It is estimated that 42 million American adults and about 3 million middle and high school students
smoke cigarettes. Clinical and pre-clinical studies show that fetal nicotine exposure occurring as a result of
mothers' tobacco use during pregnancy is associated with premature birth, sudden infant death, and an
increase in the risk for cognitive disabilities. Whereas maternal nicotine use and the consequent fetal nicotine
exposure are undeniable concerns, in reality, smoking is more prevalent among men (21.6%) than women
(16.5%). However, whether a father's use of tobacco products has consequences for his offspring is not clear.
To address this unexplored issue, we developed a mouse model of paternal nicotine exposure, in which adult
male mice were exposed to nicotine in drinking water. While the nicotine exposure was ongoing, the male mice
were bred with drug naïve female mice. Quite unexpectedly, we found that the offspring of the nicotine-
exposed fathers had significant hyperactivity and attention deficits. Equally unexpectedly, although the
hyperactivity was present in both the male and female offspring, the attention deficits were present only in the
male offspring. Moreover, the expression of genes for dopamine receptors (dopamine is the neurotransmitter
that regulates motor and cognitive function) in the frontal cortex and striatum (two brain regions together
involved in motor and cognitive function) showed brain region-specific and sex-specific changes in the
offspring descending from the nicotine-exposed male mice. These surprising and novel observations led us to
investigate epigenetic modifications in the father's spermatozoal DNA as a potential mechanism underlying the
transmission of the behavioral and molecular phenotypes from the nicotine-exposed father to his offspring. We
found significant changes in global DNA methylation status and the methylation status of the dopamine D2
receptor in the fathers' spermatozoa. Finally, F2 generation (grandchildren of the nicotine-exposed F0 males)
derived by crossing F1 males from nicotine-exposed fathers with drug naïve females also showed sex-specific
attention deficits and dopamine receptor gene expression changes in the brain, suggesting that the effects of
nicotine exposure were evident in multiple generations of descendants. Based on these observations, we
propose the hypothesis that offspring sired by nicotine-exposed fathers show significant and sex-specific
behavioral and molecular phenotypes, and that nicotine-induced epigenetic modification of the DNA of the
fathers' spermatozoa is a mechanism for heritability of the phenotypes. We will test this hypothesis by
performing behavioral analyses, whole-genome bisulfite sequencing to assay methylation status of the DNA,
and RNA sequencing to assay changes in the transcriptome. The proposed studies offer excellent training
opportunities for undergraduate and graduate students and strengthen the research base at our institution.
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