Lineage- and Sex-Dependent Behavioral and Biochemical Transgenerational Consequences of Developmental Exposure to Lead, Prenatal Stress, and Combined Lead and Prenatal Stress in Mice

Abstract

Background:

Lead (Pb) exposure and prenatal stress (PS) during development are co-occurring risk factors with shared biological substrates. PS has been associated with transgenerational passage of altered behavioral phenotypes, whereas the transgenerational behavioral or biochemical consequences of Pb exposure, and modification of any such effects by PS, is unknown.

Objectives:

The present study sought to determine whether Pb, PS, or combined Pb and PS exposures produced adverse transgenerational consequences on brain and behavior.

Methods:

Maternal Pb and PS exposures were carried out in F0 mice. Outside breeders were used at each subsequent breeding, producing four F1-F2 lineages: [F1 female-F2 female (FF), FM (male), MF, and MM]. F3 offspring were generated from each of these lineages and examined for outcomes previously found to be altered by Pb, PS, or combined Pb and PS in F1 offspring: behavioral performance [fixed-interval (FI) schedule of food reward, locomotor activity, and anxiety-like behavior], dopamine function [striatal expression of tyrosine hydroxylase (Th)], glucocorticoid receptor (GR) and plasma corticosterone, as well as brain-derived neurotrophic factor (BDNF) and total percent DNA methylation of Th and Bdnf genes in the frontal cortex and hippocampus.

Results:

Maternal F0 Pb exposure produced runting in F3 offspring. Considered across lineages, F3 females exhibited Pb-related alterations in behavior, striatal BDNF levels, frontal cortical Th total percentage DNA methylation levels and serum corticosterone levels, whereas F3 males showed Pb- and PS-related alterations in behavior and total percent DNA methylation of hippocampal Bdnf. However, numerous lineage-specific effects were observed, most of greater magnitude than those observed across lineages, with outcomes differing by F3 sex.

Discussion:

These findings support the possibility that exposures of previous generations to Pb or PS may influence the brain and behavior of future generations. Observed changes were sex-dependent, with F3 females showing multiple changes through Pb-exposed lineages. Lineage effects may occur through maternal responses to pregnancy, altered maternal behavior, epigenetic modifications, or a combination of mechanisms, but they have significant public health ramifications regardless of mechanism. https://doi.org/10.1289/EHP4977

Introduction

Developmental exposures to lead (Pb) and to prenatal stress (PS) remain significant risk factors for subsequent adverse behavioral outcomes in children (Bock et al. 2015; Canfield et al. 2003, 2004; Jusko et al. 2008; Nigg et al. 2008; Pallarés and Antonelli 2017), including cognitive dysfunction, reduced IQ, and attention deficits (Canfield et al. 2004; Geier et al. 2018; Gutteling et al. 2006; Ji et al. 2018; Lamb et al. 2014; Lanphear et al. 2005; Zhu et al. 2015). These consequences can even persist across the life span (Needleman et al. 1990; Schwabe et al. 2012). Furthermore, in combination, Pb and PS can result in enhanced neurotoxicity as has been seen in both human studies (Tamayo y Ortiz et al. 2017) and animal models (Rossi-George et al. 2011; Sobolewski et al. 2018a, 2018b; Virgolini et al. 2006; Weston et al. 2014), an outcome likely attributable to the shared biological targets of these two factors, that is, the hypothalamic–pituitary–adrenal (HPA) axis, as well as the brain mesocorticolimbic (MESO) system, a network that includes the frontal cortex, nucleus accumbens, and hippocampus (Barros et al. 2004; Berger et al. 2002; Jung et al. 2019; Martínez-Telléz et al. 2009; Rossi-George et al. 2011; Virgolini et al. 2008b). The potential for combined effects of Pb and PS may be further augmented by the fact that HPA axis and brain MESO neurotransmitter circuits have significant interactions critical to the mediation of executive functions and operant behaviors (Bahari et al. 2018; Hernaus et al. 2018). Accordingly, a variety of outcomes along the MESO dopamine and stress pathways have been shown sensitive to Pb, PS, or combined Pb and PS during lifelong exposure and developmental exposures in F1 rat offspring (Barros et al. 2004; Berger et al. 2002; Cory-Slechta et al. 1998, 1999; Martínez-Telléz et al. 2009; Rossi-George et al. 2011; Virgolini et al. 2008b), including alterations in HPA axis function in rats and mice [serum corticosterone and glucocorticoid receptor (GR) concentrations], in dopaminergic function [alterations in tyrosine hydroxylase (Th), the rate-limiting enzyme in dopamine synthesis], in brain-derived neurotrophic factor (BDNF) concentrations [critical to cognitive function and neuronal differentiation during development], and in behavior [learning of fixed-interval (FI) schedule-controlled behavior] (Sobolewski et al. 2018b; Virgolini et al. 2006, 2008b; Weston et al. 2014). Recently, questions have arisen as to the potential for these phenotypic changes to span multiple generations, particularly questioning whether transgenerational inheritance of such developmental neural reprograming is possible.

Developmental programing occurs when exposure to a parent (F0) results in a change in the offspring (F1). Given that female gametes are exposed during fetal life, multigenerational programing occurs when the second generation (F2) is altered. Full transgenerational inheritance, particularly for females, is defined as the occurrence of a specific phenotype in a generation not directly exposed, that is, into a third (F3) generation (Figure 1). Several mechanisms exist by which transgenerational effects and programing can occur (Bale 2015; Gapp et al. 2014; Skinner 2014). For example, an F0 exposure can result in transmission of information through parental gametes. However, other mechanisms of transgenerational passage occur as well, for example, via alterations in the gestational environment, maternal behavior, or social learning, which can influence generational passage of brain and behavioral phenotypes (Jablonka and Raz 2009).

Figure 1.

Schematic of breeding scheme. Breeding scheme and experimental design for the transgenerational study. Exposures to lead (Pb), prenatal stress (PS), or combined Pb and PS occurred in the F0 generation. Offspring from each subsequent generation were bred to unexposed age-matched males or females from Jackson Laboratories; this allowed us to follow each specific sex lineage from F1 to F3 separately based on F1 exposure to either dam or sire. Single pups/sex/dam were used for breeding to preclude litter-specific effects. Germ cells were collected from the F1-F2 offspring, as were measures of litter size and weight. Behavioral testing began in a randomly chosen subset of F3 offspring at about 60 d of age. Note: FF, F1 female F2 female; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FM, F1 female F2 male; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; MF, F1 male F2 female; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MM, F1 male F2 male; MMM, F1 male F2 male F3 male.

Multigenerational effects of PS following maternal parent generation (F0) exposures have been observed in the subsequent first filial (F1) and second filial (F2) offspring, leading to passage of an altered phenotype. Multigenerational effects of unpredictable maternal separation with or without unpredictable maternal stress in mice were seen in F2 offspring as shown by altered approach avoidance behavior (Weiss et al. 2011). In another multigenerational study (Dias and Ressler 2014), the exposure of F0 female mice to fear conditioning based on odor stimuli increased behavioral sensitivity to this odor in F1 and F2 offspring, with F2 inheritance related to parental gamete transmission. Transgenerational studies have also demonstrated that the male F3 offspring of F1 male mice that had been subjected to chronic and unpredictable maternal separation showed depressive-like behavioral deficits similar to those observed in the F1 males, despite the fact that F3 males were reared in a normal environmental setting (Franklin et al. 2010). In a subsequent study, also based on early chronic and unpredictable maternal separation, alterations in social recognition memory were increased in F1 males as well as in F2 and F3 females, findings that could not be attributed to alterations in olfactory recognition (Franklin et al. 2011).

In the human environment, PS may be particularly impactful in low socioeconomic status communities (Farah 2017), where such stress can occur across generations, consistent with the cyclical nature of poverty. A series of studies in rats (Kiss et al. 2016; McCreary et al. 2016) mimicked this feature of the human environment by imposing PS consisting of restraint stress and forced swimming in a semi-random sequence in an F0, F1, and F2 generation and subsequently examined multiple consequences in female F3 offspring. Under these conditions, increases in basal corticosterone levels were seen across generations, with almost 2-fold increases between the F2 and F3 generations. In addition, F3 females exhibited increases in distance traveled in an open field as well as increases in time spent in the margins of the open field, interpreted by some as an anxiety-emotional phenotype. Magnetic resonance imaging analyses indicated reductions in both whole-brain and hippocampal volumes. Based on an absolute gray volume index, F3 females also exhibited reductions in both cortical and hippocampal neuronal density. In a comparison of paw use as a measure of brain laterality, multigenerationally exposed, but not transgenerationally exposed, F4 males exhibited increases in left paw preference, indicative of altered brain laterality (Ambeskovic et al. 2017).

