Developmental Origins Of Adult Disease Research Articles

Morphometric analysis of the intergenerational effects of protein restriction on nephron endowment in mice

BackgroundParental nutritional status is crucial in shaping offspring's kidney development. However, the association between a protein-restrictive diet and its intergenerational impact on kidney development remains unclear. MethodsWe conducted multigenerational morphometric measurements to investigate the effects of parental protein deprivation on offspring kidney development across four generations. F0 mice were divided into two groups and fed a normal protein diet (NPD) or a low-protein diet (LPD) for three weeks before mating and continued these diets throughout gestation and lactation. Body weight (BW), kidney weight (KW), KW/BW ratio, nephron counts, and blood pressure were assessed in F1 pups. To examine paternal effects, we bred CD1 females on an NPD with males on an LPD. BW, KW, KW/BW, and nephron counts were measured at P20. To measure the transgenerational effect of parental LPD on kidney development, F1 offspring (from parents on LPD) were fed NPD upon weaning. These F1 offspring were bred at 6 weeks of age to produce F2, F3 and F4 generations. Kidney metrics were evaluated across generations. ResultsThe average body weight of P0 pups from parents on NPD was 1.61g, while pups from parental LPD weighed an average of 0.869g, a decrease of 54 % (p = 6.9e-11, Wilcoxon test). F1 from parental LPD have significantly smaller kidneys than the control, with an average combined kidney weight of 0.0082g versus 0.0129g, a 37 % decrease (p = 3.2e-02, Wilcoxon test). P20 BW and KW remained low in LPD offspring. These effects persisted for 4 generations (F1 to F4) with an average glomerular count reduction of roughly 20 %. F3 and F4 showed wider variability in glomerular counts but were not statistically significant compared to controls. ConclusionsBoth maternal and paternal LPD significantly affected offspring nephron endowment. Our study underscores the complex nature of nutritional transgenerational effects on kidney development, emphasizing the importance of both maternal and paternal dietary impacts on kidney development and the developmental origin of adult disease.

Epigenetic impact on children’s future

The adult diet and lifestyle are conventionally considered at the origin of cardiovascular and metabolic diseases that occur during life time. The hypothesis that supports the link between intrauterine malnutrition, prematurity, maternal stress, and the occurrence of type 2 diabetes and ischemic heart disease in adults/elderly, called Developmental Origins of Adult Disease, has been supported by numerous clinical studies. The concept of fetal phenotype programming gained consistency through epigenetic studies that showed how DNA methylation changes, histone changes, maintained over time are at the origin of chronic adult diseases. Initially demonstrated through studies on experimental animals and later through research on human subjects exposed to extreme situations, epigenetic changes were identified in severe maternal malnutrition, folic acid deficiency, smoking and maternal obesity or contact with environmental toxins. It is possible for some of the epigenetic changes to be transmitted transgenerationally, their argumentation subject to objective limitations.

Cardiometabolic Risk Factors in Adolescent Women’s Health

Despite significant advances in diagnosis and treatment, cardiovascular disease (CVD) remains a major cause of mortality and morbidity for men and women. While disease rates are somewhat declining for men, risk for women, and earlier age at onset, is increasing. As with many common conditions, prevention and/or early intervention for those at risk, leads to better long-term outcome than treatment and secondary prevention. In order to better achieve reduction in disease in adulthood, we need to recognize critical periods of vulnerability for disease development. These include broadly the maternal environment, but also early childhood and adolescence, two periods with rapid growth and development, where environmental “insults” may have long-lasting impact. As suggested by the Developmental Origins of Adult Disease (DoAD) and the Dutch famine studies, the in-utero environment is a critical window. And maternal health leading into pregnancy, maternal health during pregnancy and any potential environmental exposures, will all impact childhood and likely adult health. Careful studies across childhood, particularly with emphasis on BMI and BMI trajectories and adverse social situations and abuse, are producing informative data regarding the importance of this critical time-period for both awareness and intervention. And, interventions, as simple as improving diet and sleep, may improve our ability for early intervention in simple ways to improve women’s health and longevity.

Developmental origins of adult diseases.

