Who’s your daddy? Behavioral and epigenetic consequences of paternal drug exposure

Abstract

Substance use disorders (SUDs) reflect genetic and environmental factors. While identifying reliable genetic variants that predispose individuals to developing SUDs has been challenging, epigenetic factors may also contribute to the heritability of SUDs. Familial drug use associates with a wide range of problems in children, including an increased risk for developing a SUD. The implications of maternal drug use on offspring development are a well-studied area; however, paternal drug use prior to conception has received relatively little attention. Paternal exposure to several environmental stimuli (i.e. stress or diet manipulations) results in behavioral and epigenetic changes in offspring. The purpose of this review is to determine the state of the preclinical literature on the behavioral and epigenetic consequences of paternal drug exposure. Drug-sired offspring show several developmental and physiological abnormalities. These offspring also show deficits in cognitive and emotional domains. Examining sensitivity to drugs in offspring is a growing area of research. Drug-sired offspring are resistant to the rewarding and reinforcing properties of drugs. However, greater paternal motivation for the drug, combined with high drug intake, can result in addiction-like behaviors in offspring. Drug-sired offspring also show altered histone modifications and DNA methylation levels of imprinted genes and microRNAs; epigenetic-mediated changes were also noted in genes related to glutamatergic and neurotrophic factor signaling. In some instances, drug use resulted in aberrant epigenetic modifications in sire sperm, and these changes were maintained in the brains of offspring. Thus, paternal drug exposure has long-lasting consequences that include altered drug sensitivity in subsequent generations. We discuss factors (i.e. maternal behaviors) that may moderate these paternal drug-induced effects as well as ideas for future directions.

1. Substance use disorders

Substance use disorders (SUDs) occur when the recurrent use of alcohol/drugs leads to clinical and functional impairments that are detrimental to a person’s health, or the welfare of others [1]. According to the 2015 National Survey on Drug Use and Health, SUDs are highly prevalent in the United States with ~20 million adults meeting clinical criteria for a substance or alcohol use disorder. In addition, the economic costs associated with SUDs are greater than 740 billion/year [2], largely due to costs associated with crime, health care, and lost work productivity. Chronic drug and alcohol use increases the risk of many negative health consequences, including cardiovascular and neurological problems and cancer [2]. Given the substantial economic and individual costs, it is essential to identify risk factors that predispose individuals to developing a SUD.

Genetics play a prominent role in the development of SUDs independent of the environment. Family, twin, and adoption studies find that SUD heritability ranges from 40% for hallucinogens to 72% for cocaine [3]. Parental drug use has long-lasting ramifications on child outcomes. The consequences of maternal drug use during pregnancy are a well-studied area. In animal and human studies, maternal drug use associates with several developmental, cognitive, and emotional impairments in offspring [4–7]. In contrast, the consequences of paternal drug use, especially in periods prior to conception, have received relatively little attention. This is unfortunate given that drugs and alcohol can modify sperm in humans and animals [8–12]; studies in the latter show that these changes can be passed to future generations. Thus, drug or alcohol exposure can have long-lasting implications for subsequent generations.

Given this significant genetic influence, candidate gene and genome wide association studies have aimed to identify genetic variants that contribute to SUDs. These investigations have been challenging given the polygenic nature of SUDs. Considering these challenges, hypothesis-driven candidate gene studies have identified several genes involved in drug metabolism and the monoamine and serotonin systems [3]. Genome-wide association studies have identified novel variants that associate with smoking behaviors. However, genome wide association studies have been less successful in identifying loci associated with other substances, particularly alcohol [3]. Thus, there may be other factors that contribute to this missing heritability. For instance, rare variants of strong effect remain unidentified [13]. Another possibility is the growing attention to molecular epigenetic factors in human diseases, including addiction [14–16]. The primary goal of this review is to determine the current state of the preclinical literature on the inter- and trans-generational consequences of paternal drug exposure, as well as to highlight areas for further study that may improve prevention and treatment approaches for SUDs.

2. Epigenetics

Epigenetics refers to a range of mitotically and meiotically heritable molecular modifications that alter gene expression without changing the underlying DNA sequence [15]. Several related epigenetic mechanisms regulate gene expression: chromatin remodeling, DNA methylation, and non-coding RNAs. These mechanisms are essential to normal cell function allowing diverse cell types to emerge from a single genome. Additionally, some epigenetic alterations can have an acute onset (1 hr) and offset (24 hr), while others have a more stable profile reflecting events from prior decades [17]. The epigenetic mechanisms described below work collectively to regulate gene expression and a wide array of biological functions.

2.1. Chromatin remodeling

Chromatin consist of a complex of DNA and histone proteins. DNA is tightly wrapped around eight core histone proteins, two copies each of H2A, H2B, H3, and H4, within a nucleosome. Histone tails that project from the histone core are the sites for post-translational modifications. Covalent modifications, such as acetylation, methylation, and phosphorylation, at histone tails modify the chromatin structure leading to open (active) or closed (repressive transcriptional state), or a somewhere in between these two states [18, 19]. An open chromatin state, or euchromatin, enhances gene expression and occurs when acetyl groups attach to lysine residues located on histone tails [20, 21]. Acetylation loosens the electrostatic bond between histones and DNA, providing transcription factors access to promoter regions. Histone acetyltransferases increase acetylation and histone deacetylases maintain it [21]. Relative to acetylation, histone methylation is a more complex histone modification system that, depending on the site and number of methyl groups bonded, can facilitate or repress gene transcription. Histone methylation is controlled by both histone methyltransferases and histone demethylases. Some methyl marks are found in inactive chromatin (i.e., H3K27me3), while others are found in transcriptionally active chromatin (i.e., H3K4me3) [22]. In addition, phosphorylated histones are found in both active and inactive chromatin [23]. It is important to note that these and other histone modifications, such as SUMOylation, ubiquitination, citullination, and ADP-ribosylation form a “histone code” to govern gene expression [21].

