Developmental toxicant exposures and sex-specific effects on epigenetic programming and cardiovascular health across generations

Abstract Despite substantial strides in diagnosis and treatment, cardiovascular diseases (CVDs) continue to represent the leading cause of death in the USA and around the world, resulting in significant morbidity and loss of productive years of life. It is increasingly evident that environmental exposures during early development can influence CVD risk across the life course. CVDs exhibit marked sexual dimorphism, but how sex interacts with environmental exposures to affect cardiovascular health is a critical and understudied area of environmental health. Emerging evidence suggests that developmental exposures may have multi- and transgenerational effects on cardiovascular health, with potential sex differences; however, further research in this important area is urgently needed. Lead (Pb), phthalate plasticizers, and perfluoroalkyl substances (PFAS) are ubiquitous environmental contaminants with numerous adverse human health effects. Notably, recent evidence suggests that developmental exposure to each of these toxicants has sex-specific effects on cardiovascular outcomes, but the underlying mechanisms, and their effects on future generations, require further investigation. This review article will highlight the role for the developmental environment in influencing cardiovascular health across generations, with a particular emphasis on sex differences and epigenetic mechanisms. In particular, we will focus on the current evidence for adverse multi and transgenerational effects of developmental exposures to Pb, phthalates, and PFAS and highlight areas where further research is needed.


Developmental environment and cardiovascular disease
The developmental origins of health and disease (DOHaD) hypothesis posits that environmental insults during critical windows of development can lead to diseases later in life [1,2]. While the majority of DOHaD studies have focused on pregnancy and lactation, development continues throughout childhood and puberty. Thus, vulnerability to environmental exposures under the DOHaD paradigm spans well beyond pregnancy. Likewise, a role for paternal environmental exposures in the programming of offspring health is increasingly evident but remains less studied [3,4]. Early environmental insults have been linked to an increased risk of several non-communicable diseases later in life, including diabetes, cancer, neurodegenerative diseases, and the focus of this reviewcardiovascular diseases (CVDs) [2,[5][6][7]. In this review article, we focus on CVDs, which refer to a broad array of health conditions, including atherosclerosis, myocardial infarction, stroke, cardiac arrhythmia, hypertension, heart failure, heart valve disease, and congenital heart defects [2,8]. In many cases, as are noted throughout the manuscript, direct links between various chemicals and CVDs may not yet be established. However, we have noted that some are linked to conditions that are not classically considered CVD but are nevertheless intimately linked to cardiovascular health, such as obesity, disruptions in hormone signaling, and hyperglycemia [2,8,9]. For this reason, we have chosen to include these in the manuscript where relevant. It is important to note that, because CVDs are multifactorial, it is often difficult to ascertain whether effects of chemical exposures on cardiovascular health are direct or are secondary to effects on body weight, endocrine function, inflammation, and other factors. In vitro studies using traditional and new approach methodologies will likely shed light on this important distinction but are beyond the scope of this review. The developing cardiovascular system is exquisitely sensitive to exogenous influences. Several decades ago, Dr David Barker observed that adverse conditions in utero increased the risk of coronary heart disease later in life, highlighting the importance of this critical stage of life in influencing long-term health trajectory. One of the most famous examples of Dr Barker's work illustrating how early developmental environment affects cardiovascular health is the Dutch hunger winter of 1944-45, during which there was a restriction of food to the western part of the Netherlands as a result of World War II [10]. Women who were pregnant during this period were thus exposed to severe caloric restriction. Because medical records and food rations were well-documented, the effects of the famine on children born to women during this time have been extensively investigated. The Dutch Famine birth cohort documented that nutrient restriction in utero in the first trimester was associated with increased risk of obesity in women and dyslipidemia and cardiovascular disease in both sexes. Restriction in the third trimester, on the other hand, was associated with decreased risk of obesity in men, independent of birth weight [10][11][12]. Other studies in animal models have shown consistent effects of in utero nutrient restriction on epigenetic programming of genes controlling metabolic homeostasis and CVD risk [13]. Subsequent research has demonstrated that maternal obesity, hyperglycemia, and poor diet (high fat, high sucrose, or low protein) influence CVD risk in offspring [14][15][16][17]. Likewise, adverse childhood experiences have also been linked to poor cardiovascular health later in life [18][19][20]. In addition to nutritional and psychosocial factors, early exposure to environmental toxicants can also influence cardiovascular development and disease risk across the life course. A number of toxicants have been investigated in this regard, including air pollution [21], metals [22][23][24], phthalates [25,26], perfluoroalkyl substances (PFAS) [27], pesticides [28], and hormone mimics such as diethylstilbestrol (DES) [29]. A summary of the factors discussed here can be found in Table 1.

Sex differences in environment-induced cardiovascular disease
Although the early developmental environment plays a role in CVD risk, the effects of many environmental influences differ based on sex in both animals and humans [30][31][32][33][34][35]. However, the sex-specific effects of the developmental environment on cardiac health have received little attention. It is well-established that the incidence, pathogenesis, and prognosis of CVDs differ substantially between sexes [36,37]. For example, although acute myocardial infarction is one of the leading causes of death in both men and women, women experience this condition later in life and exhibit atypical symptoms, resulting in delayed diagnosis [36]. Likewise, while ischemic heart disease is frequently characterized by coronary artery occlusion in men, women are more likely to exhibit microvascular dysfunction [37]. Hypertension is more common among men in young adults, but this disparity shifts in advanced age, where a greater number of women experience hypertension [38,39]. Other conditions such as heart failure, genetic cardiomyopathies, and drug-induced arrhythmias also exhibit sexual dimorphism [37,40,41]. It is therefore imperative that we understand the sex differential effects that the early environment has on CVD risk and pathogenesis. Many factors noted above, including maternal obesity, diet, and diabetes [16,[42][43][44], psychosocial stressors [45], and toxicant exposures [46][47][48], show sex-specific effects on cardiovascular health. In this review, we will discuss what is known regarding the sex-specific multi and transgenerational effects of toxicant exposures on cardiac health. Each section contains a summary table of the studies discussed, which includes information on sex differences, if any, observed in each study.