The occurrence and mechanisms of developmental adult diseases have gradually attracted attention in recent years. Exposure of gametes and embryos to adverse environments, especially during plastic development, canalter the expression of certain tissue-specific genes, leading to increased susceptibility to certain diseases in adulthood, such as diabetes, cardiovascular disease, neuropsychiatric, and reproductive system diseases, etc. The occurrence of chronic disease in adulthood is partly due to genetic factors, and the remaining risk is partly dueto environmental-dependent epigenetic information alteration, including DNA methylation, histone modifications, and noncoding RNAs. Changes in this epigenetic information potentially damage our health, which has also been supported by numerous epidemiological and animal studies in recent years. Environmental factors functionally affect embryo development through epimutation, transmitting diseases to offspring and even later generations. This review mainly elaborated on the concept of developmental origins of adult diseases, and revealed the epigenetic mechanisms underlying these events, discussed the theoretical basis for the prevention and treatment of related diseases.

Genomic map of candidate human imprint control regions: the imprintome

ABSTRACT Imprinted genes – critical for growth, metabolism, and neuronal function – are expressed from one parental allele. Parent-of-origin-dependent CpG methylation regulates this expression at imprint control regions (ICRs). Since ICRs are established before tissue specification, these methylation marks are similar across cell types. Thus, they are attractive for investigating the developmental origins of adult diseases using accessible tissues, but remain unknown. We determined genome-wide candidate ICRs in humans by performing whole-genome bisulphite sequencing (WGBS) of DNA derived from the three germ layers and from gametes. We identified 1,488 hemi-methylated candidate ICRs, including 19 of 25 previously characterized ICRs (https://humanicr.org/). Gamete methylation approached 0% or 100% in 332 ICRs (178 paternally and 154 maternally methylated), supporting parent-of-origin-specific methylation, and 65% were in well-described CTCF-binding or DNaseI hypersensitive regions. This draft of the human imprintome will allow for the systematic determination of the role of early-acquired imprinting dysregulation in the pathogenesis of human diseases and developmental and behavioural disorders.

The Reproduction of Shame: Pregnancy, Nutrition and Body Weight in the Translation of Developmental Origins of Adult Disease

Developmental origins of health and disease (DOHaD) and epigenetics have expanded understanding of how the environment affects the health of women before and during pregnancy—with lifelong health consequences for the fetus. This has translated to a narrow focus on women’s lifestyle during pregnancy, especially for women classified as obese. In this study, we show that psychosocial harms such as distress or shame felt by pregnant women are rarely countenanced in these endeavors. To demonstrate this, we examine published documents about a large set of trials of lifestyle interventions united through an international consortium. Yet there is now a literature in which pregnant women with large bodies report feeling humiliated and a wider literature on the stigma of obesity. We argue that shame is produced and reproduced through the discursive and material knowledge-making scientific practices of DOHaD translation. Interventions that intensify the shame of large body size in pregnancy may be stressful, and neurophysiological stress pathways are well-known within DOHaD to have consequences for fetal development, so these interventions potentially undermine the very processes they set out to protect. A feminist response may protect women from shame and redirect attention to the social and structural determinants of health.

Development and Utility of a Novel Intergenerational Health Knowledgebase

ObjectiveOur long‐term goal is the development of a clinically‐relevant, validated enterprise data warehouse for research. The Intergenerational Health Knowledgebase (IHK) will advance and accelerate the study of the developmental origins of adult disease. We sought to harmonize data from the electronic health record (EHR) and external data sources and efficiently synthesize key variables from pregnancy and early life in the IHK. The objective was to make the IHK be efficient, effective, flexible and able to integrate with participation information for on‐campus biobanks.MethodsThe development of the IHK is an iterative process involving multiple steps: 1) clinical data team (CDT) members identify and locate clinically significant distinct clinical variables in the front‐facing EHR pertaining to obstetrics and pediatrics. 2) The Biomedical Informatics group in the Institute for Clinical and Translational Science (ICTS‐BMI) group locates these variables in the transactional database of the EHR. 3) The CDT members validate the extracted variables. Extracted data is compared with expected outcomes from the EHR. Inconsistencies are reviewed by the CDT and ICTS‐BMI group to pinpoint potential causes. 4) With confirmed data variables, ICTS‐BMI develops novel data tables and user‐friendly SQL queries. The resultant OBstetrics Data Integration Architecture structure integrates multiple data sources within the institution which includes, but is not limited to, imaging data, diagnoses, vital sign information, medications, medication administration, and procedures for the maternal‐fetal dyad. Powering this architecture is a common semantic layer that uses established standardized vocabularies and value sets. This includes storing patient demographic data, patient diagnoses using ICD9‐10 codes, storing laboratory results using LOINC codes, and problems using ICD9‐10/SNOMED‐CT codes. In our EHR, all data is joined using pregnancy episode identification numbers linking maternal and neonatal data under a singular pregnancy instance. ICTS‐BMI developed a secure web‐based Knowledgebase viewer for performing queries of the IHK without expert SQL knowledge.ResultsThe IHK contains indexed maternal and child data. The IHK is frequently updated to contain every pregnancy cared for at the University of Iowa Hospitals & Clinics (UIHC) (currently 62,000+ pregnancies). Because of our team’s complementary expertise, they were able to anticipate needed information, identify and validate key variables in the EMR, and plan pre‐defined queries. As a result, we have quickly provided data for studies ranging from the effects of maternal environmental exposures, the role of patient distance to the hospital in pregnancy outcomes, medication use during pregnancy, and the association of depression with adverse outcomes.ConclusionThe development of the novel IHK has facilitated pregnancy‐related research and identified new targets for improving maternal health and following long‐term health outcomes of parents and children. Further, integration of the IHK with the UIHC Women’s Health Tissue Repository facilitates using biological specimens for research.