2.2. DNA methylation

DNA methylation is the most well studied epigenetic modification and is involved in regulating gene expression by marking genes for silencing or activation. Specifically, DNA methylation occurs when methyl groups attach to the 5’ pyrimidine ring via DNA methyltransferases (DNMT) and methyl CpG-binding protein 2 (Mecp2) enzymes [24]. DNMT3a and DNMT3b are involved in de novo DNA methylation, while DNMT1 maintains DNA methylation after DNA replication. DNA methylation occurs often at cytosine:guanine dinucleotides (CpG) to form 5’-methylcytosine guanine dinucleotides (mCG). [24–28]; however, DNA methylation can also occur at other dinucleotide pairings [29, 30]. Promoter regions of genes contain a high density of CpG dinucleotides called “CpG islands” [31]. A substantial percentage (~70%) of CpG islands are methylated while a smaller percentage (~2%) are unmethylated [32]. Typically, DNA methylation near transcription start sites represses gene transcription while methylation within the gene body activates gene transcription [25, 27, 28, 33–38]. DNA demethylation (e.g. hydroxymethylation) is facilitated by ten eleven translocation (TET) proteins and typically activates transcription [39, 40]. Normal developmental processes, such as genomic imprinting and X chromosome inactivation, rely on DNA methylation.

DNA methylation can also interact with histone modifying enzymes to affect chromatin. MeCP2 binds DNA at methylated cytosines to inhibit transcription [41–45]. Additionally, MeCP2 may recruit histone deacetylases to deacetylate proximal histones, thereby attenuating gene expression [46–48]. Conversely, MeCP2 may be involved in recruiting transcription factors, such as CREB in active promoters [49].

2.3. Non-coding RNA’s

Non-coding RNAs can also alter gene expression. MicroRNA’s (miRNA’s) are short (~20 nucleotides) non-coding RNA’s that are involved in post-transcriptional silencing. miRNA’s are transcribed from genomic DNA and a single strand can suppress protein translation of dozens of genes [50]. The literature examining the role of miRNA’s in the intergenerational effects of drugs is limited. However, non-coding RNAs are hypothesized to be passed down to future generations via the male germ line [51].

2.4. Epigenetic reprogramming in male gametes

It is becoming clearer that male germ cells do more than passively carry genetic information. Sperm can alter the epigenetic profile and regulate the expression of hundreds of genes in embryos [52]. Mammalian germ cells undergo two rounds of epigenetic reprogramming throughout the lifecycle, 1) during preimplantation development and 2) during germ cell development [53–57]. Reprogramming during the former is important for naïve pluripotency in the zygote epigenome while the latter erases parental and somatic epigenetic marks and enables gametogenesis [58, 59]. During the early embryonic period, the primordial germ cells that give rise to spermatogenic cells in males demethylate from around 70% to 4% as they migrate and colonize the gonadal region [60, 61]. At this point, even imprinted loci are hypomethylated. Chromatin modifications maintain genomic integrity during this period of demethylation. For example, repressive chromatin modifications suppress retrotransposon activity [62]. Eventually, methylation is reestablished in a sex-dependent manner, ~embryonic day 13.5 for males and after birth for females [58].

Some genomic loci can escape global demethylation. Most of these loci are associated with retrotransposons [61, 63, 64] while others are found in pericentromeric satellite repeats [65] and in subtelomeric regions [63]. In addition, single-copy sequences and genes expressed in the brain and ubiquitously can also escape global demethylation [61, 63–65]. It is important to note that preserved methylation at these sites is not necessarily maladaptive and may be important for maintaining chromosome stability and chromosome alignment and segregation during mitosis [62].

2.5. Inter- and trans-generational consequences of paternal drug exposure

Paternal exposure to environmental stimuli can result in several intergenerational consequences. At the preclinical level, paternal diet manipulations alter glucose metabolism and brain development in offspring [66–68]. Sires exposed to stress paradigms have offspring with blunted stress responses and greater depression- and anxiety-like behaviors [69–71]. Conditioned fear to odors is also enhanced in offspring of sires exposed to olfactory fear conditioning [72]. Some of the behavioral and physiological effects seen in offspring are accompanied by changes in DNA methylation levels [68, 72, 73]. In each section below, we will review the behavioral and epigenetic consequences of paternal drug exposure (Table 1).

Table 1.