In contrast, the extent to which multigenerational or transgenerational consequences occur in response to Pb exposure is unknown. One study suggested multigenerational consequences of Pb based on an observed correspondence between DNA methylation changes in dried neonatal blood spots from mother–infant pairs to the grandmother’s blood lead levels (Sen et al. 2015). Given that the cycle of poverty and associated stressors, as well as the cycle of Pb exposure continues, transmission across generations of adverse effects of these environmental factors is of public health relevance, regardless of the mechanism by which it occurs.

The present study sought to determine whether Pb, PS, or combined Pb and PS could produce adverse transgenerational consequences on brain and behavior using a mouse model. The breeding scheme employed Pb, PS, or combined Pb and PS treatment of F0 female mice followed by observation of F3 offspring using an experimental design that allowed tracking of effects by specific lineages across the generations. A primary question to be addressed in a transgenerational study on Pb and PS exposure is whether the same neural networks, chemical, and behavioral targets shown to be sensitive to Pb, PS, or combined Pb and PS in F1 offspring (developmental exposures) are still impacted into the F3 generation. As such, our initial outcome measures include alterations in the function of key regions in the MESO network (frontal cortex, hippocampus and striatum), of BDNF and Th protein concentrations, as well as changes in HPA axis function including serum corticosterone and GR concentrations and functional behavioral outcomes such as reward-mediated behaviors. In addition, locomotor assessments and elevated plus maze were examined to inform behavioral mechanisms and domains potentially altered. Finally, given human data suggesting that Pb altered DNA methylation patterns multigenerationally (Sen et al. 2015), alterations in DNA methylation of genes of these noted targets, Bdnf, Th, and Gr were assessed, in addition to Igf2/H19 and Esr1, as potential markers of alterations in methylation patterns.

Methods

Animals and Breeding and Pb and PS Exposures

Eight-week-old C57BL/6J mice were obtained from Jackson Laboratories for F0 exposures and subsequent breeding (Figure 1). Mice were housed in standard mouse caging, pair housed until breeding, under a 12-h light–dark cycle maintained at $22\pm 2°\mathrm{C}$ fed standard rodent chow (LabDiet Autoclavable Diet 5010) with approximately $3\mathrm{mm}$ high performance bedding (BioFresh) at the University of Rochester Medical Center. Forty randomly chosen young adult females were assigned to receive distilled deionized drinking water or $100\text{\hspace{0.17em} ppm}$ Pb acetate dissolved in distilled deionized drinking water beginning 2 months prior to mating to ensure a body burden of Pb (e.g., skeletal Pb accumulation) consistent with human Pb exposure. These exposure levels are consistent with our prior studies, allowing direct comparisons, and associated with blood Pb levels in mouse offspring at postnatal day 6–7 (PND6–7) of approximately $10–15\mathrm{\mu }\mathrm{g}/\text{dL}$ (Sobolewski et al. 2018b; Weston et al. 2014), consistent with the Centers for Disease Control and Prevention definition of a level of concern of $10\mathrm{\mu }\mathrm{g}/\text{dL}$ prior to 2012 (CDC 2019b) and within the range of human exposures (Pirkle et al. 1994). Relative to the generational time represented in this study, blood leads of $24–36\mathrm{\mu }\mathrm{g}/\text{dL}$, with the highest exposed children at nearly $60\mathrm{\mu }\mathrm{g}/\text{dL}$ in 1979, were reported in neurotoxicological studies (Needleman et al. 1979). Following 2 months of Pb exposure, a 1:1 breeding scheme was used, with 40 unexposed male mice. Gestation day 0 (GD0) was designated as the day when a vaginal plug was detected, at which point females were moved to individual housing for the duration of pregnancy and lactation. Half of the females in the 0- and the $100\text{-ppm}$ Pb exposure groups, randomly chosen, underwent prenatal immobilization restraint stress (PS) in a 30-min session on GD15–18, a critical window for glucocorticoid sensitivity (Cintra et al. 1993; Noorlander et al. 2006), whereas the others remained in their home cages. This yielded four groups of F0 dams: 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$, 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$), and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$)].

The F1 females and males were thus developmentally exposed to Pb, PS, or the combination of Pb and PS. At PND60, they were bred to unexposed C57BL/6J mice to generate the F2 offspring. Subsequently, the F2 offspring were bred at PND60 to age-matched unexposed C57BL/6J mice to generate the F3 generation of offspring. At least 12 dams were generated for each of these lineages at each generation (Figure 1). This design allowed us to follow sex-specific differences ($\mathrm{F}=\text{female}$; $\mathrm{M}=\text{male}$) in each lineage across the experiment from F1 to F3, resulting in four lineages for each F3 sex ($\text{female \hspace{0.17em}}\mathrm{F}3\text{\hspace{0.17em} offspring}=\text{FFF}$, FMF, MMF, and MFF; $\text{male \hspace{0.17em}}\mathrm{F}3\text{\hspace{0.17em} offspring}=\text{MMM}$, MFM, FFM, FMM). A total of 8–10 dams were used to generate each of the F3 lineage offspring, with a single pup/sex/dam included in each outcome measure. Germ cells were collected from male and female offspring from F1 and F2 generations and stored at $-80°\mathrm{C}$ for future analysis. Mature sperm were taken from the cauda epididymis of 10- to 12-week-old male mice. For oocytes, germinal vesicle (GV)-stage oocytes were retrieved from six female mice per group also at 10–12 weeks of age.

Litter size and total litter weight were measured from PND1 until weaning (PND23–27). Runts were counted at weaning and defined as pups that weighed less than half the average weight at weaning (F3: F: $14\mathrm{g}$ and M: $16\mathrm{g}$). Any litter that had one runted pup or more was defined as a runted litter, this was a conservative determination to focus on the dam as the independent unit of analysis (see Figure S1). Pups were pair housed by sex/treatment group for the F1, F2, and F3 offspring. F3 offspring groups included subsets that underwent behavioral testing as well as those that did not undergo behavioral assessments (nonbehavioral controls). For each dam in each of the F0-F1-F2 breedings, a single male and female pup were used to preclude litter-specific effects. In the F3 generation, single pups/sex/dam were also used to generate groups for behaviorally tested and nonbehaviorally tested offspring. Sample sizes depend upon the assay, and all animals were randomly chosen in subsets from the full cohort for use in different assays. All experimental activities were approved by the University of Rochester Institutional Animal Care and Use Committee.

Behavioral Outcomes

Behavioral testing on the FI schedule relies on food reinforcement and thus required mice to be food-motivated. For that purpose, caloric restriction was initiated at approximately 60 d of age in all F3 offspring regardless of behavioral testing status to maintain uniform conditions across all mice. Mice were restricted to approximately 85% of their ad libitum feeding weights for the duration of behavioral testing. Animals were tested in a counterbalanced order in all paradigms, and these tests were carried out at a constant time of day.

Fixed-interval schedule-controlled behavior.

Assessment of FI schedule-controlled behavior was conducted in operant chambers (Med Associates) housed in sound-attenuating cabinets equipped with white noise for attenuation of distracting sounds and fans for ventilation. Three response levers were located horizontally across the back wall of the chamber (left, center, right), with a liquid dipper and dual pellet dispenser for reinforcer delivery on the front (opposite wall). Mice were initially trained to press the left lever via an overnight autoshaping program previously developed in our laboratory (Cory-Slechta and Weiss 1985). It began with a 20-min period during which food rewards were delivered independently of behavior on a random time basis, whereas a response on the left lever could also produce a food reward. Following the 20-min period, free food deliveries ceased and only a response on the left lever produced a sucrose pellet reward ($20\mathrm{mg}$; Bio-Serv) and this schedule remained in effect until 50 reinforcers were earned. Subsequently, an FI 60-s schedule of reinforcement (FI60) was imposed, which was programed to reward the first left lever response that occurred after a fixed interval of time (60-s) had elapsed. Reward delivery also initiated the next 60-s interval. Responses during the 60-s interval had no programed consequences; responses on either the center or right lever never provided reward. A total of 30 FI schedule behavioral test sessions, each of which were 30 min in duration, were carried out 5 d/week (Monday–Friday). Behavioral measures of FI performance included overall response rates (total responses/total session time), postreinforcement pause time (PRP; time to the first response in the interval), run rate ($\text{total \hspace{0.17em} responses}/\text{total \hspace{0.17em} time}–\text{postreinforcement \hspace{0.17em} pause \hspace{0.17em} time}$), and inter-response times (IRTs), that is, time between each successive response (Rossi-George et al. 2011).