Epigenetics and multi and transgenerational inheritance
Epigenetics refers to the study of mitotically heritable changes in gene regulation that do not involve an alteration to the DNA sequence itself. Several mechanisms of epigenetic regulation have been identified, including modifications to the 5-position of cytosine bases in DNA (5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine, and 5-formylcytosine), modifications to histone proteins (methylation, acetylation, phosphorylation, and ubiquitination), and actions of non-coding RNA. The most extensively studied epigenetic modification is methylation of the 5-position of cytosine bases [5-methylcytosine (5mC)], which plays a role in X-inactivation, genomic imprinting, and repression of transposable elements [49][50][51][52]. The regulation of gene expression by DNA methylation is associated with genomic location. For example, DNA methylation at promoters is generally associated with repression of genes, while intragenic DNA methylation is associated with gene activation [53,54]. The 5hydroxymethylcytosine (5hmC) modification plays a critical role post-fertilization and in primordial germ cells (PGCs) during the dynamic reprogramming of DNA methylation [55][56][57] but has also been shown to be a stable epigenetic mark present in a variety of mammalian tissues [58][59][60], and we have shown that it is stably reprogrammed by perinatal exposures in mice and humans [61,62].
Two distinct waves of epigenetic reprogramming occur during early development, making this period vulnerable to environmental perturbations [63,64]. The first wave of reprogramming occurs post-fertilization, in which parental epigenetic marks are erased and the somatic epigenetic marks of the developing embryo are established [65,66]. The second wave of reprogramming occurs in the fetal primordial germ cells, in which sex and parent of originspecific epigenetic patterning are established [67]. Environmental disruptions in the normal epigenetic patterning in germ cells that are not corrected during the development of the future generation may result in multi and transgenerational effects [68]. In a pregnant (F0) individual, toxicant-induced epigenetic changes may occur in their somatic and germ cells, as well as in the somatic and primordial germ cells of the developing fetus ( Fig. 1) [68]. Toxicant exposure-induced effects occurring in the offspring (F1) and grand offspring (F2) generations are termed multigenerational effects, as F0 and F1 generations receive direct exposure during the pregnancy, and the primordial germ cells of the F2 generation are also directly exposed ( Fig. 1) [68]. In contrast, transgenerational epigenetic inheritance refers to the transmission of environmental effects to future generations that did not receive the exposure directly through embryonic development or germ cells, i.e. F3 generations and beyond ( Fig. 1) [68]. In males and non-gestating females, the F0 individual and F1 offspring are directly exposed, and transgenerational effects are observed in the F2 generation and beyond (Fig. 1). Transgenerational effects of environmental exposures have been reported in nematodes, plants, fruit flies, and mammals [68][69][70][71]. However, the prevalence of this phenomenon and its relevance to health, in particular human health, is the subject of significant debate and ongoing study [72,73].