Intergenerational effects of paternal drug exposure

Drug	Species	Dose/Route	Exposure Period	Findings in Offspring	Reference
Developmental findings
Alcohol	LE rats and SW mice	Liquid diet	9 weeks	No change in birth weight or weight at weaning	[100]
	SW mice	Liquid diet	7 weeks	No change in litter size, birth weight, or weight at weaning	[77]
	SD and LE rats	Liquid diet	3–4 weeks	↓ litter size; No change in pup mortality	[87]
	C57/BL6 (Cast7) mice	Voluntary consumption of 10% alcohol in 0.066% Sweet N Low	4 hr daily access to over 70 days	Fetal growth restrictions	[84]
	C57/BL6J (B6) mice	Vapor	CIE for 8 hrs day/ 5 days a week over 6 weeks	↓ body weight at weaning in males	[95]
	C57/BL6J	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	No change in weight at weaning ↑ body weight at 8 weeks in males (Experiment 2)	[94]
	CD1 mice	Voluntary ad libitum access to 11% alcohol	60 days	No change in perinatal mortality, litter size, and number of dead-born pups ↓ body weight in adulthood	[91]
	C57/BL6J	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	No change in litter size; ↑ weight in males from 4–6 weeks of age	[11]
	SW mice	5 g/kg via IP injection	Single injection 12 h before mating	↓ body weight at birth; ↑ runts; ↓ survival	[81]
	C57BL/6J mice	3 g/kg via gavage	Daily intubations over 60 days	No change in body weight at birth	[97]
	SW mice	Liquid diet	56–61 days	No change in litter size, birth weight, or weight at PD 21 or PD55;	[92]
	C3H mice	Liquid diet	30 days	No changes in prenatal mortality, fetal weight, sex ratios, or soft tissue malformations	[93]
	SD rats	6 g/kg via gavage	Three times per week over 9 weeks	No change in litter size; ↓ fetal weight; ↑ runts	[80]
	SD rats	Liquid diet	60 days	↓ litter size; ↑ birth weight; ↑ male-to-female pup ratio	[74]
	Italian Wistar rats	10–20% alcohol in drinking water	13 weeks	↓ birth and adult weight in males	[96]
	SD rats	2–6 g/kg via gavage	Single intubation	No change in fetal litter weight, ↑ runts; ↑ malformations	[82]
	SD rats	0–5 g/kg via gavage	Daily intubations over 3–9 weeks	↑ fetal weight; ↑ male-to-female ratio	[86]
	SD rats	5 g/kg via IP injection	Single injection	↓ litter size; ↑ pup mortality	[75, 76]
	SD rats	0–3 g/kg via gavage	Twice daily intubations over 9 weeks	No change in birth weights; ↑ runts; ↓ male-to-female ratio	[83]
	SD rats	Liquid diet	52 days	No change in birth weight or weight at weaning	[88]
	SD rats	10–30% alcohol in drinking water	6 weeks	No change in fetal weights, litter size, or male-to-female ratio	[89]
	SD rats	2–6 g/kg via IP injection	60 days	No change in litter size	[90]
	LE rats	0–20% alcohol in drinking water	60 days	↓ litter size; ↓ litter and pup weight ↑ malformations	[78]
	Wistar rats	0–30% alcohol in drinking water	100–137 days	↓ litter size; ↓ body weight;	[79]
Cocaine	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	No change in litter size, sex ratio, or growth curves	[107]
	SD rats	15 mg/kg via SC injection	Daily or twice weekly over 100–150 days	↓ birth weight	[108]
	LE rats	30 mg/kg via SC injection	Daily over 72 days	No change on litter size, birth weight, or weight at weaning	[100]
Morphine	SD rats	Pellets implanted between shoulder blades	Adolescence (PD 25–27)	↓ litter size	[117]
	Wistar rats	5–60 mg/kg via IP injection	Twice daily over 35 days beginning in adolescence (PD 42)	↑ birth weight	[121]
	SD rats	Escalating dose regimen (2.5–25 mg/kg) via SC injection	Twice daily over 10 days	No change in birth weight or mortality	[119]
	SD rats	25 mg/kg via IP injection	Single treatment in adulthood	↓ litter size; ↑ mortality	[118]
	CD1 mice	Escalating dose regimen (up to 240 mg/kg) via SC injection	5–8.5-day treatment period	birth weight; ↓ body weights; ↑ developmental delays through 4 generations	[120]
Methadone	Fischer rats	5 mg/kg via SC injection	5-day treatment period	No change in litter weights or body weights before PD28. In adulthood, ↓ body weights	[122]
Nicotine	C57/BL6J	200 μg/ml nicotine in drinking water	5 weeks	No change in litter size or sex ratio	[125]
THC	SD rats	2 mg/kg via oral gavage	Daily gavages over a 12-day treatment period	No change in litter size, sex, ratio, or birth and weaning body weights in THC-sired offspring.	[129]
Learning and activity findings
Alcohol	LE rats and SW mice	Liquid diet	9 weeks	↓ locomotor activity in rats and mice	[100]
	C57/BL6J mice	Vapor	CIE for 8 hrs day/ 5 days a week over 6 weeks	No change in locomotor activity on open field or motor coordination on rotarod at baseline or after alcohol (1.0 g/kg)	[95]
	C57/BL6 mice	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	No change on locomotor activity in open field at baseline or after alcohol (1.0 g/kg); ↑ motor coordination on rotarod after alcohol in males	[11]
	SW mice	Liquid diet	7 weeks	↓ activity at PD 20	[87]
	Italian Wistar rats	10–20% alcohol in drinking water	13 weeks	↑ in locomotor activity; ↑ novelty seeking	[96]
	SW mice	Liquid diet	56–61 days	↓ activity at PD 20 and PD 24 ↓ passive avoidance learning ↓ learning on T maze	[92]
	SD rats	0–3 g/kg via gavage	Twice daily intubations for 7 months	↓ passive avoidance learning in males at PD 18, no change in adulthood; ↑ locomotor activity in adult males	[98]
	SD rats	0–3 g/kg via gavage	Twice daily intubations over 13 weeks	↑ amphetamine-locomotor activity	[83]
	SD rats	Liquid diet	39 days beginning at PD 30	↓ learning on radial arm maze and T maze	[99]
	SD and LE rats	Liquid diet	3–4 weeks	↓ activity in LE rats; ↑ and ↓ activity in SD rats; No change in passive avoidance learning	[77]
	SD rats	Liquid diet	52 days	↓ activity in males; ↓ active avoidance learning in females; No change in passive avoidance learning	[88]
	SD rats	Vapor	6 weeks	No change in motor coordination on rotarod; No change in operant and avoidance learning	[101]
Cocaine	C57BL/6J mice	20 mg/kg via IP injection	Daily cocaine hydrochloride (20 mg/kg) or saline injections over 75 days	↑ cocaine (10 mg/kg)- and amphetamine (2 mg/kg)-induced activity levels in both sexes. No change on Morris water maze, Y maze, or social behavior in both sexes.	[109]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↓ long-term object memory in males ↓ hippocampal long-term potentiation in males	[107]
