with child abuse clustered in genome regions of at least one megabase. The observations for child abuse showed little overlap with methylation patterns associated with socioeconomic position. CONCLUSIONS: Our observed genome-wide methylation profiles in adult DNA associated with childhood abuse justify the further exploration of epigenetic regulation as a mediating mechanism for long-term health outcomes.

Knockdown of RARB2 identifies a dual role in cancer.

Two chemoprevention trials have shown that retinoic acid (RA) may be harmful in patients at risk for lung cancer, and RA administration to this high-risk group results in RARB2 reactivation. Although RARB2 is thought to possess tumor suppressive activity, its expression has recently been correlated with poorer prognosis in patients with nonsmall cell lung cancer. We hypothesized that RARB2 expression is necessary for the growth and maintenance of the oncogenic phenotype in lung cancer cells in which RARB2 has not been inactivated. We tested various antisense oligodeoxynucleotides (ASO) against RARB2 in multiple lung cancer cell lines and used microarray technology to compare the patterns of gene expression following ASO treatment versus RA treatment in the A-549 lung cancer cell line. We show that ASO treatment reduces proliferation and causes apoptosis in 3 RARB2-expressing lung cancer cell lines but has no apparent effect in at least two other lung cancer cells lines having lost RARB2 expression or one normal lung RARB2-expressing cell line; we demonstrate a correlation between resulting RARB2 expression levels and cell growth; and identify transcriptional effects related to both RA and RARB2 signaling. In particular, five genes known to contribute to carcinogenesis or chemotherapeutic resistance are down-regulated following ASO treatment: three of these are up-regulated following RA treatment. This work demonstrates a dual role for RARB2 (tumor suppression and tumor promotion) and identifies a challenge with respect to using RARB2 as a target for treatment or prevention strategies.

A modified protocol for bisulfite genomic sequencing of difficult samples.

The bisulfite genomic sequencing protocol is a widely used method for analyzing DNA methylation. It relies on the deamination of unmethylated cytosine residues to uracil; however, its high rates of DNA degradation and incomplete cytosine to uracil conversion often lead to failed experiments, uninformative results, and false positives. Here, we report the addition of a single-step multiple restriction enzyme digestion (MRED) designed to differentially digest polymerase chain reaction products amplified from unconverted DNA while leaving those of converted DNA intact. We show that for our model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor. We believe that this method may easily be adapted for analyzing other genes and provide guidelines for selecting the most appropriate MRED restriction enzymes.

Allelic methylation bias of the RARB2 tumor suppressor gene promoter in cancer.

Retinoic acid receptor B2 (RARB2) is frequently inactivated in cancer. Methylation in the 5'-untranslated region and first exon is known to play a role; however, few studies have analyzed the detailed methylation pattern of the promoter region. We show that hypo- and hypermethylated alleles coexist in 5/11 cell lines in which RARB2 is inactivated. We present evidence supporting the mitotic transmission of these divergent methylation patterns and find a correlation between methylation divergence and heterozygosity at the 3p24 loci, suggesting an allelic methylation bias in these lines. Using a newly devised strategy based on allelic identification via methylation-sensitive restriction enzyme digestion combined with the use of a single nucleotide polymorphism, rs755661, we demonstrate that such a bias exists in three cancer cell specimens heterozygous at rs755661 and therefore amenable to this study. This previously unreported phenomenon of allelic methylation bias suggests that a promoter methylation-independent mechanism may be responsible for inactivation at the hypomethylated allele and this inactivation is reminiscent of an aberrant form of de novo imprinting. Approaches to interpreting methylation data should incorporate the notion of allelic methylation bias.