Effects of environment on cardiovascular health across generations
Several animal and human studies provide evidence that ancestral environmental exposures may induce cardiovascular health effects that span generations (Fig. 2). A summary of the manuscripts reviewed in this section can be found in Table 2. In animal studies, F1, F2, and F3 male progeny of rats exposed to global nutrient and calorie restriction during their entire pregnancy exhibited cardiovascular dysfunction, including high blood pressure, altered nitric oxide production, and impaired vasodilation in response to acetylcholine [74]. Maternal obesity may also produce transgenerational effects on cardiac health. Obese female mice produced both male and female offspring from F1 Only females were evaluated [28] Diethylstilbestrol Gestation Human Increased incidence of coronary artery disease and myocardial infarction Only females were evaluated [29] Figure 1: multigenerational vs. transgenerational effects. Left: In a pregnant (F0) individual exposed to a toxicant, the F1 and F2 generations will also receive direct toxicant exposure, resulting in multigenerational effects. Transgenerational effects are those that occur in generations not directly exposed to the toxicant, i.e. F3 and beyond. Right: In males and non-gestating females, the F0 individual and F1 offspring are directly exposed (via germ cells), and transgenerational effects are observed in the F2 generation and beyond. The sex-specific effects of toxicants on transgenerational epigenetic inheritance are poorly understood Figure 2: Diet and exposure to toxicants such as Pb, PFAS, and phthalates can affect the epigenome of the parental germ cells, as well as the somatic and germline cells of a developing fetus, resulting in multigenerational and transgenerational effects on cardiovascular disease risk. The sex-specific generational effects of Pb, PFAS, and phthalates have not been adequately investigated to F3 with cardiac defects, including mitochondrial dysfunction and increased left ventricular mass [16]. Epigenetic or other mechanisms were not investigated in either of these studies, however, so the underlying molecular basis for their observations remains unclear. In zebrafish, paternal exposure to bisphenol A resulted in cardiac defects (pericardial edema, malformations in both cardiac chambers, ectopic heartbeats, and arrhythmias) in F1 progeny as well as altered expression of cardiac developmental genes [75]. Cardiac edema was also observed in F2 progeny exposed to the highest dose of BPA [75]. The authors observed no significant changes in global DNA methylation in sperm and testicular tissue from control vs. treated animals; however, other epigenetic or site-specific DNA methylation changes were not investigated [75]. Human studies provide further support for the hypothesis that the cardiovascular effects of the environment can span multiple generations. Research conducted on three cohorts of people born in 1890, 1905, and 1920 in Overkalix, a Municipality in Sweden, revealed that a dearth of food during a father's slow growth period (the period between ages 9 and 12 years) resulted in a reduced risk of mortality from cardiovascular disease in the children [76]. However, if the paternal grandfather was exposed to an abundance of food during this critical period, then his grandchildren had a higher risk of mortality from diabetes [76]. Further investigation of this cohort of people showed that if a paternal grandmother lived through a period of sharp changes in food availability in early life up to puberty, then the daughters of her sons exhibited an increased risk for cardiovascular mortality [77]. In a separate study of a population exposed to the Chinese Famine (1959-61), prenatal exposure to famine was associated with an increased risk of hyperglycemia in adulthood for the exposed, as well as their offspring [78]. The effects on offspring hyperglycemia risk were independent of sex and occurred in offspring of both exposed mothers and fathers [78]. The molecular mechanisms underlying these effects are unclear; however, studies have demonstrated that epigenetic factors such as DNA methylation may underlie the transmission of stress across generations [79,80].
Multi-and transgenerational effects of toxicant exposures have been reported for a wide variety of chemicals, including BPA [81,82], phthalates [82], parabens, pesticides [83] and dioxin [84], fungicides [85], and jet fuel [86]. The transgenerational effects of chemical exposures on long-term cardiovascular health are poorly understood, and given the significant burden of morbidity and mortality posed by CVDs, further investigation into this important topic is urgently needed. Although a relatively small number of studies have investigated the transgenerational effects of environment specifically on cardiac health, significant animal and human evidence also links ancestral environment to conditions closely associated with CVD, including obesity, diabetes, and reproductive dysfunction. For example, ancestral exposure to BPA and phthalates [82], pesticides/herbicides [83,87], tributyltin [6,88], and high-fat diet [89] have been linked to an increased risk of obesity or adiposity, and some of these exposures exhibited sex differences. Obesity induced by methoxychlor was present in both F3 males and females, with a greater incidence in males [83]. Offspring consumption of a high-fat diet led to obesity in male but not female F4 mice after ancestral exposure to tributyltin [6]. A maternal high-fat diet led to increased body weight in F3 female mice, which was transmitted through the paternal line [89]. On the other hand, glyphosate and plastics-induced obesity was present in both F3 males and females at roughly equal frequency [82,87]. These studies identified chemical-induced epigenetic changes that were present in the generations that did not receive direct exposure, providing evidence that transgenerational inheritance may be mediated by changes to the germline epigenome. Indeed, methoxychlor and BPA/phthalate-induced phenotypes were accompanied by changes in DNA methylation in the sperm of F3 mice [83,84]. Glyphosate exposure led to altered DNA methylation in F1-F3 sperm [87]. Effects of ancestral exposure to tributyltin were associated with changes in DNA methylation and chromatin accessibility in F3 and F4 sperm and adipose tissue [6]. Increased body weight as a result of maternal high-fat diet was accompanied by changes in expression of paternally expressed imprinted genes, suggesting that stable epigenetic modifications at these genes may have been responsible for the observed effects on gene expression and body weight [89].
In the following sections, we will describe the sex-specific effects of developmental exposure to Pb, PFAS, and phthalates on cardiovascular health and epigenetic programming in human and animal studies, as well as the current evidence available regarding the transgenerational effects of these chemicals on cardiovascular health.

Developmental Pb exposure and cardiovascular health
The metal Pb is a ubiquitous environmental contaminant with a large number of deleterious human health effects. Sources of human Pb exposure include soil, water, food, contaminated household dust, consumer goods, folk remedies, smoking, and industrial sources [90,91]. Human exposure to Pb occurs primarily through ingestion or inhalation, where it causes adverse neurological, hematological, renal, and cardiovascular effects [91]. A summary of the cardiovascular and epigenetic effects of developmental Pb exposure can be found in Tables 3 and 4, respectively.

Cardiovascular effects of Pb exposure in animals
Pb exposure in animals outside of the developmental period is widely reported to cause cardiotoxicity, heart failure, and hypertension [92][93][94][95]. Developmental exposures have been less extensively studied, but several have demonstrated adverse effects on blood pressure, cardiac function, and metabolic parameters. First, developmental Pb exposure in rats leads to hypertension in both sexes [96] or in male offspring (females were not included in the study) [97]. Exposure to Pb is also associated with altered angiogenesis in mouse embryos, suggesting that some Pb-induced developmental defects may be mediated by impaired blood flow [98]. Pb exposure during lactation impaired cardiac mitochondrial function and depleted antioxidant capacity in male rats [22], consistent with data in humans demonstrating direct effects of Pb on cardiac function [99]. Several additional studies have demonstrated that Pb exposure causes sex-specific changes in metabolic parameters that are closely linked to cardiovascular health, including weight, food intake, insulin signaling, blood glucose levels, and circulating IGF-1 [100][101][102]. Several additional Pb exposure studies have been conducted in young, recently weaned animals, a developmental period in which significant growth and hormonal changes are still occurring. Newly weaned male rabbits treated with Pb for 8 weeks showed serum and histological evidence of cardiac injury [103]. Young male rats exposed to Pb exhibited pathological changes in the structure of the aorta and myocardium [104]. Chronic Pb exposure in young male mice led to increased cardiac inotropy and blood pressure, concomitant with changes in circulating enzymes critical for blood pressure regulation [105].