	C57BL/6J mice	20 mg/kg via IP injection	Daily cocaine injections over 20 weeks	No change on novel object discrimination or Morris water maze in both sexes	[111]
	CD1 mice	Inhalation	Twenty 2-hr sessions	↓ sustained visuospatial attention and spatial working memory 5 arm maze in males and females	[110]
	LE rats	30 mg/kg via SC injection	Daily over 72 days	↑ hyperactivity	[100]
Morphine	SD rats	Escalating dose regimen (2.5–25 mg/kg) via SC injection	10-day exposure period in adults	No change in open field or Morris water maze	[119]
Morphine	CD1 mice	Escalating dose regimen (up to 240 mg/kg) via SC injection	5–8.5-day treatment period	↓ Morris water maze performance and learning when preceded by foot shock	[120]
Methadone	Fischer rats	5 mg/kg via SC injection	5-day treatment period	↓ open field activity in both sexes ↑ activity in locomotor chambers in both sexes; ↓ latencies in passive avoidance; ↑ avoidance in males during active avoidance ↓ avoidance in females during active avoidance; ↓ motor coordination on rotarod in males, not females	[122]
Nicotine	C57/BL6J mice	0.2 mg/100g via IP injection	Four daily injections over 5 weeks	↑ locomotor activity; No change on novel object recognition test	[126]
THC	SD rats	2 mg/kg via oral gavage	Daily gavages over a 12-day treatment period	No change in object recognition test of episodic memory in THC-sired offspring No change is spatial memory in THC-sired offspring ↑ habituation of locomotor activity in THC-sired offspring ↓ sustained attention in THC-sired offspring	[129]
WIN55,212-2	Wistar rats	1.2 mg/kg via IP injections	Daily injections over a 20-day period	No change in locomotor activity at baseline or after stress in WIN-sired offspring No change in object recognition test of episodic memory in WIN-sired offspring	[130]
Affective findings
Alcohol	C57/BL6J (B6) mice	Vapor	CIE for 8 hrs day/ 5 days a week over 6 weeks	↑ anxiolytic phenotype after alcohol (1.0 g/kg) on EPM in males	[95]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	↑ anxiolytic phenotype after alcohol (1.0 g/kg) on EPM in males	[11]
	SW mice	5 g/kg via IP injection	Single injection 12 h before mating	↑ aggression; ↓ fear	[81]
	LE rats and SW mice	Liquid diet	7–14 weeks	↑ immobility on FST in mice ↓ immobility on FST in rats	[103]
	LE rats	Liquid diet	9 weeks	↓ novelty-induced grooming;	[102]
	Italian Wistar rats	10–20% alcohol in drinking water	13 weeks	↓ anxiety-like behaviors on light-dark box	[96]
Cocaine	C57BL/6J mice	20 mg/kg via IP injection	Daily cocaine injections over 75 days	↑ anxiety-like behavior in males on EPM; No change on depression-like behavior on FST	[109]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine over 60 days	↑ anxiety-like behavior in males on novelty-induced hypophagia and marble burying tests; No change on cocaine-induced effects on anxiety-like behavior in males; No change on depression-like behavior on FST in males	[112]
	C57BL/6J mice	20 mg/kg via IP injections	Daily cocaine injections over 20 weeks	↑ depression-like behavior on TST in both sexes; No change on EPM or open field in both sexes	[111]
Morphine	Wistar rats	10 mg/kg via SC injections	Twice daily over 14 days in adults	No change on anxiety-like behavior on EPM or sucrose preference in both sexes	[123]
	SD rats	Escalating dose regimen (2.5–25 mg/kg) via SC injections	10-day exposure period in adults	↑ anxiety-like behavior on SPM	[119]
Nicotine	C57/BL6J	0.2 mg/100g via IP injections	Four daily injections over 5 weeks	↓ depression-like behavior on FST; No change on EPM	[126]
	C57/BL6J	200 μg/ml	Nicotine in drinking water over 5 weeks	No change on EPM	[125]
THC	SD rats	2 mg/kg via oral gavage	Daily gavages over a 12-day treatment period	No change in anxiety-like behavior on OF or fear response in THC-sired offspring.	[129]
WIN55,212-2	Wistar rats	1.2 mg/kg via IP injections	Daily injections over a 20-day period	No change in anxiety-like behavior on EPM in WIN-sired offspring ↑ stress-induced anxiety-like behavior in WIN-sired offspring	[130]
Reward-related findings
Alcohol	C57/BL6J (B6) mice	Vapor	CIE for 8 hrs day/ 5 days a week over 6 weeks	↓ preference at 3% and 6% in males	[95]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	No change in preference for 8% alcohol in males ↓ CVS-induced polydipsia in males	[94]
	CD1 mice	11% alcohol in drinking water	Ad libitum access over 60 days	↑ alcohol (0.5 mg/kg) CPP in males ↑ alcohol (1.5 mg/kg) CPA in males	[91]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	↓ alcohol (3%, 6%, & 9%) preference in males; ↓ alcohol (9% &12%) consumption in males; No change in alcohol clearance; No change in saccharin or quinine consumption in males or females	[11]
Cocaine	C57BL/6J mice	20 mg/kg via IP injections	Daily cocaine hydrochloride (20 mg/kg) or saline injections over 75 days	↓ cocaine (5 mg/kg) CPP in females	[109]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	Delayed acquisition of 0.5 and 1.0 mg/kg cocaine self-administration in males ↓ breakpoints for 1.0 mg/kg cocaine	[10]
	SD rats	15mg via IP injections 0.75 mg/kg via IV self-administration	Every 12-h over 7days (IP injections) Self-administration took place over 13 days	↓ cocaine self-administration in F1 and F2 offspring of high cocaine intake sires ↑ cocaine self-administration in F1 and F2 offspring of sires with high cocaine motivation	[114]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↓ cocaine sensitization in male offspring; No change in nicotine self-administration	[113]
Morphine	Wistar rats	5–60 mg/kg via IP injections	Twice daily over 35 days beginning in adolescence (PD 42)	↑ analgesic effects of morphine; ↑ morphine dependence	[121]
	SD rats	25 mg/kg via IP injection	Single treatment in adulthood	↑ analgesic effects of morphine in males, but not females	[118]
	Wistar rats	10 mg/kg via SC injections	Twice daily over 14 days in adults	No change in voluntary morphine consumption in both sexes	[123]
Nicotine	C57/BL6J	200 μg/ml	Nicotine in drinking water over 5 weeks	No change in nicotine self-administration; ↑ survival in males after toxic nicotine doses; ↑ survival in males after toxic cocaine doses	[125]
Molecular and physiological findings