Abstract WMP109: Prenatal Alcohol Exposure Worsens Stroke Outcomes In Young Rats And Exacerbates Brain Damage In Ischemic Middle-aged Rats

Background and Purpose: Endocrine imbalances and cardiovascular diseases in post-natal life have been linked to adverse prenatal environment. There is very little information on the developmental origin of adult diseases, particularly prenatal alcohol exposure and stroke outcomes. Recent studies indicate that adults with prenatal adult exposure (PAE) are at agreater risk for obesity, hypertension and hyperglycemia, all of which increase the risk for severe stroke. The present study tested the hypothesis that prenatal alcohol exposure (PAE) elevates stroke severity in young and middle-aged rats. Methods: Pregnant Sprague Dawley rats (bred in-house) were exposed to 10% alcohol or air, for 1 hour from gestational day 8 through18 using the rodent alcohol inhalation system. Plasma IGF-1 levels were determined using an ELISA kit. Adult offspring (5 and 12 mo old) were subjected to endothelin-1 induced MCAo and outcomes were evaluated after 2 days. Acute stroke-induced disabilities were assessed by neurological scores and vibrissae evoked fore-limb placement task as well as the extent of infarction. Results: While plasma IGF-1 levels were higher in young control males than females, levels were significantly reduced in young-PAE males (p<0.04) compared to control males. MCAo significantly increased mortality in young PAE-males compared to control-males or PAE and control females. Behavioral analysis indicates that young-PAE animals exhibited greater neurological deficits as determined by their higher composite neurological score compared to controls (p<0.03), although their infarct volume was not significantly different from control animals. However, MCAo in the middle-aged PAE rats caused significant behavioral deficits: higher neurological scores (p<0.03), greater cross midline impairment (p<0.03), and increased brain infarction (p<0.04) compared to control stroke rats. Conclusions: The present findings support the hypothesis that PAE contributes to adverse effects on stroke outcome as indicated by greater mortality and poor neurological function in young rats, which is exacerbated by age during the acute phase.(Funding support, NIAAA, AA026756)

Invited Perspective: Air Pollution and Dementia: Challenges and Opportunities.

Invited Perspective: Air Pollution and Dementia: Challenges and Opportunities.

Reducing embryonic mtDNA copy number alters epigenetic profile of key hepatic lipolytic genes and causes abnormal lipid accumulation in adult mice.

Adverse fetal environment, in particular a shortage or excess of nutrients, is associated with increased risks of metabolic diseases later in life. However, the molecular mechanisms underlying this developmental origin of adult diseases remain unclear. Here, we directly tested the role of mitochondrial stress in mediating fetal programming in mice by enzymatically depleting mtDNA in zygotes. mtDNA-targeted plasmid microinjection is used to reduce embryonic mtDNA copy number directly, followed by embryo transfer. Mice with reduced zygote mtDNA copy number were born morphologically normal and showed no accelerated body weight gain. However, at 5 months of age these mice showed markedly increased hepatic lipidosis and became glucose-intolerant. Hepatic mRNA and protein expressions of peroxisome proliferator-activated receptor α (Pparα), a key transcriptional regulator of lipid metabolism, were significantly decreased as a result of increased DNA methylation in its proximal regulatory region. These results indicate that perturbation of mitochondrial function around the periconceptional period causes hypermethylation and thus suppressed expression of PPARα in fetal liver, leading to impaired hepatic lipid metabolism. Our findings provide the first direct evidence that mitochondrial stress mediates epigenetic changes associated with fetal programming of adult diseases in a mammalian system.