Cardiovascular effects of Pb exposure in humans
Although investigations into the effects of Pb on health have focused primarily on neurological outcomes, numerous studies also link Pb exposure to adverse cardiovascular outcomes, including high blood pressure, myocardial infarction, coronary artery disease, cardiac arrhythmias, heart failure, atherosclerosis, and stroke [99,[106][107][108]. Developmental exposures to Pb have also been linked to cardiovascular diseases in humans. First, there Bisphenol A (100 and 2000 μg/l)

Zebrafish
No changes in global DNA methylation in testicular cells or spermatozoa Cardiac edema and malformations (defects in both heart chambers) in high dose exposed 7 dpf larvae (F1 and F2) Not reported [75] Food availability Slow growth period (before pre-pubertal peak in growth velocity) in males Human Not investigated Increased risk of death from diabetes mellitus in grandchildren of men exposed to abundant food during the slow growth period; decreased risk of death from CVD among grandchildren of men exposed to famine during the slow growth period Not reported [76] Food availability Period prior to puberty in females Human Not investigated Increased risk of cardiovascular mortality in daughters of the sons from women exposed to sharp changes in food supply before puberty    Not reported [112] Pb levels in first meconium samples of newborns Gestation Human Not investigated Increased Pb levels in meconium of infants with conotruncal heart defects Not reported [113] Pb levels in blood of mothers with infants under 6 months of age Gestation Human Not investigated Increased blood Pb levels in mothers of infants with congenital heart defects Effects observed in both males and females [114] Pb levels in prenatal cord blood and early childhood Gestation and early childhood Human Not investigated Elevated cord blood Pb associated with increased systolic blood pressure; elevated early childhood Pb associated with increased peripheral vascular resistance and decreased stroke volume in response to acute stress Not reported [115] Blood Pb levels in childhood (9-11 years of age) Early childhood Human Not investigated Impaired response to cardiovascular stress (reduced stroke volume and increased peripheral vascular resistance) Not reported [116] Pb levels in maternal toenail samples collected at ∼28 weeks gestation and/or 6 weeks postpartum  Yes [146] is evidence that Pb alters normal cardiac development. Children exposed to Pb in e-waste had smaller left ventricles, decreased left ventricular function, and increased markers of inflammation, effects that were independent of sex [109]. Maternal Pb exposure has also been associated with congenital heart defects in several studies, although sex differences were not investigated [110][111][112][113], or effects did not differ by sex [114]. Cardiovascular function and reactivity are tightly regulated by the autonomic nervous system, and several lines of evidence link Pb exposure to altered autonomic regulation. Blood Pb levels were associated with significant autonomic and cardiovascular dysregulation in response to acute psychological stress in children, with some sex differences [115,116]. Like adults, studies have also demonstrated that Pb exposure may lead to increased blood pressure in children. In utero exposure to Pb is associated with increased baseline blood pressure in children [47,117,118], with sex-dependent [47] and independent [117] associations observed. Importantly, some reported effects of Pb were significant at Pb levels well below 10 μg/dl, levels that are still prevalent in the USA, particularly among urban minority populations [47,117,119]. Cardiovascular and renal functions are intimately linked, and Pb exposure was linked to altered kidney volume in children, particularly in females [120]. Finally, Pb may also impact cardiac health through alterations in blood lipids, an effect observed only in male children [121]. Although developmental Pb exposure has been linked to neurological defects later in life [5], the effects of early Pb exposure on adult cardiovascular health have not been investigated. Importantly, individuals in the USA who were highly exposed to Pb as children in the 1960s, 1970s, and early 1980s [122] are now in middle age, a stage of life when cardiovascular diseases emerge. It is thus imperative that the scientific community better understand the effects of early Pb exposure on long-term cardiovascular health.

Effects of developmental Pb exposure on the epigenome
Pb exposure throughout the life course has been linked to alterations in DNA methylation, histone modifications, and non-coding RNAs in various tissues [123]. Significant evidence also links developmental Pb exposure to epigenetic alterations in offspring. Human studies investigating the effects of developmental Pb exposure on the epigenome have focused primarily on the measurement of Pb in maternal blood, maternal tibia or patella, cord blood, or neonatal blood spots as proxies for Pb exposure during pregnancy. These studies have repeatedly demonstrated that Pb exposure is associated with changes in DNA methylation and hydroxymethylation in offspring blood [62,[124][125][126], and sex differences have been reported [125][126][127]. Moreover, recent work suggests that maternal Pb-induced changes in DNA methylation may be transmitted to grandchildren [128]. Few studies to date, however, have investigated the effects of Pb on the epigenome in the context of the cardiovascular system and cardiovascular disease. Pb-associated changes in DNA methylation have been identified at genes relevant to cardiovascular disease, although the long-term implications of these findings for cardiovascular health are unclear. For example, developmental Pb exposure leads to a sex-independent reduction in methylation of the glycoprotein VI gene, which plays an important role in platelet activation and blood clotting [129]. Likewise, maternal blood Pb exposure was associated with altered cord blood DNA methylation at numerous CpGs, particularly in female infants, including several associated with cardiovascular development and diseases such as NOTCH1 and GNAS [130][131][132].
Developmental Pb exposure-induced changes in DNA methylation at imprinted genes, which play a critical role in growth and cardiometabolic health, have also been reported. In addition to changes in DNA methylation at the imprinted GNAS locus noted above [132], maternal blood Pb levels were associated with altered DNA methylation of the MEG3 imprinted gene in cord blood, concomitant with reduced birth weight and rapid increases in adiposity, both of which are associated with long-term risk of cardiovascular disease [133]. Given the small sample size, sex differences were not identified. Roles for MEG3 have been identified in the regulation of cardiac and vascular remodeling in the context of cardiac fibrosis and angiogenesis [134,135]. Prenatal Pb exposure is also associated with changes in DNA methylation in the imprinted gene IGF2 [136], which have also been implicated in cardiovascular disease [137].
Perinatal Pb exposure has also been linked to epigenetic changes in animal models, although effects on cardiovascular tissues have not been adequately investigated. Pb exposure affects Dnmt expression and activity in zebrafish embryos [138,139]. In particular, Pb exposure and thermal stress cooperate to affect heart rate in zebrafish embryos, concomitant with altered expression of Dnmt1 and Dnmt3b [139]. Although these studies did not address cardiovascular disease directly, modulation of the functions of Dnmt1, Dnmt3a, and Dnmt3b has been linked to atherosclerosis and heart failure in animal models [140,141], and single nucleotide polymorphisms in DNMT1 are associated with coronary artery disease risk in humans [142]. In mice, Pb exposure during pregnancy and lactation led to sex and tissue-specific alterations in DNA methylation at murine IAP transposons, although heart tissue was not examined [143]. Given the established role for Pb as a neurotoxicant, multiple studies have demonstrated that perinatal Pb exposure causes epigenetic changes in rodent brain [144,145]. Few studies, however, have investigated the effects of Pb on the cardiac epigenome. We recently discovered that perinatal Pb exposure results in sex-specific changes in DNA methylation in the hearts of offspring mice that are present in adulthood, long after cessation of the exposure [146]. The implications of these changes for cardiac health are under investigation.
Developmental PFAS exposure and cardiovascular health This study investigated pregnant women [159] (continued)