Alcohol	C57/BL6J (B6) mice	Vapor	CIE for 8 hrs day/ 5 days a week over 6 weeks	↑ Bdnf mRNA in VTA in males No change in CORT response to 15 min restraint stress	[95]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	↓ corticosterone levels 30 min after 15 min restraint stress in males	[94]
	Kunming mice	0, 1.1, and 3.3 g/kg via gavage	Every 2 days over 4 weeks	No change in Peg3 mRNA in cerebral cortices	[106]
	CD1 mice	11% alcohol in drinking water	Ad libitum access over 60 days	↓ NGF protein levels at baseline in frontal cortex and kidneys and after alcohol (0.5 g/kg) in frontal cortex, hippocampus, olfactory lobes, hypothalamus in males; ↓ BDNF protein levels at baseline in kidneys and after alcohol exposure in frontal cortex and olfactory lobes in males; ↑ hippocampal TRKA protein levels at baseline and after alcohol in males ↓ frontal cortex p75 protein levels at baseline No change in DAT, D1 and D2 receptors, pro-NGF, pro-BDNF protein levels in hippocampus, hypothalamus, frontal cortex, and olfactory lobes in males.	[91]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	↑ Bdnf exon IXa mRNA expression in VTA of males; No change in Bdnf exon IV or Delta like non-canonical notch ligand 1 in VTA of males or females No change in Bdnf exon IXa or IV mRNA levels in mPFC of males or females	[11]
	SW mice	Liquid diet	7 weeks	↑ ocular infections	[105]
	SW mice	Liquid diet	56–61 days	↑ thymus weight; ↓ testosterone at PD 55	[92]
	SD and LE rats	Liquid diet	3–4 weeks	↓ testosterone	[87]
	SD rats	Liquid diet	60 days	No change in insulin-like growth factor-1 at PD 10; ↑ leptin levels at PD 10	[74]
	Italian Wistar rats	10–20% alcohol in drinking water	13 weeks	↓ glial enolase; ↓ superoxide dismutase; ↓ glutamine synthetase	[96]
	SD rats	3 g/kg via gavage	Twice daily intubations over 9 weeks	↑ adrenal weight at birth; ↓ spleen weight at PD 21	[83]
	SD rats	Vapor	6 weeks	Alterations in norepinephrine, 5-hydroxytryptamine, and Met-enkephalin levels	[101]
	SD rats	2–6 g/kg via IP injection	60 days	↑ cerebral weights	[90]
	Kunming mice	1.1 and 3.3 g/kg via gavage	Every 2 days over 4 weeks	↑ deafness	[106]
Cocaine	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↓ hippocampal D-serine glutamine and glutamate levels in males ↓ hippocampal D-amino oxidase 1 mRNA in males	[107]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine over 60 days	↑ hippocampal Corticotropin releasing hormone receptor 2 mRNA and CRF receptor 2 protein levels in males	[112]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↑ Bdnf exon IV mRNA and BDNF protein levels in the mPFC in males	[10]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↓ H3K24me2, H3K18ac, H3K20me2, H3K27me2; ↑ H3K14AC	[113]
Morphine	SD rats	Pellets implanted between shoulder blades	Adolescence (PD 25–27)	↓ testosterone in males; ↓ luteinizing hormone in males; ↑ adrenal weights in males; ↑ corticosterone in females; ↑ beta endorphin in females	[117]
	Wistar rats	32 mg/kg via gavage	Twice daily over 5 days	↓ long-term potentiation induction in both sexes	[124]
	SD rats	Escalating dose regimen (2.5–25 mg/kg) via SC injections	10-day exposure period in adults	↓ decreased dendritic length and branching; No change in spine density; ↓ Insulin-growth factor 2 mRNA and protein in hippocampus	[119]
	SD rats	25 mg/kg via IP injection	Single treatment in adulthood	No change in pain thresholds in males;	[118]
	Wistar rats	5–60 mg/kg via IP injections	Twice daily over 35 days beginning in adolescence (PD 42)	↓ synaptophysin in grand offspring;	[121]
Methadone	Fischer rats	5 mg/kg via SC injection	5-day treatment period	No change in thymus, adrenal, or gonadal weight at PD4; ↑ adrenal weights in adult females; ↓ thymus weight in adults of both sexes	[122]
Nicotine	C57/BL6J	200 μg/ml nicotine in drinking water	5 weeks	↑ expression of hepatic xenobiotic metabolizing and drug clearance genes	[125]
Nicotine	C57/BL6J	0.2 mg/100g via SC injection	Four daily injections over 5 weeks	↑ thalamic Wnt family member 4 mRNA levels	[126]
WIN55,212-2	Wistar rats	1.2 mg/kg via IP injections	Daily injections over a 20-day period	No change in stress-induced corticosterone levels ↑ DNMT1 mRNA levels in the prefrontal cortex of non-stressed WIN-sired offspring ↑ DNMT3a mRNA levels in the prefrontal cortex of stressed WIN-sired offspring	[130]
Epigenetic findings
Alcohol	C57/BL6 (Cast7) mice	Voluntary consumption of 10% alcohol in 0.066% Sweet N Low	4 hr daily access to alcohol over 70 days	No change in sperm-inherited DNA methylation profile	[84]
	Kunming mice	1.1 and 3.3 g/kg via gavage	Every 2 days over 4 weeks	↓ H19 (paternally imprinted gene) methylation in sperm of sires ↑ Peg3 methylation in sperm of sires ↑ Peg3 methylation at specific CpG sites in cerebral cortices of offspring No changes in DNA methylation of Necdin and Small nuclear ribonucleoprotein peptide N in sperm of sires or cerebral cortices of offspring	[106]
	C57/BL6	Vapor	CIE for 8 hrs day/ 5 days a week over 5 weeks	↓ DNA methylation of Bdnf exon IX in motile sperm and VTA of male and female offspring;	[11]
Cocaine	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↑ global hippocampal histone 3 acetylation at transcriptional start site and downstream of D-amino oxidase 1 in males; ↑ H3K4me1; ↑ H3K9ac; ↑ H3K18me1; ↑ H3K23me1; ↑ H3K27me1; ↑ H4K16ac in males	[107]
	SD rats	0.25 mg/infusion via IV injection	Sires self-administered cocaine during daily 2-h sessions over 60 days.	↑ histone 3 acetylation and BDNF exon IV association in MPFC of males ↑ histone 3 acetylation and BDNF exon IV association in MPFC of males	[10]
	SD rats	15mg via IP injections 0.75 mg/kg via IV self-administration	Every 12-h over 7days (IP injections) Self-administration took place over 13 days	Several DNA methylation changes found that differentiate sires with high cocaine intake from sires with high cocaine motivation in sperm and nucleus accumbens of offspring. Specifically, ↑ DNA methylation levels of Btg2 and Nr4a.	[114]
Nicotine	C57/BL6J	0.2 mg/100g via IP injection	Four daily injections over 5 weeks	↑ DNA methylation of mmu-miR-15b in sire sperm and thalamus of offspring	[126]
WIN55,212-2	Wistar rats	1.2 mg/kg via IP injections	Daily injections over a 20-day period	↑ stress-induced global DNA methylation in the prefrontal cortex of WIN-sired offspring	[130]

