Cannabis use in the United States is increasing, with highest consumption among men at their peak reproductive years. We previously demonstrated widespread changes in sperm DNA methylation with cannabis exposure in humans and rats, including genes important in neurodevelopment. Here, we use an in vitro human spermatogenesis model to recapitulate chronic cannabis use and assess DNA methylation at imprinted and autism spectrum disorder (ASD) candidate genes in spermatogonial stem cell (SSC)- and spermatid-like cells. Methylation at maternally imprinted genes SGCE and GRB10 was significantly altered in SSC- and spermatid-like cells, respectively, while PEG3 was significantly differentially methylated in spermatid-like cells. Two of ten randomly selected ASD candidate genes, HCN1 and NR4A2, had significantly altered methylation with cannabis exposure in SSC-like cells. These results support our findings in human cohorts and provide a new tool with which to gain mechanistic insights into the association between paternal cannabis use and risk of ASD in offspring.

Sex-linked differences in mitochondrial ATP production, enzyme activities, and reactive oxygen species generation have been reported in multiple tissue and cell types. While the effects of reproductive hormones underlie many of these differences, regulation of sexually dimorphic mitochondrial function has not been fully characterized. We hypothesized that sex-specific DNA methylation contributes to sex-specific expression of nuclear genes that influence mitochondrial function. Herein, we analysed DNA methylation data specifically focused on nuclear-encoded mitochondrial genes in 191 males and 190 females. We found 596 differentially methylated sites (DMSs) (FDR p < 0.05), corresponding to 324 genes, with at least a 1% difference in methylation between sexes. To investigate the potential functional significance, we utilized gene expression microarray data. Of the 324 genes containing DMSs, 17 showed differences in gene expression by sex. Particularly striking was that ATP5G2, encoding subunit C of ATP synthase, contains seven DMSs and exhibits a sex difference in expression (p = 0.04). Finally, we also found that alterations in DNA methylation associated with in utero tobacco smoke exposure were sex-specific in these nuclear-encoded mitochondrial genes. Interestingly, the level of sex differences in DNA methylation at nuclear-encoded mitochondrial genes and the level of methylation changes associated with smoke exposure were less prominent than that of other genes. This suggests more conservative regulation of DNA methylation at these nuclear-encoded mitochondrial genes as compared to others. Overall, our findings suggest that sex-specific DNA methylation may help establish sex differences in expression and function and that sex-specific alterations in DNA methylation in response to exposures could contribute to sex-variable toxicological responses.

Mixtures of Pb ²⁺ and two mechanistically distinct mitochondrial toxicants, ruthenium red and carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP), resulted in greater than additive Pb ²⁺ -induced cytotoxicity in HepG2 (human liver) cells.

Mitochondria are vital for cellular energy production, as these organelles generate most of the cellular energy required for various metabolic processes. Mitochondria contain their own circular DNA, which is present in multiple copies and is exclusively maternally inherited. Cellular energy in the form of adenosine 5′-triphosphate is produced via oxidative phosphorylation and involves the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes. Mitochondrial DNA itself is replicated by a dedicated set of nuclear-encoded proteins composed of the DNA polymerase gamma, the Twinkle helicase, the mitochondrial single-stranded DNA binding protein, as well as several accessory factors. Mutations in these genes, as well as in the genes involved in nucleotide metabolism, are associated with a spectrum of mitochondrial disorders that can affect individuals from infancy to old age. Additionally, mitochondrial disease can arise as a result of point mutations, deletions, or depletion in the mitochondrial DNA or in genes involved in mitochondrial transcription, replication, maintenance, and repair. Although a cure for mitochondrial diseases is currently elusive, several treatment options have been explored. In this review, we explore the molecular insights of the core mitochondrial replisome proteins that have aided our understanding of mitochondrial diseases and influenced current therapies.