Cardiovascular effects of developmental PFAS exposure in animals
Numerous animal studies, conducted in rodents, zebrafish, Xenopus, and chicken embryos, have demonstrated that developmental PFAS exposures are associated with adverse cardiovascular outcomes. PFOA exposure in chicken embryo cardiomyocytes (exposures occurring while in the embryo or ex-vivo) led to reduced viability, altered cell morphology, and de-regulated intracellular calcium levels [148]. Additional work demonstrated that developmental exposure to PFOA, or the PFOA replacement, hexafluoropropylene oxide dimer acid (HFPO-DA, GenX) in chicken embryos led to altered cardiac function and morphology [149,150]. Developmental PFAS exposures have been shown to cause cardiac developmental defects in Xenopus embryos [151] or in weanling rats [152] or mice [153], although the sex-specific effects of these chemicals were not investigated. At a molecular level, single cell analysis of zebrafish embryos revealed that developmental PFOA exposure disrupts the expression of genes associated with cardiac contractility and differentiation in the cardiac cell population [154]. Additional work has demonstrated that gestational exposure to PFAS alter maternal and offspring lipids, with potential implications for long-term cardiovascular health. Gestational GenX exposure led to altered maternal and offspring glucose and lipid metabolism in rats, although sex-specific effects were unclear [155]. Low-dose developmental PFOA exposure in female mice led to an increased rate of weight gain and an increase in serum leptin and insulin in mid-life [156]. Collectively, these data suggest that PFAS exposures in early life may have adverse cardiovascular consequences. However, given substantial differences in PFAS dosing and toxicokinetics between humans and animals, the drawing of direct parallels between species must be done with caution.

Cardiovascular effects of developmental PFAS exposure in humans
Early life PFAS exposures are linked to adverse cardiovascular effects in humans, although some studies have produced conflicting and difficult to interpret findings. Several endpoints have been investigated, including maternal metabolic and cardiovascular effects, as well as offspring congenital heart defects, blood pressure, blood glucose, lipid profiles, levels of hormones regulating appetite and obesity, BMI, and growth. In pregnant women, PFAS exposures are linked to adverse metabolic and cardiovascular effects. Maternal levels of PFAS are positively associated with total cholesterol and triglycerides [157], as well as higher blood glucose and increased risk of gestational diabetes mellitus [158]. PFAS exposures during pregnancy are also associated with an increased risk of hypertension or preeclampsia [159,160], with differences observed based on the sex of the fetus [160]. Although the implications for the long-term health of the offspring are unclear, adverse effects of maternal diabetes, hyperglycemia, preeclampsia, and high cholesterol on offspring cardiovascular health have been reported in human observational studies and experimental models [14,15,[161][162][163][164]].
An increasing number of studies have begun to assess the effects of PFAS on children and adolescents. With regard to PFAS and congenital heart defects in humans, data are limited, with conflicting results. One study found an association between maternal levels of several PFAS and congenital heart defects in both sexes [165], while a second study also found a weak association between PFOA and congenital heart defects [166]. However, other studies have found no association [167,168]. Effects of PFAS on blood pressure have also been reported, with several studies showing modest increases in blood pressure with PFAS exposure. For example, PFAS have been associated with increased blood pressure in several different populations of children and adolescents, with some studies identifying sex differences [48] and others not [169,170]. Other studies, however, have observed no effects on blood pressure [171,172].
PFAS exposures at multiple time points during early life have also been linked to altered blood glucose, lipids, and metabolic homeostasis in children and adolescents. Maternal blood levels of several PFAS were linked to increased lipids in cord blood [173]. Gestational exposure to PFOA and perfluorohexanesulfonic acid (PFHxS) were associated with unfavorable cardiometabolic risk scores in adolescence, driven by altered insulin levels and insulin resistance, blood pressure, waist circumference, and other factors [27]. Several studies have demonstrated that blood PFAS levels in children and adolescents are associated with increased blood glucose and/or alterations in lipids and amino acids [170,174,175], with some investigations identifying sex differences [175]. Other researchers found no effects of PFAS on blood lipids or glucose [171]. PFHxS levels in children were associated with altered levels of adipokines, including adiponectin and leptin, with effects on adiponectin found only in boys [176]. Effects of developmental exposure to various PFAS on childhood BMI and growth have been investigated, with PFAS being associated with increased BMI or growth [177,178], decreased BMI or growth [179][180][181], no change [182], or effects that differed based on age [183]. Sex differences in the effects of these chemicals on BMI have been reported [177,180].
Conflicting research findings into the effects of PFAS on cardiovascular health are likely due to differences in the toxicokinetics and toxicodynamics of the various PFAS, the timing of outcome measurement (pre vs. post-puberty), the timing of exposure, the experimental methods employed, sample sizes, as well as lifestyle habits and other confounding factors among the populations studied. Therefore, further investigations into the effects of developmental PFAS on human cardiovascular health are necessary. Several human studies have demonstrated associations between blood levels of PFAS and DNA methylation at various loci, including LINE-1 elements, which are linked to cardiovascular diseases [184]. Notably, recent work has also linked PFAS exposures to altered expression of miRNAs, where exposure in women was associated with changes in miRNAs that have functions in cardiovascular and Alzheimer's diseases [185]. Among the predicted target genes for the miRNAs was DNMT3A, suggesting potential crosstalk between miRNA and DNA methylation [185]. A second study in this group identified PFAS-associated changes in DNA methylation in several biological pathways, including cardiac hypertrophy [186]. Investigations into the effects of developmental exposure to PFAS on the human epigenome have utilized maternal or offspring blood, cord blood, and blood spots. PFAS levels in maternal serum were associated with changes in DNA methylation in cord blood at several loci implicated in cardiovascular disease and development [187][188][189][190][191]. Additional work has produced similar findings in cord blood global or site-specific DNA methylation [192][193][194], with some studies identifying sex differences [192] and others finding no evidence of sex specificity [193,194]. Concentrations of PFOS and PFOA in infant blood spots were associated with sexspecific changes in DNA methylation at a small number of loci, including the gene PTBP1, related to cardiovascular disease and development [195].