LE, Long Evans; SW, Swiss Webster; SD, Sprague-Dawley; C3H, C3H/HeJ; IP, intraperitoneal; SC, subcutaneous; EPM, elevated plus maze; FST, forced swim test; CpG, cytosine:guanine dinucleotides; VTA, ventral tegmental area; mPFC, medial prefrontal cortex; Bdnf, Brain-derived neurotrophic factor; Btg2, BTG gamily member 2; Igf2, Insulin-growth factor 2; Nr4a1, Nuclear Receptor Subfamily 4 Group A Member ; Peg3, Paternally expressed gene 3

3. Alcohol

3.1. Developmental findings

Paternal alcohol exposure induces several developmental aberrations. It reduces litter sizes [74–79] and increases the number of runts [80–84], malformations [78, 82], and pup mortality [75, 76, 81] in rats and mice. Litters from alcohol-exposed sires also exhibit increased [85, 86] or decreased male-to-female ratios [83]. Yet, several groups find that these litter parameters are unaltered in rats [80, 82, 87–90] and mice [11, 91–93]. Alcohol-sired offspring also display increased [11, 74, 94], decreased [78–81, 91, 95, 96], and no change [77, 82, 83, 87–89, 92, 93, 97] in body weights at birth, weaning, or adulthood. At times, changes in body weights occur in a sex-dependent manner. Overall, paternal alcohol exposure alters several developmental parameters across strains in rats and mice, but results are inconsistent across studies.

3.2. Learning and locomotor activity findings

Paternal alcohol exposure alters learning and memory and locomotor activity in offspring. Alcohol-sired offspring exhibit greater impairments in inhibitory [98] and active avoidance [88], and working memory [92, 99]. Across several studies using rats and mice, alcohol-sired offspring exhibit hyperactivity [77, 96, 98], hypoactivity [77, 87, 88, 92, 100], and unaltered [11, 95] activity levels when measured in pre-adolescence or adulthood. Alcohol-sired offspring also show greater amphetamine-induced hyperactivity [83]. Alcohol-sired offspring display normal motor coordination on the rotarod [101], but male offspring are less sensitive to alcohol-induced impairment in motor coordination [11].

3.3. Affective findings

Paternal alcohol exposure alters baseline and alcohol-induced affective behaviors, sometimes in a species-dependent manner. Swiss Webster alcohol-sired males exhibit greater aggression and less fear behaviors [81]. Alcohol-sired males show less anxiety-like behavior at baseline [96, 102] and after alcohol administration [11, 95]. C57/BL6J alcohol-sired offspring display greater depression-like behavior; however, Long Evans alcohol-sired offspring display less depression-like behavior [103].

3.4. Reward-related findings

Paternal alcohol alters sensitivity to the rewarding properties of alcohol in a sex-dependent manner. In two-bottle choice procedures, C57/BL6J alcohol-sired males consume less alcohol [11, 95]. CD1 alcohol-sired males also exhibit greater place preference at a lower alcohol dose, while place aversion is seen at a higher dose that induced a preference in control-sired offspring [91]. Thus, paternal alcohol exposure may confer a phenotype that is protective against alcohol-motivated behaviors in male offspring or lead to a leftward shift in the alcohol dose response function. Studies using rats and operant self-administration procedures are lacking.

3.5. Molecular and physiological findings

Paternal alcohol exposure results in several molecular and physiological abnormalities in offspring, such as alterations in organ weights, gonadal hormones, neurotransmitter and stress systems, and neurotrophic factors. Alcohol-sired offspring display greater brain [90], thymus [92], and adrenal weights [83]; while spleen weights are lower [83]. Alcohol-sired male offspring have lower testosterone levels [77, 92]. Preadolescent alcohol-sired offspring have greater leptin levels. The glutamate, serotonin, norepinephrine, and opioid systems are also altered in alcohol-sired offspring [96, 101]. Alcohol-sired male offspring have greater Brain-derived neurotrophic factor (Bdnf) mRNA expression in the ventral tegmental area [11, 95], while protein levels are lower in the kidneys, frontal cortex, and olfactory lobes at baseline and after alcohol [91]. Nerve growth factor protein levels are also lower at baseline and after alcohol exposure in alcohol-sired males [91]. In response to acute restraint stress, alcohol-sired males show lower [94] or unaltered corticosterone levels [95]. Paternal alcohol exposure also increases deafness [104] and susceptibility to ocular infections in offspring [105].