In memory of Michelle Lane: 1970 - 2020.

In memory of Michelle Lane: 1970 - 2020.

Mechanisms of intergenerational transmission of polycystic ovary syndrome.

Developmental origins of adult disease (DoHAD) refers to critical gestational ages during human fetal development and beyond when the endocrine metabolic status of the mother can permanently program the physiology and/or morphology of the fetus, modifying its susceptibility to disease after birth. The aim of this review is to address how DoHAD plays an important role in the phenotypic expression of polycystic ovary syndrome (PCOS), the most common endocrinopathy of women characterized by hyperandrogenism, oligo-anovulation and polycystic ovarian morphology. Clinical studies of PCOS women are integrated with findings from relevant animal models to show how intergenerational transmission of these central components of PCOS are programmed through an altered maternal endocrine-metabolic environment that adversely affects the female fetus and long-term offspring health. Prenatal testosterone treatment in monkeys and sheep have been particularly crucial in our understanding of developmental programming of PCOS because organ system differentiation in these species, as in humans, occurs during fetal life. These animal models, along with altricial rodents, produce permanent PCOS-like phenotypes variably characterized by LH hypersecretion from reduced steroid-negative feedback, hyperandrogenism, ovulatory dysfunction, increased adiposity, impaired glucose-insulin homeostasis and other metabolic abnormalities. The review concludes that DoHAD underlies the phenotypic expression of PCOS through an altered maternal endocrine-metabolic environment that can induce epigenetic modifications of fetal genetic susceptibility to PCOS after birth. It calls for improved maternal endocrine-metabolic health of PCOS women to lower their risks of pregnancy-related complications and to potentially reduce intergenerational susceptibility to PCOS and its metabolic derangements in offspring.

Neurodevelopmental concepts of schizophrenia in the genome-wide association era: AKT/mTOR signaling as a pathological mediator of genetic and environmental programming during development

Normative brain development is contingent on the complex interplay between genes and environment. Schizophrenia (SCZ) is considered a highly polygenic, neurodevelopmental disorder associated with impaired neural circuit development, neurocognitive function and variations in neurotransmitter signaling systems, including dopamine. Significant evidence, accumulated over the last 30 years indicates a role for the in utero environment in SCZ pathophysiology. Emerging data suggests that changes in placental programming and function may mediate the link between genetic risk, early life complications (ELC) and adverse neurodevelopmental outcomes, with risk highlighted in key developmental drivers that converge on AKT/mTOR signaling. In this article we overview select risk genes identified through recent genome-wide association studies of SCZ including AKT3, miR-137, DRD2, and AKT1 itself. We propose that through convergence on AKT/mTOR signaling, these genes are critical factors directing both placentation and neurodevelopment, influencing risk for SCZ through dysregulation of placental function, metabolism and early brain development. We discuss association of risk genes in the context of their known roles in neurodevelopment, placental expression and their possible mechanistic links to SCZ in the broad context of the ‘developmental origins of adult disease’ construct. Understanding how common genetic variation impacts early fetal programming may advance our knowledge of disease etiology and identify early critical developmental windows for prevention and intervention.

Early lifecycle UV-exposure calibrates adult vitamin D metabolism: Evidence for a developmentally originated vitamin D homeostat that may alter related adult phenotypes.

Within the Developmental Origins of Adult Disease (DOHaD) model, early life environmental exposures can confer a long-term legacy on human health. This mechanism may be adaptive or maladaptive depending on lifestyle circumstances. This article examines the role of first trimester UV-exposure on late-life vitamin D levels, and potentially related adaptive and maladaptive phenotypes (height and osteoporosis respectively). Six hundred and forty nine subjects were examined for vitamin D2 and D3 (HPLC) and height (stadiometer). Osteoporosis was assessed with an extensive medical history questionnaire. Solar irradiance over the first 90 days postconception correlated positively with late-life vitamin D3 (R2 = .0140; P = .0082; β = .1075), but not vitamin D2 levels. It also correlated positively with female adult height (R2 = .170; P = .0103; β = .1291) and negatively with the occurrence of female osteoporosis (P = .0495). All data were adjusted for age and gender as appropriate (unadjusted data also provided). From a contemporary perspective, vitamin D levels varied significantly according to season of blood sampling as might be predicted (P = .0009). Increased solar irradiance/UV exposure during the first trimester of pregnancy calibrates adult vitamin D metabolism, which is an important hormone in maintaining calcium balance. This may explain how very early lifecycle UV exposure can influence skeletal development (adult height) and modify risk for the skeletal degenerative disorder osteoporosis. The data demonstrate humans are tuned to the world (exposome) in ways we have not yet fully considered, and which are entrained at the earliest phase of the lifecycle.