Locomotor activity.

A single 60-min test session was carried out to examine locomotor activity in automated chambers equipped with 48-channel infrared photobeams (Med Associates). Photobeam breaks were recorded in 5-min bins to assess horizontal, vertical, and ambulatory movements across time (resulting in 12 bins for a 1-h locomotor activity session). Ambulatory counts were defined as the number of beam breaks, whereas in ambulatory movement; ambulatory episodes were defined by a minimum number of ambulatory counts (at least three successive photobeam breaks). The total ambulatory time refers to the time in ambulatory movement status, whereas distance represents the Euclidean distance of all ambulatory episodes, which together can also be used to determine speed (average velocity). Vertical activity was defined as movement that broke photobeams placed in the z-axis with breaks in the x and y-axes as well. Resting time was defined as time spent with no new photobeam breaks. Stereotypic counts and stereotypic time measure activity within a defined space in the arena. Jump counts are measured as the number of time periods that no x or y-axis beam breaks were detected, with a break in the z-axis, whereas jump time reflects the total duration in seconds of the jump counts.

Elevated plus maze.

The elevated plus maze (EPM) measured time spent in enclosed versus open (nonpreferred) arms (Hogg 1996). Mice were initially placed in a center arena of the apparatus. Behavior was recorded as either time spent in the closed arms (with sides) or time spent in the attached open arm and center (no sides) platform over a 5-min period. Performance was reported in total duration of time (in seconds) spent in either condition. Sessions were videorecorded and subsequently scored by an observer blinded to treatment status using Observer XT (version 13.0; Noldus).

Tissue collection.

Tissue was collected from both behaviorally tested and nonbehaviorally tested F3 offspring after the conclusion of behavioral testing at approximately 120 d of age. Mice were weighed and then sacrificed by cervical dislocation without the use of sedatives and fresh brains removed and hemisected. Randomization across groups was used during sacrifice to prohibit circadian variation occurring in a single group. The striatum, frontal cortex, and hippocampus were dissected from each brain after which they were flash-frozen. Trunk (whole) blood was collected into pre-chilled centrifuge tubes and centrifuged for 20 min at $\mathrm{3,500}\times g$ to obtain serum. Serum samples were stored at $-20°\mathrm{C}$ until day of assay.

Pb determinations in F1 and F3 blood and F3 bone.

Blood Pb (PbB) measurements were determined from trunk blood of F1 PND5–6 offspring and F3 120-d offspring using a PerkinElmer PinAAcle™ 900Z atomic absorption spectrophotometer equipped with longitudinal Zeeman background correction and a transverse heated graphite furnace (PerkinElmer Life and Analytical Sciences). Lead absorption was measured at $283.3\text{\hspace{0.17em} nm}$ using a PerkinElmer System 2 electrodeless discharge lamp source. In a $1.5\text{-}\mathrm{mL}$ microcentrifuge tube, $50\text{-}\mathrm{\mu }\mathrm{L}$ blood samples were diluted 1:10 with chemical modifier solution ($450\mathrm{\mu }\mathrm{L}$) containing 0.2% wt/vol ammonium phosphate [$\left({\text{NH}}_4\right){3\text{PO}}_4$]; 0.5% (vol/vol) Triton™ X-100; and 0.2% nitric acid (${\text{HNO}}_3$). Sample aliquots ($15\mathrm{\mu }\mathrm{L}$) of the diluted blood were deposited in the graphite furnace with an auto sampler.

Bone was isolated from 120-d-old F3 offspring and digested with ${\text{HNO}}_3$ and brought to a constant volume for determination of Pb content using a PerkinElmer PinAAcle™ 900Z atomic absorption spectrophotometer equipped with longitudinal Zeeman background correction and a transverse heated graphite furnace (PerkinElmer Life and Analytical Sciences). Lead absorption was measured at $283.3\text{\hspace{0.17em} nm}$ using a PerkinElmer System 2 electrodeless discharge lamp source. A bone matrix modifier of $\left({\text{NH}}_4\right){3\text{PO}}_4$ and calcium nitrate [$\mathrm{Ca}{\left({\mathrm{NO}}_3\right)}_2$] was used to stabilize lead during the pyrolysis furnace step. The modifier solution contained 2% ${\text{HNO}}_3$, 0.4% (wt/vol) ammonium dihydrogen phosphate (${\text{NH}}_4{\mathrm{H}}_2{\text{PO}}_4$), and $0.4\mathrm{g}/\mathrm{L}$ calcium as $\mathrm{Ca}{\left({\mathrm{NO}}_3\right)}_2$.

Serum corticosterone determinations.

Serum corticosterone was measured in duplicate using a commercially available enzyme immunoassay kit (Arbor Assays) according to manufacturer’s specifications. Sample replicates with coefficient of variation (CVs) higher than 15% were excluded from analysis.

Levels of striatal Th, GR, and BDNF.

Protein expression levels of Th, GR, and BDNF were assessed in frontal cortex and hippocampal nuclear and cytosolic brain fractions. Nuclear and cytoplasmic fractions were extracted from samples in $50\text{-mM}$ Tris buffer with 10% sucrose, 0.5 M sodium chloride (NaCl), and 1 Complete™ Mini Protease Inhibitor Cocktail tablet (Roche) per $10\mathrm{mL}$ buffer, using repeated homogenization and centrifugation on ice. Total protein concentration was quantified by standard bicinchoninic acid assay (Pierce). Sample volume required for $10\mathrm{\mu }\mathrm{g}$ of protein was diluted 1:1 with loading buffer (Bio-Rad Laemmli Sample Buffer, 5% $\mathrm{\beta }\text{-mercaptoethanol}$), denatured for 5 min in a 100°C water bath, and loaded onto 7–10% Tris-glycine. Sample analyses were counterbalanced by treatment and behavioral group across all gels. Gels were run using Tris/glycine/sodium dodecyl sulfate buffer (Bio-Rad) transferred to a $0.2\text{-}\mathrm{\mu }\mathrm{m}$ nitrocellulose membrane using a semi-dry transfer apparatus at $15\mathrm{V}$ for 30 min. Membranes were then washed in Tris-buffered saline (TBS) containing 1% Tween- 20 (TBST), blocked in TBST with 5% nonfat milk and incubated overnight in primary antibody as follows: GR, 1:500 dilution (catalog no. MA1-510; Thermo Scientific); actin, 1:10,000 dilution (catalog no. A1978; Sigma-Aldrich); BDNF, dilution 1:300 (catalog no. AB1534; Millipore); and Th, dilution 1:2000 (catalog no. AB152; Millipore). All primary and secondary antibodies were validated before use and delivered in TBST with 2% nonfat milk. Blots were developed using $1\mathrm{mL}$ per membrane of Clarity Western ECL Substrate (Bio-Rad) for 5 min and visualized using a Bio-Rad ChemiDoc MP Imaging System. Images were transferred to ImageJ software (Schneider et al. 2012) and bands for each sample were normalized to $\mathrm{\beta }\text{-actin}$ loading controls. Data were then expressed as normalized relative optical density (ROD). The proteins were detected at the following molecular weights: GR: 97kDa, BDNF: 18 kDa with homodimer at $\sim 27–30\text{\hspace{0.17em} kDa}$ (detected at around 30), and Th: 62 kDa.

DNA methylation.