3.6. Epigenetic findings

Paternal alcohol exposure alters DNA methylation levels of paternally imprinted and neurotrophic factor genes. Alcohol exposure increases Paternally expressed gene 3 (Peg3) [106] and decreases Bdnf [11] methylation levels in the sperm of sires. These changes are maintained in the cerebral cortices (Peg3) and ventral tegmental area (VTA; Bdnf) in the brains of offspring [11, 106]. Bdnf methylation and mRNA changes in VTA associate with lower sensitivity to alcohol-induced anxiolysis and lower alcohol consumption in male offspring [11]. However, a recent study in mice found no changes in sperm-inherited DNA methylation in sires after voluntary alcohol consumption [84]. Thus, in some instances, alcohol-induced changes to the sperm epigenome can have long-term functional consequences in male offspring.

4. Cocaine

4.1.1. Developmental findings

There has been little research examining for developmental consequences of paternal cocaine exposure. Studies in Sprague-Dawley and Long Evans rats that have passively received cocaine or self-administered cocaine from 2–2.5 months show no changes in several developmental outcomes including litter size, sex ratio, and weights at birth and weaning [87, 107]. Lower birth weights are found after more than 3 months of paternal cocaine exposure in Sprague-Dawley rats [108]. Thus, longer durations of paternal cocaine exposure may impact developmental outcomes.

4.1.2. Learning and locomotor activity findings

Cocaine-sired offspring also show deficits in learning and memory tests and greater hyperactivity. In Sprague-Dawley rats, cocaine-sired male offspring display impaired long-term object memory and decreased hippocampal long-term potentiation [109]. In CD 1 mice, cocaine-sired offspring of both sexes show impaired sustained visuospatial attention and spatial working memory [110]. In addition, C57/BL6J and Long Evans cocaine-sired offspring display greater hyperactivity at baseline and after psychostimulant exposure [87, 109]. No changes have been seen in C57/BL6J cocaine-sired offspring on spatial and working memory, novel object discrimination, and social behavior [109, 111]. Overall, paternal cocaine exposure induces learning and memory deficits and increased baseline and psychostimulant-induced activity in offspring. Impairments in learning and memory appear to be strain-specific in mice.

4.1.3. Affective findings

Cocaine-sired offspring also show altered affective behaviors. Paternal cocaine exposure increases anxiety-like behavior in Sprague-Dawley and C57/BL6J male offspring on the elevated plus maze, novelty-induced hypophagia, and marble burying tests [109, 112]. It should be noted that findings in mice have been inconsistent as anxiety-like behavior is unchanged on open field and elevated plus maze in cocaine-sired offspring [111]. Additionally, C57/BL6J cocaine-sired offspring show greater depression-like behaviors on the tail suspension test [111]; however, no change in depression-like behavior on the forced swim test has been observed in C57/BL6J and Sprague-Dawley cocaine-sired offspring [109, 112]. Taken together, paternal cocaine treatments result in an anxiogenic phenotype in male offspring across rodent species, but findings on depression-like behavior are inconsistent.

4.1.4. Reward-related findings

Mice and rat studies show that paternal cocaine exposure alters sensitivity to cocaine in offspring. Male Sprague-Dawley cocaine-sired offspring show reduced cocaine sensitization [113]. C57/BL6J cocaine-sired females display lower cocaine place preference [109]. Sprague-Dawley cocaine-sired offspring exhibit delayed acquisition and motivation during cocaine self-administration [10, 114] but unaltered nicotine self-administration [113]. It is possible that the intergenerational effects of paternal cocaine exposure on reward measures are cocaine-specific. While a recent study shows that cocaine-sired grand offspring (F2 generation) exhibit normal cocaine self-administration [113], another finds that sires that both self-administer high amounts of cocaine and display greater levels of cocaine motivation have male offspring and grand offspring that exhibit addiction-like behaviors [114]. Thus, high cocaine intake alone, but not a high motivation + high intake combination, confers a protective effect against the rewarding and reinforcing properties of cocaine in offspring.

4.1.5. Molecular and physiological findings

Cocaine-sired offspring show altered neurotransmitter levels and expression of genes related to amino acid degradation and the stress axis. Sprague-Dawley cocaine-sired male offspring show lower levels of hippocampal D-serine glutamine, glutamate, D-amino oxidase 1 mRNA, and Corticotropin releasing hormone receptor 2 mRNA and protein levels [107, 112]. In the medial prefrontal cortex, cocaine-sired males have greater Bdnf exon IV mRNA and protein levels [10, 114]. Increased levels of BDNF protein in the mPFC correlate with cocaine intake in sires and not cocaine motivation [114]. In summary, paternal cocaine treatments alter gene expression and protein levels in the hippocampus and medial prefrontal cortex in male, but not female, offspring.

4.1.6. Epigenetic findings

Paternal cocaine exposure induces several histone modifications in the brains of offspring. Sprague-Dawley cocaine-sired males show greater global histone 3 acetylation downstream of D-amino oxidase 1, H3k4me1 (histone 3 lysine 4 methylation), H3K9ac (histone 3 lysine 9 acetylation), H3K18me1, H3K23me1, H3K27me1, and H4K16ac in the hippocampus [107]. These epigenetic changes associate with deficits in a hippocampal memory task and synaptic plasticity. Interestingly, these deficits were reversed by hippocampal administration of the NMDA receptor co-agonist D-serine; however, it is unclear whether D-serine reversed the epigenetic marks on the histone proteins. In the medial prefrontal cortex, Sprague-Dawley cocaine-sired males also display greater histone 3 acetylation and Bdnf exon IV associations, in addition to lower cocaine self-administration [10]. Importantly, these functional and epigenetic changes are not a result of altered maternal behavior [10]. The blunted cocaine sensitization in Sprague-Dawley cocaine-sired male offspring was accompanied by lower abundance of H3K4me2, H3K20me2, H3K27me2, and H3K18ac and increased abundance of H3K14ac in the nucleus accumbens [113]. Interestingly, differential methylation in sperm exists between sires that show high cocaine motivation + high cocaine intake versus high cocaine intake alone [114]. Hundreds (~475) of differentially methylated CpG sites were maintained in F1 offspring, primarily at transcription start sites (± 2,000 base pairs) and intergenic regions. Specifically, this resulted in greater methylation of BTG family member 2 (Btg2) and Nuclear receptor subfamily 4 group A member 1 (Nr4a1) promoters in sperm of high cocaine motivated sires and their offspring. Both genes have been implicated in neurogenesis and other brain functions [115, 116]. A similar epigenetic profile was found in the nucleus accumbens of cocaine sires and offspring. Overall, paternal cocaine exposure induces histone and DNA methylation changes that alter expression of glutamate-related, stress, neurogenesis, and neurotrophic factor genes. These epigenetic changes are accompanied by hippocampal memory deficits at baseline and lower sensitivity to the reinforcing effects of cocaine, primarily in male offspring. However, differential behavioral and DNA methylation patterns emerge when cocaine motivation in sires is considered.