Effects of developmental PFAS exposure on the epigenome
Several additional studies in vitro and in animal models have demonstrated PFAS-induced epigenetic changes; however, few investigations into the effects of PFAS on the cardiac epigenome have been conducted thus far. Some in vitro evidence suggests that PFAS exposures modulate the epigenome in undifferentiated cells. PFOS exposure leads to increased oxidative stress, altered expression of DNA methyltransferases and sirtuins, and decreased DNA methylation in human trophoblast cells [196]. Likewise, PFOS exposure altered DNA methylation in differentiating adipocytes [197]. In mouse embryoid bodies, PFOS increased expression of the Polycomb complex members and markers of pluripotency and decreased expression of factors associated with differentiation [198]. Exposure to a mixture of PFAS along with the polychlorinated biphenyl PCB126 led to increased expression of dnmt1 in zebrafish embryos [199]. The effects of these chemicals on cardiac differentiation in vitro or in vivo, however, have not been investigated.

Developmental phthalate exposure and cardiovascular health
Phthalates are a large class of chemicals that are divided into two groups based on their molecular weight and chemical properties. High molecular weight phthalates are found in medical tubing, food packaging, vinyl toys, and building products, while low molecular weight phthalates are found in personal care products such as perfumes, lotions, nail polish, and shampoos [200]. Human exposure to phthalates occurs through inhalation, skin absorption, ingestion, and intravenous injection [200]. Phthalate exposures are linked to a wide array of adverse metabolic, neurodevelopmental, reproductive, and cardiovascular health effects [201]. A summary of the cardiovascular and epigenetic effects of developmental phthalate exposures can be found in Tables 7  and 8, respectively.

Cardiovascular effects of developmental phthalate exposure in animals
Animal and in vitro studies provide substantial evidence that phthalate exposures are deleterious to cardiovascular health. In mice, maternal exposure to diethylhexyl phthalate (DEHP) caused cardiac developmental defects [25,202]. Work in zebrafish yielded similar findings, where exposure to DEHP [203], butyl benzyl phthalate (BBP) [204], or dibutyl phthalate (DBP) [205] during Only females were evaluated [206] DEHP administered to dams (1, 10, and 100 mg/kg/day) or olive oil control by oral gavage Lactation-postpartum days 1-21

Rat
Not investigated Increased blood glucose, decreased glucose uptake and oxidation, and decreased insulin receptor expression in male hearts at post-natal day 22 Only males were evaluated [207] DEHP (1, 10, 100, and 300 mg/kg) administered intragastric under anesthesia   [217] Maternal urinary phthalates in 1st, 2 nd , and 3rd trimester Human Not investigated 3rd trimester high molecular weight phthalates associated with lower systolic and diastolic blood pressure in girls (mean age 9.7 years); no associations observed for boys Yes [46] Urinary phthalate metabolites in children 6-8 years of age Childhood Human Not investigated Significant positive association between several phthalate metabolites and blood pressure z-score, pulse pressure, mean arterial pressure; monomethyl phthalate associated with increased risk of high blood pressure Not reported [218] Urinary metabolite concentrations of three phthalates (low molecular weight, high molecular weight, and DEHP)  Not reported [235] development led to cardiac developmental defects. However, whether there are sex differences in phthalate-induced developmental defects is unclear. Additional work has linked lactational exposure to DEHP to systemic and cardiac-specific changes in insulin signaling in female offspring rats [206] and in male offspring [207], and young male mice exposed to DEHP developed cardiac mitochondrial dysfunction [208]. These in vivo changes in metabolism have also been observed in vitro, where DEHP exposure in neonatal rat cardiomyocytes led to shifts in cellular metabolism associated with cardiac disease states [209]. Developmental exposure to DEHP led to decreased blood pressure in adult male rats, suggesting that DEHP can cause long-term changes in cardiovascular physiology [210]. Finally, work in cell lines and animal tissue demonstrates that phthalates interfere with synthesis of prostaglandins, lipid signaling molecules with important roles in cardiovascular health [211].