Targeted DNA methylation analysis focused on the Bdnf and Th genes in the frontal cortex and Nr3C1, Bdnf, Th, Esr1 and insulin-like growth factor 2 Igf2/H19 in the hippocampus. Hippocampal genomic DNA was extracted using the DNeasy Blood and Tissue DNA extraction kit (Qiagen) according to manufacturer’s instruction and quantified using the NanoDrop spectrophotometer. In addition, frontal cortex genomic DNA and total RNA were simultaneously extracted using the AllPrep DNA/RNA extraction kit (Qiagen) according to manufacturer’s instruction and nucleic acids quantified as described for hippocampal DNA. One microgram genomic DNA was bisulfite-treated using the EpiTect Bisulfite Conversion kit (Qiagen) and $20\text{\hspace{0.17em} ng}$ of bisulfite-converted DNA used for polymerase chain reaction (PCR) amplification. We designed PCR and sequencing primers using the Pyromark Q24 Assay Design Software. PCR primer information is described in Table S1. Amplified PCR products were sequenced using a Pyromark Q24 Advanced Pyrosequencer and analyzed using the PyroMark Q24. Four CpG sites within the exon IV gene body of Bdnf were assessed; CpG site 1 of 4 is a cAMP-response element binding protein (CREB) binding site, a transcription factor essential for Bdnf transcription (Kundakovic et al. 2015). Six CpG sites within the imprinting control region of the imprinted gene locus, Igf2/H19 were analyzed because the sites are sensitive to environmental perturbation and also considered a site for DNA methylation alterations (Rivera et al. 2008; Susiarjo et al. 2013). For Nr3c1, two CpG sites within the promoter that are established nerve growth factor-inducible protein (NGFI-A) binding sites were examined as sites critical for Nr3c1 transcription (Liberman et al. 2012). Six CpG sites within the promoter of Th were assessed; these sites were altered by isolation stress, resulting in altered Th gene expression in a model of isolation stress (Niwa et al. 2013).

Statistical Analyses

Two approaches to data analyses were included given that few studies include both sex and lineage as factors in transgenerational inheritance. To determine whether transgenerational F3 effects were present, analyses of all outcome measures were undertaken using F3 sex as a factor, that is, collapsed across lineages. In addition, to examine potential lineage-based effects, analyses of all outcome measures were done by specific lineage (F1-F2-F3). Repeated measures analyses of variance (ANOVA) were used to evaluate measures of FI performance and locomotor activity, with Pb and PS as between group factors; and time (FI: across sessions; locomotor activity: across 5-min bins within the 60-min session) as the within group factor. Based upon our innumerable prior observations of differential effects of both Pb and PS by sex (Cory-Slechta et al. 2017, 2010, 2013a; Schneider et al. 2016; Varma et al. 2017; Virgolini et al. 2006, 2008a), these analyses were carried out separately by sex. Significant main effects or interactions were followed by post hoc t-tests or two-factor ANOVA as appropriate. Elevated plus maze data, as well as striatal protein expression levels and DNA methylation changes, were analyzed separately by sex using two-factor ANOVA with Pb and PS as between group factors. Significant main effects or interactions were followed by post hoc t-tests. Cases where $\mathrm{Pb}+\text{\hspace{0.17em} PS}$ (100-PS) were solely significantly different from control in post hoc tests are indicated on graphs.

Statistical analyses of potential lineage-based differences across 0-NS control groups were also examined for all behavioral measures to determine whether control group differences might underlie any apparent F3 transgenerational effect. Assessment of changes in numbers of runted litters in F3 offspring was determined using chi-square analyses. A $p\le 0.05$ was considered to confirm statistical significance; trends (i.e., $p>0.05$), where relevant, are indicated.

Results

Clinical Outcomes

No differences in maternal weight gain, successful pregnancies, litter size, or offspring weight at weaning (with all runts excluded) were found in the F1 and F2 breeding (see Table S2). However, in F3 offspring, a significant increase in runting was found in litters derived from Pb exposure, particularly from the combined Pb and PS exposures of the F0 generation [see Figure S1; chi-square Pearson $\text{value}=27.75$, $p=0.023$, (1/80 litters for control and 8/76 for Pb; specifically, 1 from control, 1 from Pb-only, and 7 from combined Pb- and PS-exposed F0 conditions)]. Runting did not appear to be lineage specific—with one runted litter from the control FM (F1-F2) lineage, one from the Pb-only exposed MF lineage, two from the combined Pb and PS exposed FF lineage, three from the combined Pb and PS exposed MF lineage, and two from the combined Pb and PS exposed MM lineage—but, rather, occurred more frequently in the $\mathrm{Pb}+\text{\hspace{0.17em} PS}$ lineages. These runts were excluded from any further testing.

Lead Determinations

Blood Pb values of F1 pups at PND6–7 were significantly elevated compared with control pups [$\mathrm{F}\left(\mathrm{3,20}\right)=488.3$, $p<0.001$; $0.0\mathrm{\mu }\mathrm{g}/\text{dL}$ vs. $12.5\pm 0.34\mathrm{\mu }\mathrm{g}/\text{dL}$, respectively]. In F3 offspring, no increases in either blood Pb or bone Pb were found above background Pb concentrations [blood: $\mathrm{F}\left(\mathrm{3,34}\right)=0.94$, $p=0.34$; bone: $\mathrm{F}\left(\mathrm{3,19}\right)=0.16$, $p=0.69$]. Specifically, F3 bone Pb means and standard errors (SEs) were $0.00\mathrm{\mu }\mathrm{g}/\text{dL}$ and $5.2\pm 5.2\text{\hspace{0.17em} ng}/\mathrm{g}$ for control and $0.4\pm 0.4\mathrm{\mu }\mathrm{g}/\text{dL}$ and $10.3\pm 5.1\text{\hspace{0.17em} ng}/\mathrm{g}$ for Pb-treated mice.

Transgenerational Effects

Findings are presented in the main text in both narrative and graphical formats primarily for statistically significant outcomes, many of which were lineage specific. In the interest of clarity, readability, and transparency, all data, including nonsignificant outcomes and data by lineage or across lineage are provided in Table 1 or available in the Supplemental Material as described below.

Table 1.

Summary of statistical outcomes by lineage.

Brain and behavioral outcomesa	F3 Females				F3 Males
	F1 Dam		F1 Sire		F1 Dam		F1 Sire
	FFF	FMF	MFF	MMF	FFM	FMM	MFM	MMM
Fixed interval performance
Overall response rate	$\mathrm{Pb}\times \text{PS}\times \mathrm{T}$ (I)	$\mathrm{Pb}\times \text{PS}$ (I)	—	$\mathrm{Pb}\times \text{PS}\times \mathrm{T}$ (I)	Pb (I)	—	—	—
Run rate	$\mathrm{Pb}\times \text{PS}$ (I)	$\mathrm{Pb}\times \text{PS}$ (I)	—	—	—	—	—	PS (I)
Mean postreinforcement pause	—	—	—	—	$\mathrm{Pb}\times \text{PS}$	$\mathrm{Pb}\times \mathrm{T}$; $\text{PS}\times \mathrm{T}$	$\mathrm{Pb}\times \text{PS}\times \mathrm{T}$	—
Elevated plus maze
Closed	—	—	Pb (D)	—	—	—	—	—
Open	—	—	Pb (I)	—	—	—	—	—
Locomotor time course
Ambulatory distance	—	—	—	—	$\text{PS}\times \mathrm{T}$	—	—	—
Ambulatory time	—	PS; $\mathrm{Pb}\times \text{PS}$	—	—	$\text{PS}\times \mathrm{T}$	$\mathrm{Pb}\times \mathrm{T}$	—	—
Ambulatory counts	—	$\mathrm{Pb}\times \text{PS}$	—	—	$\text{PS}\times \mathrm{T}$	—	—	—
Stereotypic time	—	$\mathrm{Pb}\times \mathrm{T}$	—	—	—	—	—	—
Stereotypic counts	—	$\mathrm{Pb}\times \mathrm{T}$	—	—	—	$\mathrm{Pb}\times \mathrm{T}$	$\mathrm{Pb}\times \text{PS}\times \mathrm{T}$	—
Resting time	—	$\mathrm{Pb}\times \text{PS}$	—	—	$\text{PS}\times \mathrm{T}$	$\mathrm{Pb}\times \mathrm{T}$	$\mathrm{Pb}\times \mathrm{T}$; $\mathrm{Pb}\times \text{PS}\times \mathrm{T}$	—
Vertical counts	—	—	—	—	—	$\mathrm{Pb}\times \mathrm{T}$	—	—
Vertical time	—	—	—	—	$\mathrm{Pb}\times \mathrm{T}$	$\mathrm{Pb}\times \mathrm{T}$	—	—
Serum corticosterone
Serum corticosterone	—	—	∼Pb (D)	∼PS (D)	—	—	—	—
Striatal protein (nuclear and nuclear/cytosolic)
Tyrosine hydroxylase (Th)	Pb (I)	—	$\mathrm{Pb}\times \text{PS}$	∼$\mathrm{Pb}\times \text{PS}$ (I)	—	Pb (D)	∼$\mathrm{Pb}\times \text{PS}$	Pb (I)
Brain-derived neurotrophic factor (BDNF)	PS (I)	—	$\mathrm{Pb}\times \text{PS}$ (I)	Pb (I)	—	—	—	—
Glucocorticoid receptor (GR)	∼Pb (I)	—	PS (D)	—	PS (I)	—	—	—
Total DNA methylation
Frontal cortex Th	—	$\mathrm{Pb}\times \text{PS}$	—	—	—	—	—	—
Frontal cortex BDNF	—	—	∼Pb (I)	—	—	—	—	—
Hippocampus Th	Pb (I)	—	—	—	Pb (I)	∼PS (D)	Pb (I)	—
Hippocampus BDNF	—	—	—	∼PS (D)	—	PS (D)	—	Pb (D)

Note: ANOVA, analysis of variance; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MMM, F1 male F2 male F3 male; Pb, lead; PS, prenatal stress; T, time (minute, block, sessions).