Dysfunctional microglia:neuron interactions with significant female bias in a developmental gene x environment rodent model of Alzheimer's disease

Signaling between microglia and neurons is poorly characterized in the pathophysiology of Alzheimer's disease (AD), particularly with regards to gene and environmental (GxE) interactions early in life. This study investigated the maladaptation of microglia:neuron signaling and subsequent susceptibility to neurodegeneration using a developmental origin of adult disease (DOAD) model of AD, characterized previously. Here, we report that postnatal exposure to lead (Pb) in a transgenic (Tg) rodent model of AD resulted in significant female bias consequent to GxE interactions. Atypical, non-neuroprotective microglial phenotypes were observed months after cessation of Pb exposure, as well as evidence for neuronal compensation, that was not observed in WT mice. Specifically, microglia from Pb-exposed Tg (GxE) females exhibited atypical polarization profiles for activation earlier and more severely than males and WT mice, that persisted over time to become contextually maladaptive. By postnatal day (PND) 240, microglia from GxE females also sequestered less neurotoxic iron in the hippocampus. In the same GxE female population, measures of neuronal parameters, such as hippocampal TrkB expression, revealed evidence of disharmonious and compensatory interactions with microglia within the pathological progression. Likewise, GxE interactions resulted in female-biased, late-life changes to key synaptic proteins crucial to synapse dynamics and microglial signaling. These incongruent microglia:neuronal dynamics were observed in GxE males at later ages compared to females, and not observed in either gene- or environment-only populations. Altogether, our results support a gene x environment model of female-biased microglial susceptibility to later-life development of AD, and highlight markers for maladaptive microglia:neuron signaling and compensation.

Measuring growth and medium- and longer-term outcomes in malnourished children.

Severe and moderate acute malnutrition are among the leading causes of mortality among children in low- and middle-income countries. There is strong evidence that growth assessed anthropometrically from conception to 2years of age marks later risk of ill health. This is central to the concept of the developmental origins of adult disease and is presumed to be related to modification of developmental processes during critical "window(s)" of vulnerability. Interventions to treat acute malnutrition have resulted in dramatic increase in the number of affected children surviving. Ensuring that these children thrive to fulfil their full physical and cognitive potential is a new challenge. Integral to this challenge is the need to be able to measure how earlier insults relate to the ability to survive and thrive to productive adulthood. Despite its obvious value, routine anthropometry does not adequately indicate how earlier adverse exposures affect more refined aspects of growth. Anthropometry is inadequate for predicting how disruption of healthy growth might modulate risk of disease or any subsequent interventions that correct this risk. A clear characterisation of healthy child growth is needed for determining which component best predicts later outcomes. The extent to which postnatal acute malnutrition is a consequence of maternal factors acting preconception or in utero and their relationship to postnatal health and long-term risk of non-communicable diseases is not clear. Body-composition measurement has significant untapped potential allowing us to translate and better understand the relationship between early insults and interventions on early growth in the short-term and long-term health outcomes.

The metabolic costs 30 years after preterm birth

The “developmental origins of adult disease” is a well-accepted hypothesis, linking early exposures to various disease states. However, whether prematurity itself contributes to metabolic and cardiovascular disease in adulthood is unknown. One limitation has been the lack of data from a population-based, prospective study of these infants as adults. Fortuitously, in 1986, every VLBW neonate born in New Zealand and admitted to a neonatal unit was prospectively enrolled in an audit of retinopathy of prematurity. In this volume of The Journal, Darlow et al report the physical wellbeing of 229 of these infants at ~30-year follow-up. These data are important. A majority of the measures of physical wellbeing and metabolic health were not different between former VLBW and term controls. However, although differences in metabolic syndrome were not statistically different between VLBW and control, some disturbing trends were noted. Logistic regression demonstrated a significant relationship between extremely preterm (<28 wks) birth and metabolic syndrome. Furthermore, a full 22% of former male VLBW infants met criteria for metabolic syndrome, compared with 11% of controls. It remains to be determined if that difference did not reach significance due to sample size. Regardless, these data demonstrate the need to continue to follow these high-risk neonates through adulthood so that we may better understand the long-term implications of preterm birth, and provide the proper screening and preventive measures to this unique population. Article page 128 ▶ Metabolic Syndrome in Very Low Birth Weight Young Adults and Controls: The New Zealand 1986 VLBW StudyThe Journal of PediatricsVol. 206PreviewTo assess the physical well-being and components of the metabolic syndrome in a national cohort of very low birth weight (VLBW) young adults and same age controls. Full-Text PDF

