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Additional file 2: Figure S2. Pyrosequencing validation of WGBS CpG sites. Bisulfite pyrosequencing of CpG sites initially identified via WGBS as significantly differentially methylated between late exposed sperm and controls for A Hoxb9; B Mettl11b; C Sycp3; D Cit; E Slit2; F Lrp1; G Cit; and early exposed and controls H Grin2a; I Syn3. CpG sites are identified as A-D on the x-axis, and the recorded methylation level is on the y-axis. Each point is an individual sperm. Black = controls, green = late exposed, blue = early exposed. *p < 0.05.

Additional file 1: Figure S1. Pyrosequencing Validation Curves. Defined mixtures of bisulfite modified fully methylated and unmethylated rat genomic DNAs were analyzed for linearity in ability to detect increasing amounts of methylation. X-axis, the input (expected) level of methylation, y-axis, the measured level of methylation. R2 and p-values values are indicated.

Additional file 3: Figure S3. Relationship between DNA methylation at CpG site 1 and gene expression for Mtss1l in the hippocampus. Relationship between DNA methylation and gene expression for Mtss1l in A all sexes combined; B males only; and C females only. Pearson correlation R square and p-values are reported. Control offspring = black with solid regression line; early exposed offspring = blue with dotted regression line.

Diazinon is an organophosphate pesticide that has a history of wide use. Developmental exposures to organophosphates lead to neurobehavioral changes that emerge early in life and can persist into adulthood. However, preclinical studies have generally evaluated changes through young adulthood, whereas the persistence or progression of deficits into middle age remain poorly understood. The current study evaluated the effects of maternal diazinon exposure on behavior and neurochemistry in middle age, at 1 year postpartum, comparing the results to our previous studies of outcomes at adolescence and in young adulthood (4 months of age) (Hawkey 2020). Female rats received 0, 0.5 or 1.0 mg/kg/day of diazinon via osmotic minipump throughout gestation and into the postpartum period. The offspring were tested on a battery of locomotor, affective, and cognitive tests at young adulthood and during middle age. Some of the neurobehavioral consequences of developmental DZN seen during adolescence and young adulthood faded with continued aging, whereas other neurobehavioral effects emerged with aging. At middle age, the rats showed few locomotor effects, in contrast to the locomotor hyperactivity that had been observed in adolescence. Notably, though, DZN exposure during development impaired reference memory performance in middle-aged males, an effect that had not been seen in the younger animals. Likewise, middle-aged females exposed to DZN showed deficient attentional accuracy, an effect not seen in young adults. Across adulthood, the continued potential for behavioral defects was associated with altered dopaminergic function, characterized by enhanced dopamine utilization that was regionally-selective (striatum but not frontal/parietal cortex). This study shows that the neurobehavioral impairments from maternal low dose exposure to diazinon not only persist, but may continue to evolve as animals enter middle age.

Tobacco smoke contains polycyclic aromatic hydrocarbons (PAHs) in addition to nicotine. We compared the developmental neurotoxicity of nicotine to that of the PAH archetype, benzo[a]pyrene (BaP), and also evaluated the effects of combined exposure to assess whether PAHs might exacerbate the adverse effects of nicotine. Pregnant rats were treated preconception through the first postnatal week, modeling nicotine concentrations in smokers and a low BaP dose devoid of systemic effects. We conducted evaluations of acetylcholine (ACh) and serotonin (5-hydroxytryptamine, 5HT) systems in brain regions from adolescence through full adulthood. Nicotine or BaP alone impaired indices of ACh presynaptic activity, accompanied by upregulation of nicotinic ACh receptors and 5HT receptors. Combined treatment elicited a greater deficit in ACh presynaptic activity than that seen with either agent alone, and upregulation of nAChRs and 5HT receptors was impaired or absent. The individual effects of nicotine and BaP accounted for only 60% of the combination effects, which thus displayed unique properties. Importantly, the combined nicotine + BaP exposure recapitulated the effects of tobacco smoke, distinct from nicotine. Our results show that the effects of nicotine on development of ACh and 5HT systems are worsened by BaP coexposure, and that combination of the two agents contributes to the greater impact of tobacco smoke on the developing brain. These results have important implications for the relative safety in pregnancy of nicotine-containing products compared with combusted tobacco, both for active maternal smoking and secondhand exposure, and for the effects of such agents in "dirty" environments with high PAH coexposure.