^aDirections of effect are indicated for main effect by $\left(\mathrm{I}\right)=\text{increase}$, $\left(\mathrm{D}\right)=\text{decrease}$. Directions of effect are generally not indicated for interaction effects where direction differs across groups; where consistent across groups, direction is indicated. Presence of text within a cell indicates significance ($p<0.05$); ∼, significance ($p<0.10$); —, nonstatistically significant effects as determined via repeated measures or factorial ANOVAs.

Behavioral Changes

FI schedule-controlled behavior.

Pb exposure resulted in a significant increase in overall response rates on the FI schedule of reward in F3 female offspring when considered across all lineages [main effect of Pb: $\mathrm{F}\left(\mathrm{1,149}\right)=4.92$, $p=0.028$]. Figure 2A displays these effects for the F3 female offspring across behavioral test sessions. No statistically significant effects of Pb, PS, or combined Pb and PS on F3 male offspring FI overall response rates were seen (Figure 2B). Effects on overall response rate on the FI schedule, however, differed by lineage and F3 sex, as can be seen in the FMF lineage (Figure 2C), where greater magnitude rate increases in response to Pb were seen in FMF F3 females [Pb by PS by sessions: $\mathrm{F}\left(\mathrm{3,29}\right)=9.98$, $p=0.0408$; Pb by sessions: $\mathrm{F}\left(\mathrm{1,38}\right)=5.59$, $p=0.023$], which at peak values were approximately 50% higher than those of control (0-NS) FMF females. Despite the absence of effects on overall response rates across lineages, moreover, F3 males from the FM lineage (Figure 2D) likewise showed increases in FI overall response rates [FMM, Pb by PS: $\mathrm{F}\left(\mathrm{1,33}\right)=4.87$, $p=0.034$], which averaged approximately 20% higher in Pb than in control F3 males.

Figure 2.

Fixed-interval (FI) response rates. (A,B) Group $\text{mean}\pm \text{SE}$ overall response rates (responses per minute) on the FI schedule for F3 female (A) and male (B) offspring derived from the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$), and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$) groups collapsed across lineages over the course of behavioral test sessions. $\text{Sample \hspace{0.17em} sizes \hspace{0.17em} of \hspace{0.17em}}n=32–40/\text{group}$. (C,D) Group $\text{mean}\pm \text{SE}$ FI overall response rates for F3 0-NS, 0-PS, 100-NS, and 100-PS groups of the FMF (C) and FMM (D) lineages, respectively. Sample sizes of $n=8–12/\text{group}$ for females; $n=7–10/\text{group}$ for males. $\mathrm{Pb}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}$ and $\mathrm{Pb}\times \text{PS}=\text{interaction \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}\text{\hspace{0.17em} by \hspace{0.17em} PS}$ following repeated measures ANOVA. *, $p\le 0.05$ compared with control; +, $p\le 0.05$ compared with PS alone; ^, $p\le 0.05$ compared with combined Pb and PS. Note: ANOVA, analysis of variance; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; Pb, lead; PS, prenatal stress; SE, standard error.

Behavioral mechanisms (determinants) underlying the increases in FI overall response rates differed by sex (see Excel Tables S1 and S2), with increases in FI overall rates of F3 females of the FMF lineage resulting primarily from increases in run rates, that is, there are faster rates of responding once responding began during the fixed interval {Pb by PS [$\mathrm{F}\left(\mathrm{1,38}\right)=6.67$, $p=0.0138$]}, with run rates reaching values that were double those of 0-NS FMF females. Although some evidence of Pb-related increases in run rates were observed in FMM males, these effects were of only marginal significance [Pb by PS: $\mathrm{F}\left(\mathrm{1,33}\right)=3.61$, $p=0.0664$]. Instead, the increases in FI overall response rates in F3 males from the FMM lineage was due to the shorter period of time pausing prior to initiating responding during the fixed interval, that is, reductions in postreinforcement pause time. Although no significant treatment-related differences in postreinforcement pause time were found in F3 FMF females, these values were significantly shortened, particularly by Pb exposure in the FMM line {Pb by PS [$\mathrm{F}\left(\mathrm{1,33}\right)=5.49$, $p=0.0253$]}.

Locomotor activity.

Small but statistically significant changes in various measures of locomotor activity were found in F3 females that appeared to derive primarily from F0 Pb exposure-induced increases in ambulation that were observed across lineages {Figure 3A; ambulatory distance: PS by block [$\mathrm{F}\left(\mathrm{11,136}\right)=2.06$, $p=0.0268$]; Figure 3B, ambulatory counts: Pb by PS [$\mathrm{F}\left(\mathrm{1,146}\right)=3.99$, $p=0.048$]}.

Figure 3.

Locomotor activity. Group $\text{mean}\pm \text{SE}$ levels of (A) ambulatory distance (cm) and (B) ambulatory counts of the female F3 offspring from the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$), and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$) groups analyzed across F3 female lineages during locomotor activity assessment; sample sizes of $n=35–40\text{\hspace{0.17em} per}$ group. Group $\text{mean}\pm \text{SE}$ levels of (C) ambulatory time and (D) ambulatory episodes in the FMF lineage; sample sizes of $n=10/\text{group}$. Group $\text{mean}\pm \text{SE}$ levels of (E) vertical counts and (F) vertical times in the FMM lineage, sample sizes of $n=8–10/\text{group}$, across 5-min bins of the 60-min assessment. PS by $\text{Block}=\text{significant \hspace{0.17em} interaction}$ at $p\le 0.05$ between prenatal stress (PS) exposure and block in statistical analyses; $\mathrm{Pb}\times \text{PS}=\text{significant \hspace{0.17em} interaction \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}\text{\hspace{0.17em} exposure \hspace{0.17em} and \hspace{0.17em} PS}$ at $p\le 0.05$ of Pb, PS following repeated measures ANOVA. Note: ANOVA, analysis of variance; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; Pb, lead; SE, standard error.

Changes in locomotor activity were lineage specific, with F3 females and males showing alterations in the FM lineage, as seen with FI behavior. Corresponding to the Pb-related changes in ambulation observed across F3 female lineages, when analyzed by lineage, increases in ambulation were seen in the FMF lineage (Figure 3C,D), again deriving primarily from F0 Pb exposure-induced increases and confirmed by significant Pb by PS interactions in statistical analyses [ambulatory time: $\mathrm{F}\left(\mathrm{1,37}\right)=7.82$, $p=0.0082$; ambulatory episodes: $\mathrm{F}\left(\mathrm{1,37}\right)=4.13$, $p=0.0495$]. Changes in locomotor behavior were also evident in F3 males from the FMM lineage (Figure 3E,F). These included Pb-related reductions in vertical counts [Pb by T $\mathrm{F}\left(\mathrm{11,23}\right)=2.71$, $p=0.021$] and vertical time [Pb by T $\mathrm{F}\left(\mathrm{11,23}\right)=2.93$, $p=0.0142$], which can be seen in both the Pb-only and combined Pb and PS groups. Summary data and data from each lineage is included in Excel Tables S3 and S4.

Elevated plus maze.

Across lineages, F3 Pb females showed reduced time spent in the closed arms [Figure 4A; $\mathrm{F}\left(3,150\right)=5.6$, $p=0.02$] and increases in time spent in the center and open arms of the elevated plus maze, as indicated by main effects of Pb [$\mathrm{F}\left(\mathrm{1,150}\right)=5.8$, $p=0.017$]. These effects appeared to derive from Pb effects in F1 sire lineages in particular [Figure 4B, closed: $\mathrm{F}\left(\mathrm{1,73}\right)=7.8$, $p=0.006$; open: $\mathrm{F}\left(\mathrm{1,73}\right)=7.8$, $p=0.006$], whereas there were no effects in F1 dam lineages [Figure 4C, closed: $\mathrm{F}\left(\mathrm{1,75}\right)=0.20$, $p=0.69$]. There were no significant changes in F3 males across all lineages or as related to F1 sire or F1 dam (Figure 4D–F).