Abstract WP153: Prenatal Alcohol Exposure Induces Glucose Intolerance and Exacerbates Stroke Severity in Middle-Aged Rats

Background and Purpose: Metabolic disorders and cardiovascular diseases in post-natal life have been linked to adverse prenatal environment. There is very little information on the developmental origin of adult diseases, particularly fetal alcohol exposure and stroke outcomes. The present study tested the hypothesis that fetal alcohol exposure induces glucose intolerance and leads to greater stroke severity in middle-aged rats. Methods: Timed-pregnant Sprague Dawley rats (GD12) purchased from Envigo laboratories were intragastrically gavaged with ethanol (3g/kg/bwt) or water twice per day from GD12 through 15. Fetal alcohol exposed (FAE) progeny (10 mo. old) were injected with glucose (1g/kg.bwt) and tested for glucose intolerance (glucose clearance rate at 15, 30 and 60 min.) Animals were subjected to endothelin-1 induced MCAo and outcomes evaluated after 2 and 5 days. Stroke induced disabilities were assessed from behavioral assays (adhesive removal, Vibrissae evoked fore-limb placement and circling) and infarct volume. Results: Glucose tolerance test showed elevated blood glucose levels in both control and FAE group at 15 and 30 min but FAE animals exhibited prolonged elevation of blood glucose (60 min., p&lt; 0.017) compared to control animals. At 2 day post-stroke, FAE animals exhibited greater neurological deficit as determined by their higher score on circling behavior (lack of muscle control, p&lt;0.031) compared to controls. No mortality was seen in the control group while MCAo resulted in 25% mortality in the FAE group. Among the survivors, brain damage assessed by infarct volume at post-stroke 5 day was not significantly different between the two groups. Conclusions: The present findings support the hypothesis that prenatal exposure to alcohol contributes to long-lasting adverse effects on adult health as indicated by poor glucose tolerance and worse neurological outcomes during the acute stage of stroke.

“Extension of the “Fetal Origin Hypothesis of Barker” towards the “Fetal Origin Hypothesis of Mental Diseases”

In this editorial we will first describe most common information about the intriguing “traditional” fetal origin hypothesis of Barker for physiological, endocrine and cardiovascular diseases (CVDs). The ‘developmental origins of adult disease’ hypothesis, often called the ‘Barker hypothesis’, states that adverse influences early in development, and particularly during intrauterine life, can result in permanent changes in physiology and metabolism, which result in increased disease risk in adulthood.

Use of 5-azacytidine in a proof-of-concept study to evaluate the impact of pre-natal and post-natal exposures, as well as within generation persistent DNA methylation changes in Daphnia

Short-term exposures at critical stages of development can lead to delayed adverse effects long after the initial stressor has been removed, a concept referred to as developmental origin of adult disease. This indicates that organisms’ phenotypes may epigenetically reflect their past exposure history as well as reflecting chemicals currently present in their environment. This concept has significant implications for environmental monitoring. However, there is as yet little or no implementation of epigenetics in environmental risk assessment. In a proof-of-principle study we exposed Daphnia magna to 5-azacytidine, a known DNA de-methylating agent. Exposures covered combinations of prenatal and postnatal exposures as well as different exposure durations and recovery stages. Growth, the transcription of genes and levels of metabolites involved in regulating DNA methylation, and methylation levels of several genes were measured. Our data shows that prenatal exposures caused significant changes in the methylome of target genes, indicating that prenatal stages of Daphnia are also susceptible to same level of change as post-natal stages of Daphnia. While the combination of pre- and postnatal exposures caused the most extreme reduction in DNA methylation compared to the control group. Furthermore, some of the changes in the methylation patterns were persistent even after the initial stressor was removed. Our results suggest that epigenetic biomarkers have the potential to be used as indicators of past chemical exposure history of organisms and provide strong support for implementing changes to the current regimes for chemical risk assessment to mimic realistic environmental scenarios.