Cardiovascular effects of developmental phthalate exposure in humans
In human epidemiologic studies, phthalate exposures in adults are linked to a variety of adverse cardiovascular outcomes, including decreased heart rate variability, coronary heart disease, and atherosclerosis [212][213][214]. Likewise, a number of human studies have demonstrated associations between early life phthalate exposure and cardiovascular diseases. First, several investigations have linked phthalates to congenital heart defects. Phthalate exposure during pregnancy cooperated with genetic factors to increase the risk of congenital heart defects in a population of Chinese children [26]. Maternal or paternal [215] or only paternal [216] occupational exposures to phthalates were associated with an increased risk of congenital heart defects. Sex-specific effects of phthalate exposures on congenital heart defects are unclear, as they were not investigated in these studies. Notably, assessments of exposure were based on questionnaires rather than measurement of phthalate levels in biological samples, so further investigation is warranted to confirm these findings. An additional investigation demonstrated that maternal urinary phthalate levels were associated with a significant increase in pericardial fat in children at 10 years of age, with no sex differences observed [217], suggesting that phthalates may alter fat deposition in organs. Phthalate exposures have also been correlated with changes in blood pressure. Maternal urinary phthalate levels were associated with decreased blood pressure in female children [46]. However, additional studies that measured phthalate concentrations in the urine of children found that metabolites of several phthalates were associated with increased blood pressure in children and adolescents [218,219]. Discrepancies in the findings are likely due to differences in the timing of exposure or outcome measurement (maternal vs. offspring), distinct toxicokinetics and toxicodynamics of the various phthalates, different doses and windows of exposure, as well as variations in genetics, diet, lifestyle and other factors of the populations investigated.
Additional work demonstrated associations between phthalate exposures and adiposity in children that were dependent upon sex, the specific phthalate, and the timing of exposure. In girls, their mothers' urinary levels of phthalate metabolites during pregnancy were associated with increased adiposity in peri-adolescence [220]. Whether these changes in body composition persist and the long-term implications of these findings for cardiovascular health are unclear. Lastly, prenatal phthalate exposures have been linked to age-dependent alterations in levels of 8-isoprostane, a marker of oxidative stress associated with CVD, in the blood of children and adolescents [221].
Findings in humans and animals thus collectively provide compelling evidence that phthalate exposures during early life are linked to adverse cardiovascular effects. However, more work is necessary to determine the sex-specific effects of these chemicals, as well as the effects of developmental phthalate exposures on cardiovascular health across the life course, including middle and old age where cardiovascular diseases are most prevalent. Moreover, as most research thus far has focused only on DEHP, further investigation into other phthalates is warranted.

Effects of developmental phthalate exposure on the epigenome
Numerous human, animal, and in vitro studies demonstrate that phthalate exposures are associated with changes in the epigenome. Similar to Pb and PFAS, the majority of the studies thus far have focused on DNA methylation, and few studies have investigated the effects of phthalates on the epigenome specifically in the cardiovascular system. Phthalate-induced epigenetic changes have been reported for both early life and adult exposures. In adolescent and young adult Taiwanese, urinary metabolites of DEHP were associated with an increase in carotid intima-media thickness (CIMT), a marker of subclinical atherosclerosis, as well as increased global DNA methylation, and the authors proposed that DNA methylation may mediate the effect of DEHP on CIMT. Although both males and females were included in the study, sex differences were not investigated [222].
Several studies have linked maternal phthalate exposures to altered DNA methylation at repetitive elements. Maternal urine levels of monoethyl phthalate or DEHP were associated with decreased DNA methylation at Alu repetitive elements in cord blood or in the blood of children at 9 years of age, respectively [223], or with decreased LINE-1 methylation in the placenta and fetal growth restriction [224]. Sex differences were not reported in either study. Although the implications of these findings for cardiovascular health are unclear, methylation of Alu and LINE-1, as well as fetal growth restriction, has all been associated with CVD [225,226]. Prenatal phthalate exposure has also been associated with sex-specific dysregulation of DNA methylation and expression of the imprinted genes H19 and IGF2 [227], both of which have important roles in cardiovascular disease and development [228,229]. Additional work has identified a link between maternal exposure to several phthalates and alterations in serum miRNA levels linked to gestational diabetes [230], a condition linked to increased risk of cardiovascular disease [162].
Additional work in vitro and in animals corroborates the findings in humans. In vitro, exposure of P19 cells to DEHP prior to cardiac differentiation led to altered expression of DNMT1 and DNMT3A before and during differentiation [231]. As in humans, few studies have investigated the effects of early life phthalate exposures specifically on the cardiovascular epigenome. Recent work from our lab demonstrated that maternal exposure to DEHP during gestation and lactation led to sex-specific changes in DNA methylation in the hearts of offspring mice at 5 months of age, long after cessation of exposure [232]. Many of the genes differentially methylated after DEHP exposure also exhibited differential methylation in cardiac tissue from human heart failure patients, suggesting that DEHP exposure alters DNA methylation at disease-relevant genes [232]. Maternal exposure to a mixture of plastics including phthalates led to sex and dose-specific alterations in puberty onset, including early onset puberty in F3 females, as well as obesity in F3 males and females [82].
Although cardiovascular outcomes were not specifically investigated in this study, precocious puberty is a phenomenon associated with obesity and cardiovascular disease in humans [233]. In rats, exposure to DEHP during pregnancy led to increased global DNA methylation and expression of Dnmt1, Dnmt3a, and Dnmt3b transcript and protein in the gastrocnemius muscle of both male and female offspring, concomitant with altered systemic glucose homeostasis [234]. The cardiovascular implications of this work are unclear; however, systemic glucose homeostasis is closely coupled to cardiovascular health [9]. Epigenetic effects of phthalates have also been demonstrated in zebrafish, where DEHP and din-butyl phthalate (DBP) exposure led to altered DNA methylation of transcription factors critical for normal cardiac development, concomitant with cardiac developmental defects [235].

Multi-and transgenerational effects of Pb, PFAS and phthalates on cardiovascular health
To date, there have only been a small number of studies that have investigated multi-or transgenerational effects of exposure to Pb, PFAS, or phthalates. A summary of the studies outlined below can be found in Table 9.