Figure 4.

Elevated plus maze. Duration of time in the closed arms of the elevated plus maze (group $\text{mean}\pm \text{SE}$) of (A–C) F3 female and (D–F) F3 male offspring of the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$) and 100-PS ($\mathrm{Pb}+\text{\hspace{0.17em} PS}$) groups across all F3 lineages (A,D: sample sizes of $n=37–40/\text{group}$), and separated by F1 sire lineages. (B) MMF and MFF collapsed for F3 females. (E) MFM and MMM for F3 males; sample sizes of $n=18–20/\text{group}$, and F1 dam lineages (C) FFF and FMF for F3 females collapsed; (F) FMM and FFM for F3 males; sample sizes of $n=18–20/\text{group}$. Pb indicates an overall main effect of lead at $p\le 0.05$ following two-way ANOVA analysis. Note: ANOVA, analysis of variance; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MMM, F1 male F2 male F3 male; Pb, lead; PS, prenatal stress; SE, standard error.

Serum Corticosterone

Serum corticosterone levels were measured in nonbehaviorally tested F3 offspring, as behavioral testing alone significantly elevated serum corticosterone concentrations [$\mathrm{F}\left(\mathrm{1,262}\right)=11.18$, $p=0.0009$]. Analyzed across all lineages, Pb decreased serum corticosterone in F3 females [Figure 5A; $\mathrm{F}\left(\mathrm{3,57}\right)=4.78$, $p=0.032$]. Effects in F3 females however, again differed by lineage (Figure 5B,C), with a Pb-related reduction in serum corticosterone in the F3 females originating from the F1 sire lineages, with a the main effect of Pb [$\mathrm{F}\left(\mathrm{3,28}\right)=4.45$, $p=0.045$]. No F3 female changes were seen in the F1 dam lineages [$\mathrm{F}\left(\mathrm{3,29}\right)=1.6$, $p=0.21$]. No treatment-related differences in serum corticosterone were seen across lineages of F3 male offspring in nonbehaviorally tested groups or in relation to F1 sire or F1 dam (Figure 5D–F). See Excel Tables S7 and S8 for summary data.

Figure 5.

Serum corticosterone. Group $\text{mean}\pm \text{SE}$ levels of corticosterone (pg/mL) analyzed across lineages of (A–C) F3 female and (D–F) F3 male offspring of the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$), and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$) with sample sizes of $n=12–18/\text{group}$ across all lineages (A,D), separated by F1 sire [(B) MMF and MFF collapsed for F3 females; (E) MFM and MMM for F3 males; sample sizes of $n=6–8/\text{group}$ and by F1 dam lineage (C) FFF and FMF for F3 females collapsed; (F) FMM and FFM for F3 males; sample sizes of $n=6–8/\text{group}$]. None of these animals had undergone behavioral testing. $\mathrm{Pb}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em} lead}$ at $p\le 0.05$ following two-way ANOVA analysis, * = significantly different from 0-NS in post hoc t-test analysis. Note: ANOVA, analysis of variance; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MMM, F1 male F2 male F3 male; Pb, lead; PS, prenatal stress; SE, standard error.

Striatal Levels of Th, GR, and BDNF Protein

When analyzed across all lineages, there were no F3 effects for nuclear or cytosolic Th or GR. However, significant differences were found by lineage, including changes in expression levels of striatal Th (Figure 6A). Analyses of Th nuclear/cytosolic fractions revealed an effect of Pb exposure in the FFF lineage [$\mathrm{F}\left(\mathrm{1,19}\right)=5.21$, $p=0.036$]. Striatal nuclear cytosolic Th expression levels were significantly reduced in the FMM lineage Pb group [$\mathrm{F}\left(\mathrm{1,19}\right)=6.25$, $p=0.0237$], and significant Pb by PS interaction was found in the MFF lineage [$\mathrm{F}\left(\mathrm{1,19}\right)=5.07$, $p=0.0389$], likely due to the reductions in the PS-only and Pb only groups, but post hoc comparisons did not reveal any significant between group differences. For striatal Th nuclear expression (Figure 6B), marginal trends for Pb by PS interactions and Pb effects were observed in the MFM and MMM lineages, respectively [$\mathrm{F}\left(\mathrm{1,19}\right)=4.18$, $p=0.057$ and $\mathrm{F}\left(\mathrm{1,19}\right)=4.14$, $p=0.051$]. Data for each lineage is shown in Figures S2 and S3 and Excel Tables S9 and S10.

Figure 6.

Striatal protein levels of tyrosine hydroxylase (Th), glucocorticoid receptor (GR), and brain-derived neurotrophic factor (BDNF). Group $\text{mean}\pm \text{SE}$ levels [relative optical density (ROD)] of expression striatal Th nuclear/cytosolic fraction in offspring of the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$), and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$) groups (sample size of $n=4–5/\text{group}$) from the FFF and MFF F3 female offspring (A) and Th nuclear fraction in FMM, MFM, and MMM male F3 offspring (B). Group $\text{mean}\pm \text{SE}$ levels (ROD) of expression of striatal (C) BDNF nuclear (nuc)/cytosolic (cyto) fraction and (D) nuclear BDNF in the FFF and MFF lineages of F3 female offspring (sample sizes of $n=5/\text{group}$). $\mathrm{Pb}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}$ at $p\le 0.05$, $\text{PS}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em} PS}$ at $p\le 0.05$, and $\mathrm{Pb}\times \text{PS}=\text{significant \hspace{0.17em} interaction \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}\text{\hspace{0.17em} and \hspace{0.17em} PS}$ at $p\le 0.05$ following two-way ANOVA analysis. $\sim \mathrm{Pb}$ by $\text{PS}=\text{marginal \hspace{0.17em} interaction \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}$ by PS in statistical analysis with a $p\le \mathrm{0.10.}$ * = significantly different from 0-NS; ^ = significantly different from 0-PS; ⁺ = significantly different from 100-NS in post hoc t-test analysis. Note: ANOVA, analysis of variance; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FMM, F1 female F2 male F3 male; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MMM, F1 male F2 male F3 male; Pb, lead; PS, prenatal stress; SE, standard error.

Through the maternal lineage for F3 females and males, nuclear GR was marginally elevated in the FFF Pb group [$\mathrm{F}\left(\mathrm{1,19}\right)=4.4$, $p=0.053$] and significantly increased in the male FFM PS group [$\mathrm{F}\left(\mathrm{1,19}\right)=10.95$, $p=0.004$]. There were no significant changes in GR expression for any additional lineages (see Excel Tables S9 and S10).

Analyzed across all lineages, levels of striatal nuclear BDNF were marginally increased by F0 PS exposure in female F3 offspring with all lineages combined (see Figure S4, top left) [$\mathrm{F}\left(\mathrm{1,78}\right)=4.16$, $p=0.07$], with highest levels in the $\mathrm{Pb}+\text{\hspace{0.17em} PS}$ group. For F3 females exposed to PS through the maternal lineage (FFF), both nuclear and nuclear/cytosolic BDNF was significantly increased {Figure 6C, main effect of PS: $\mathrm{F}\left(\text{nuclear}/\text{cytosolic}:\text{cytosolic}\mathrm{1,19}\right)=8.18$, $p=0.0114$; Figure 6D, F(nuclear: $\mathrm{1,19}=7.26$, $p=0.016$), and for BDNF nuclear/cytosolic ratios, there was also a significant interaction between Pb and PS in the MFF lineage [$\mathrm{Pb}\times \text{PS}$, $\mathrm{F}\left(\mathrm{3,19}\right)=4.72$, $p=0.045$], which subsequent post hoc tests showed to be due to the significant increases in BDNF expression levels in the combined Pb and PS group}. MMF mice derived from F0 dams treated with Pb exhibited significantly higher nuclear expression of BDNF [$\mathrm{F}\left(\mathrm{1,19}\right)=4.72$, $p=0.04$]. No changes were found in any striatal nuclear or cytosolic BDNF protein expression levels in F3 males. Data for each lineage is shown in Figures S4 and S5 and Excel Tables S9 and S10.

Frontal Cortex and Hippocampal DNA Methylation Changes

Significant changes in total DNA methylation when analyzed across lineages (Figure 7A,B) were limited to a Pb by PS interaction for frontal cortex methylation of Th in F3 females [$\mathrm{F}\left(\mathrm{3,74}\right)=4.16$, $p=0.045$], although post hoc comparisons did not provide any significant differences among treatment groups. A significant reduction in total methylation of hippocampal Bdnf in F3 males derived from F0 Pb exposure was also observed [$\mathrm{F}\left(\mathrm{3,76}\right)=4.81$, $p=0.031$].

Figure 7.

DNA methylation changes in frontal cortex and hippocampus. (A,B) Group $\text{mean}\pm \text{SE}$ levels of total percentage methylation of hippocampal Bdnf (brain-derived neurotrophic factor) across lineages of F3 male offspring [(A) sample sizes of $n=18–20/\text{group}$] and frontal cortex tyrosine hydroxylase (Th) in female F3 offspring [(B) sample sizes of $n=17–20/\text{group}$] of the F0 0-NS [control; $0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} no \hspace{0.17em} stress \hspace{0.17em}}\left(\text{NS}\right)$], 0-PS ($0\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$), 100-NS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} NS}$) and 100-PS ($100\text{\hspace{0.17em} ppm}$ $\mathrm{Pb}+\text{\hspace{0.17em} PS}$) groups. (C–E) Group $\text{mean}\pm \text{SE}$ levels of total percentage methylation of Th in the frontal cortex of FMF F3 females (C), group $\text{mean}\pm \text{SE}$ levels of total percentage DNA methylation in FFF (D) and FFM (E) F3 offspring (sample size of $n=4–5/\text{group}$). (F–H) MFM Group $\text{mean}\pm \text{SE}$ levels of total percentage DNA methylation of hippocampal Th in MFM (F), hippocampal Bdnf in the FMM (G), and hippocampal Bdnf in the MMM lineages of (H) F3 male offspring (sample size of $n=4–5/\text{group}$). $\mathrm{Pb}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}$ at $p\le 0.05$ in statistical analysis, $\text{PS}=\text{significant \hspace{0.17em} main \hspace{0.17em} effect \hspace{0.17em} of \hspace{0.17em} PS}$ at $p\le 0.05$ in statistical analysis; $\mathrm{Pb}\times \text{PS}=\text{significant \hspace{0.17em} interaction \hspace{0.17em} of \hspace{0.17em}}\mathrm{Pb}\text{\hspace{0.17em} and \hspace{0.17em} PS}$ at $p\le 0.05$ following two-way ANOVA analysis. * = significantly different from 0-NS; ^ = significantly different from 0-PS; ⁺ = significantly different from 100-NS in post hoc t-test analysis. Note: ANOVA, analysis of variance; FFF, F1 female F2 female F3 female; FFM, F1 female F2 female F3 male; FMF, F1 female F2 male F3 female; FMM, F1 female F2 male F3 male; HIPP, hippocampal; MFF, F1 male F2 female F3 female; MFM, F1 male F2 female F3 male; MMF, F1 male F2 male F3 female; MMM, F1 male F2 male F3 male; Pb, lead; PS, prenatal stress; SE, standard error.

As with other outcome measures, lineage-specific changes in total DNA methylation were observed. A Pb by PS interaction was observed for FC Th in the FMF lineage (Figure 7C) that generally mirrored the outcome across F3 female lineages [$\mathrm{F}(\mathrm{3,19})=5.5$, $p=0.032$]. Interestingly, several lineages showed Pb-related increases in percent total hippocampal Th DNA methylation in hippocampus {Figure 7D,E; FFF: [$\mathrm{F}(\mathrm{3,19})=5.41$, $p=0.033$; $\text{FFM}:\hspace{0.17em}\mathrm{F}(\mathrm{3,17})=6.46$, $p=0.024$]; and Figure 7F [$\text{MFM}:\hspace{0.17em}\mathrm{F}(3,18)=13.13$, $p=0.0025$]}, with the highest mean value as determined from post hoc tests in the combined Pb and PS group in each case.

In the FMM lineage (Figure 7G,H), PS exposure significantly reduced percent total hippocampal Bdnf DNA methylation [$\mathrm{F}(\mathrm{3,17})=6.46$, $p=0.024$], whereas an F0 Pb exposure related reduction in total hippocampal Bdnf percent DNA methylation was found in the MMM lineage [$\mathrm{F}(\mathrm{3,18})=8.06$, $p=0.0125$] that was most pronounced in the 100-PS group as indicated by post hoc t-test analysis.

Discussion

Knowledge of the neurotoxicity of Pb exposure dates back centuries (Hernberg 2000), with current understanding indicating that no safe level of Pb with respect to impaired cognitive function in children can be identified (CDC 2019a). More recent studies have documented consequences of developmental Pb exposure for brain and behavior enduring beyond childhood (Bellinger 2008; Cecil et al. 2008; Reuben et al. 2017). The consequences and nature of Pb are greater in lower socioeconomic status communities (Lefmann and Combs-Orme 2014), where a higher proportion of U.S. children younger than 6 years of age were found to have high or very high blood levels compared with those living in communities with a lower percentage of poverty (McClure et al. 2016). Because developmental Pb and PS share the HPA axis and brain MESO circuits as targets (Barros et al. 2004; Berger et al. 2002; Martínez-Telléz et al. 2009), our laboratory has studied these risk factors in combination. In these rodent studies, combined exposures led to enhanced neurotoxicity for some outcome measures as compared with Pb or PS alone (Virgolini et al. 2006, 2008a; Weston et al. 2014), findings confirmed in studies since carried out in human cohorts (Surkan et al. 2008; Tamayo y Ortiz et al. 2017). Recent studies have suggested that effects of PS may occur multigenerationally, and even transgenerationally (Gutteling et al. 2006; Schwabe et al. 2012; Weiss et al. 2011); whether similar passage occurs with Pb exposure is unknown. Using a mouse model, the present study sought to determine whether Pb, PS, or combined Pb and PS could produce transgenerational consequences for brain and behavior and to determine whether such effects differed by offspring sex and lineage.

In this study, we found that Pb, PS, and combined Pb and PS exposures produced transgenerational effects on brain and behavior. Analyzed across lineages, consistent effects that were largely associated with Pb exposure were observed in F3 females, with increases in rates of responding on the FI schedule, increases in ambulatory locomotor activity, decreases in time spent in the closed arms of the elevated plus maze, and reductions in serum corticosterone levels. For these F3 females, PS-only exposure also significantly increased striatal expression levels of BDNF. Fewer changes were seen across the F3 males, with transgenerational changes observed in the Pb group consisting of reductions in postreinforcement pause time on the FI schedule, altered locomotor activity, altered striatal Th concentrations, and decreased total percent DNA methylation of the Bdnf gene in the hippocampus.

Given the breeding strategy employed in this study, individual lineages could be tracked and specific lineages frequently showed greater magnitude effects than those observed in analyses collapsed across all lineages (summarized in Table 1). Lineage-specific analyses revealed that Pb-related increases in FI response rates of F3 females largely derived from lineages with F1 dams (grandmothers), including male parentage (FMF lineage) and mother parentage (FFF). Further, Pb-related locomotor changes were also seen in F3 female offspring from F1 dams, with male parentage (FMF lineage). In nonbehaviorally tested animals, Pb-related corticosterone reductions were seen in F3 female offspring through F1 sire lineage, with greater reductions in the MFF lineage. F3 female offspring through the F1 sire showed decreased time in the closed arms of the EPM. Increases in striatal BDNF in response to Pb alone or to combined Pb and PS were seen in F3 females from F1 sires, seen in the MFF and MMF lineages, whereas PS-based increases were found in the FFF lineage. Pb-related changes in total DNA methylation were seen in F3 females of the FFF lineage.

For F3 male offspring, lineage-specific changes were observed in FI response rates, locomotor activity, striatal Th expression, and hippocampal BDNF methylation profiles. Lineage-specific analyses revealed Pb, PS, or combined Pb and PS-related reductions in postreinforcement pause time, particularly in F3 male offspring from the MF, FM, and FF lineages. Similarly, although locomotor activity effects were relatively minor when analyzed across lineages, analyses of individual lineages revealed reductions in response to Pb, PS, or combined Pb and PS following exposures to the F1 grandmothers, particularly the FF and FM lineages. Pb-based reductions in F3 male offspring total hippocampal DNA methylation of Bdnf were primarily due to greater magnitude changes through the paternal MMM lineage. Collectively, these findings suggest that transgenerational passage is influenced by the sex composition of the lineage and that different lineages exhibit different profiles of transgenerational effects. Unfortunately, few studies of transgenerational effects include outside breeding or follow lineages other than FF and MM, yet all such lineages are present in human populations.