Pb exposure
There are currently no studies that have investigated multi-or transgenerational effects of Pb exposure on cardiovascular tissues. However, a small number of studies have identified epigenetic effects on genes linked to cardiovascular development and disease in other tissues. Using neonatal bloodspots in humans, grandmothers' exposure to Pb was associated with altered DNA methylation in the blood of grandchildren, with alterations occurring at several genes (NDRG4, APOA5, NINJ2, and TRPV2) with important functions in cardiovascular development and disease [128,[236][237][238][239]. In zebrafish, ancestral exposure to Pb resulted in neurobehavioral deficits as well as sex-specific, differential expression of genes involved in epigenetic regulation [240]. Notably, in females, cardiovascular disease-related genes were also significantly enriched among differentially expressed genes [240]. In mice, prenatal Pb exposure led to smaller F3 offspring size in addition to altered corticosterone levels in F3 females [241]. Both males and females showed region-specific alterations in DNA methylation in the brain [241]. Cardiovascular health outcomes and DNA methylation were not investigated; however, the findings of altered size at birth and glucocorticoid levels, both associated with cardiovascular disease in humans [226,242], suggest that cardiovascular effects should be investigated in future studies.

PFAS exposures
Few investigations into the multi-or transgenerational effects of PFAS exposures have been conducted to date. Among the small number of studies available, the majority have been performed in insect and worm species (summarized in Table 9). Exposure to perfluorobutane sulfonate (PFBS), a replacement for PFOS, led to transgenerational effects on reproductive function in fish [243], as well as movement defects in the F1 generation of Caenorhabditis elegans [244]. Continuous exposure to low-dose PFBS in worms ancestrally exposed to PFBS resulted in significantly shortened lifespan in F4 and F5 generations [244]. PFHxS and PFBS also caused transgenerational alterations in lipid metabolism pathways in C. elegans [70]. In zebrafish, ancestral F-53B exposure led to altered expression of several thyroid hormone regulated genes, a trend toward increased thyroxine levels, as well as impaired swim bladder inflation in animals not directly exposed to the chemical [245]. Epigenetic effects were not investigated in any of these studies.
The effects of PFAS on the germline epigenome are also poorly understood. In mice, exposure to PFOA led to significant alterations in several miRNAs in the testes [246]. In a more recent human study, an investigation into the effects of PFAS on sperm DNA methylation in men from three regions in Europe and the Arctic yielded inconclusive results [247]. No consistent changes in global DNA methylation were observed with PFAS exposures; however, small but statistically significant changes in DNA methylation were observed with specific PFAS in specific populations [247]. Moreover, the study only included partners of pregnant women, potentially selecting for men with healthier reproductive function [247]. Given the paucity of investigations into the germline and transgenerational effects of PFAS in mammals, more research in this area is urgently needed.

Phthalate exposures
Compared to Pb and PFAS, more studies have been conducted into the multi and transgenerational effects of phthalates, although they have focused primarily on hormonal functions, with effects observed in both males and females. In males, studies in rats and mice demonstrated that exposure to DEHP or DBP during pregnancy led to reduced sperm counts and mobility [248,249], as well as impaired spermatogonial stem cell function [249]. Altered sperm function in F3 rats was accompanied by lower global levels of DNA methylation [248]. As noted previously in rats, maternal exposure to a mixture of plastics (BPA and phthalates) led to reproductive alterations and obesity in F3 animals of both sexes [82].
In females, separate studies with rats and mice showed that ancestral exposure to DEHP had adverse effects on reproductive function in F3 animals, although whether epigenetic mechanisms played a role in these effects were not investigated [250,251]. Exposure of pregnant mice to a mixture of six phthalates led to premature reproductive aging in F3 females [252], a phenomenon associated with cardiovascular diseases in humans [253]. Ancestral exposure to DEHP led to female-specific changes in levels of corticosterone in response to stress, as well as altered expression of the imprinted gene, Gnas [254], which has important functions in cardiovascular health [131,255]. Further work showed multigenerational effects of DEHP exposure on DNA methylation at imprinted loci in oocytes [256], suggesting that these chemicals may have effects on germ cell function across multiple generations.
Although transgenerational effects of phthalates on cardiovascular outcomes have not been investigated, the well-established links between hormonal dysregulation and cardiovascular health [242,253,257] suggest that phthalates may also affect cardiovascular health across generations. This important question should be addressed in future studies.

Conclusion
Although environmental factors play a critical role in the etiology of cardiovascular diseases, how their effects differ by sex is poorly understood. Likewise, future studies are needed to better understand how toxicant exposures impact cardiovascular health across generations (Fig. 2). Indeed, although the studies outlined in this review and documented in Tables 1-9 demonstrate that chemical exposures during early development can have adverse cardiovascular health effects, the precise window(s) of vulnerability are still unclear. Systematic investigation into this question, Only males were evaluated [247] (continued)  [256] testing the effects of chemical exposures during different stages of development and during other potentially vulnerable points across the life course (childhood, adolescence, and reproductive senescence) is necessary to gain a deeper understanding of the threat posed by environmental toxicants to cardiovascular health. This need for additional insight into the adverse generational and sex-specific health effects of chemicals is exemplified by Pb, PFAS, and phthalates. Of note, among the studies identified in Tables 3-9 addressing the cardiovascular, epigenetic, and transgenerational effects of these toxicants, the vast majority do not provide an analysis of sex specificity. Although research practices have become more inclusive, in part through guidance from the National Institutes of Health [258], the importance of sex as a biological variable is still underappreciated in the scientific community at large [259]. Of the tens of thousands of chemicals registered for use in the USA, only a minority have undergone government safety evaluations, highlighting the significant burden on consumers and researchers to elucidate their potential health effects. Given the profound economic and health burden posed by cardiovascular diseases, more research into this important area of public health is urgently needed.