Notably, effects seen in response to Pb, PS, or combined Pb and PS in the F3 lineages are consistent with changes in behaviors and systems previously reported in developmental studies of F1 offspring. For example, Pb- as well as PS- and combined Pb and PS–induced changes in FI behavioral performance have been repeatedly noted in F1 offspring in our prior studies, with the nature of the consequences dependent upon multiple factors, including developmental period and level of Pb exposure as well as sex of offspring (Cory-Slechta and Thompson 1979; Cory-Slechta et al. 1985, 1997, 1998, 2002; Cory-Slechta 1997; Rossi-George et al. 2011; Virgolini et al. 2005, 2008a). The present study suggests that such effects could be transmitted across generations, with Pb-induced increases in FI response rates in females of the F3 generation arising from increased run rates. In addition, F3 male offspring of the FMM, FFM, and MMM lineages displayed significantly shorter postreinforcement pause times, contributing to the trend toward Pb-related increases in FI rates. Increases in FI response rates signify inefficient behavioral function given that increased response rates cannot accelerate reinforcement availability. In addition, increased response rates on FI schedules have been directly related to reductions in self-control in both infants and children (Darcheville et al. 1992, 1993). Shorter postreinforcement pause times also signify impairments in learning the timing of the interval, normally seen as a characteristic scalloped pattern of responding with little responding early in the interval and subsequent increases as time elapses. These FI changes are further notable given that they could be reflective of impulsivity and or inability to inhibit responding. All of these are features of neurodevelopmental disorders such as autism spectrum disorder and attention deficit hyperactivity disorder (Coghill et al. 2014; Hove et al. 2017; Lambrechts et al. 2018; López-Martín et al. 2015; Rubia et al. 2009; van Hulst et al. 2017).

Our prior experimental studies in rodents also show Pb, PS, and combined Pb and PS-induced changes in MESO neurotransmitter systems in developmentally exposed F1 offspring (Cory-Slechta et al. 2008, 2013a, 2013b; Weston et al. 2014) consistent with effects of these factors on brain dopamine systems. We have previously demonstrated in rats the importance of MESO dopamine to mediation of performance on the FI schedule of reward, a behavior altered by Pb and PS in rats (Cory-Slechta et al. 1998, 2002; Evans and Cory-Slechta 2000). The nature of the changes in dopamine systems, as with FI performance, is dependent upon developmental periods and concentration of Pb exposure, as well as sex of F1 offspring. These findings correspond to the observed changes in female F3 lineages in expression of striatal Th, the rate-limiting enzyme in dopamine synthesis, and with alterations in BDNF, which is critical to brain dopamine function (Bustos et al. 2004). BDNF is a protein that plays a key role in MESO system synaptic plasticity and learning/memory (Kowiański et al. 2018; von Bohlen und Halbach and von Bohlen und Halbach 2018) via mechanisms that include its control of dopamine D3 receptor function (Guillin et al. 2004). Rodent studies focused on developmental exposures to PS (stress to the F0 dam, prenatal for exposure the F1 generation) indicate that Bdnf mRNA levels are reduced in the hippocampus, with increases in DNA methylation particularly in females (Berry et al. 2015; Boersma et al. 2014; Dong et al. 2015; Seo et al. 2016). Our prior studies, moreover, have demonstrated that Pb exposure reduced BDNF protein levels in the frontal cortex, whereas PS increased BDNF in the nucleus accumbens and the hippocampus of male rats, and in females, PS exposure reduced nucleus accumbens BDNF (Weston et al. 2014). Correspondingly, in the F3 generation assessed in this study, PS-related increases in BDNF expression in the striatum were observed when analyzed across all F3 female lineages, particularly in the FFF lineage, whereas significant Pb-related increases occurred in the F1 male sire lineages (MMF and MFF lineages), particularly under conditions of combined Pb and PS. Alterations in BDNF by PS, including shifts in methylation and expression profiles, have likewise been related to neurodevelopmental disorders with sex-biased prevalence rates, including female-biased disorders such as anxiety and depression (Baker-Andresen et al. 2013; Zheng et al. 2016).

As mentioned above, our prior developmental (F1) studies have shown modification of the HPA axis function following Pb, PS, or both (Rossi-George et al. 2011; Virgolini et al. 2005, 2006, 2008a, 2008b). In the present study, female F3 offspring showed significant Pb-related reductions in corticosterone levels across lineages, particularly F3 females from F1 sires. In addition, the same group of females had significant reductions in time spent in the closed arms of the elevated plus maze, suggesting decreased fear or “anxiety-like” behavior. The strong influence in females may be due to female sensitivity, or due to the nature of gamete production. Future studies should also examine F2 males because these would be the first unexposed generation and may elicit more changes than F3 males in HPA axis function and behavior. Sex-dependent Pb-induced changes in stress responsivity need additional research on a) the functionality of the stress response because either increased or reduced serum corticosterone potentially represent disruptions to stress homeostasis (Korte et al. 2005; McEwen 2010; Peters et al. 2017), and b) additional generations of both F2 males and F4 females to determine if transgenerational effects are eliminated in subsequent unexposed generations. Furthermore, PS may have had a larger role using different types of stressors given that the nature and duration of a stressor have been shown to alter long-term, sex-specific neurophysiological responses (McEwen 2010; Peters et al. 2017).

Collectively, these data indicate that lineage and sex-specific transgenerational passage of brain and behavioral phenotypes can be induced by developmental exposures to risk factors such as Pb and PS. Importantly, these effects were seen even with full outbreeding using unexposed C57BL/6J mice, compared with transgenerational studies where treated animals are bred to each other. Although differences by sex in F3 consequences are certainly recognized (Dew-Budd et al. 2016; Zizzari et al. 2016), the contribution of lineage transfer in relation to sex, and potential dynamic changes in response to ongoing environmental conditions, have received far less attention (Burggren 2015).

What remains unknown from these studies is the mechanism by which these transgenerational effects are produced. Although transgenerational effects are primarily studied in the context of epigenetic inheritance, there are actually two major routes by which transgenerational effects and programing can occur that may include either direct molecular epigenetic inheritance or environmental passage, or both (Danchin et al. 2011), via parental, ecological, and cultural means. The fact that many of the behavioral and biochemical changes observed here were lineage dependent and that transgenerational effects were observed in both the FF and MM lineages could suggest that both types of mechanisms were operative. Further, although our prior studies of Pb, PS, and combined Pb and PS in F1 offspring revealed numerous instances of enhanced toxicity of combined Pb and PS, few such examples were found transgenerationally, with the majority of effects reflecting either Pb or PS exposures alone. Interestingly, most examples of enhanced F3 effects with combined Pb and PS exposure were seen in levels of the percent total DNA methylation of the Th and/or Bdnf gene in the frontal cortex and the hippocampus. Further, it is worth investigating whether changes in methylation patterns exist broadly and whether these changes may play a role in dysregulation of imprinted genes known to tightly regulate growth considering the increased number of litters with runted pups in F3 $\mathrm{Pb}+\text{\hspace{0.17em} PS}$ litters. Future studies investigating transgenerational mechanisms of cumulative risk should focus on multiple mechanisms of environmental transmission, including small RNAs, chromatin reprograming, and genomic imprinting and methylation, in addition to focusing on changes in maternal behavior and health and by understanding that transgenerational heredity can be directly inherited, occurring in response to inherited environments, or arise stochastically (Bale 2015; Boškovíc and Rando 2018). Understanding the differences in the transgenerational effects of Pb, PS, and combined Pb and PS, integrated across varied inheritance mechanisms, may help assist in defining differential contributions of lineage and offspring sex.

Understanding these complexities is critical, because in the human environment, Pb exposures are likely to be more complex. First, following birth, Pb and PS exposure will inevitably continue to occur. This dynamic sequence of events would likely induce epigenetic changes that could further influence a behavioral phenotype. Further, bone Pb stores are reduced during pregnancy and breastfeeding (Gulson et al. 2016), suggesting that Pb exposure is reduced across successive pregnancies. Hence, it might be expected that the magnitude of transgenerational passage would be altered based on birth order. These points are especially relevant to studies in human populations as well as to proposed reversals via epigenetic means of such transgenerational impacts.