5. Opioids

5.1. Developmental findings

Adolescent and adult paternal morphine exposure impairs offspring development in several domains. Paternal morphine treatment in Sprague-Dawley rats decreased litter size and increased offspring mortality [117, 118]. The findings on birth weight are mixed, with some studies showing greater (Wistar rats), lower (CD1 mice), and no change (Sprague-Dawley rats) in birth or adult weight [119–121]. Some developmental abnormalities are passed on over 4 generations in CD1 mice [120]. Paternal methadone treatment in Fischer rats does not alter litter weights or body weights prior to weaning; however, methadone-sired offspring exhibit lower body weights in adulthood [122]. Thus, there is emerging evidence that paternal opioid exposure can have a long-lasting impact on developmental trajectories over multiple generations.

5.2. Learning and locomotor activity findings

Few studies have examined opioid-sired offspring for changes in learning and activity domains. Locomotor activity and spatial memory are unaltered in Sprague-Dawley morphine-sired offspring [119]; however, CD1 morphine-sired offspring show impairments in learning in active avoidance and spatial memory [120]. Interestingly, paternal methadone treatment in F344 rats results in changes in learning and activity [122]. Both sexes show decreased open field activity. Both sexes also showed enhanced learning in inhibitory avoidance procedures. Male offspring exhibit enhanced learning during active avoidance, while females display impaired learning. Male, but not female, offspring also have impaired motor coordination [122]. Although few studies have examined paternal opioid treatment-induced changes in learning and activity in offspring, methadone-sired offspring show greater variations in these domains, which at times occur in a sex-dependent manner.

5.3. Affective findings

The literature on paternal morphine effects on anxiety- and depression-like behavior is mixed. Sprague-Dawley morphine-sired offspring display an anxiogenic phenotype [119], while anxiety-like behaviors in Wistar morphine-sired offspring are unchanged [123]. Pooriamehr et al. [123] also found that depression-like behavior on sucrose preference tests are unchanged in Sprague-Dawley morphine-sired offspring. Further work using a wider range of behavioral tests that model anxiety- and depression-like behaviors is needed.

5.4. Reward-related findings

Paternal morphine exposure alters sensitivity to morphine in adult offspring. Wistar morphine-sired offspring show increased sensitivity to the analgesic effects of morphine [121]. Sprague-Dawley morphine-sired male offspring, but not females, also exhibit increased sensitivity to morphine-induced analgesia [118]. Interestingly, paternal morphine exposure in Wistar rats results in increased morphine dependence [121], but voluntary morphine consumption is unchanged [123]. The timing of paternal treatment may influence these divergent findings, with sire treatment beginning in adolescence inducing morphine dependence in offspring. However, altered sensitivity to morphine-induced analgesia is seen in adolescent and adult paternally treated offspring. Overall, morphine-sired offspring show greater sensitivity to the analgesic effects of morphine with timing of paternal exposure determining responses to morphine reward.

5.5. Molecular and physiological findings

Paternal opioid treatment results in several physiological and molecular abnormalities. Sprague-Dawley morphine-sired male offspring have greater adrenal weights and lower luteinizing hormone and testosterone levels [117]. Paternal methadone treatment results in greater adrenal weights in adult females, and lower thymus weights in both sexes [122]. The findings on basal pain thresholds are strain-specific in males; Wistar rat offspring show greater pain thresholds [121] while these measures are unaltered in Sprague-Dawley offspring [118]. Greater hypothalamic beta endorphin and corticosterone levels are found in female offspring [117]. In both sexes, there is decreased hippocampal dendritic length and branching, as well as decreased Insulin-growth factor 2 mRNA and protein levels [119]. Induction of long-term potentiation is also impaired in both sexes [124]. Interestingly, grand offspring (F2) display lower synaptophysin levels, but levels of this enzyme are unchanged in their parents (F1) [121]. Taken together, there is robust evidence that paternal opioid exposure results in changes in organ weights, synaptic activity, and several hormone levels related to growth-regulation and neurotransmitter function.

5.6. Epigenetic findings

No studies found.

6. Nicotine

6.1. Developmental findings

Few studies have explored the effects of paternal nicotine use on the health of subsequent generations. In C57BL/6J mice, litter size and sex ratios are unchanged in litters sired by adolescent nicotine-exposed males. Importantly, nicotine-sires were prevented from mating with a nicotine-naïve female for one week after the 5-week exposure period, well beyond the half-life of nicotine and its metabolite cotinine [125].

6.2. Learning and locomotor activity findings

Nicotine exposure results in hypoactivity in sires but differential changes on locomotor and learning behaviors in offspring. Specifically, C57BL/6J nicotine-sired offspring display greater locomotor activity, while recognition memory is unaltered [126].

6.3. Affective findings

Nicotine exposure induces depression-like behavior in sires but promotes resilience in offspring. For example, C57BL/6J nicotine-sired offspring show lower depression-like behaviors on the forced swim test [126], but anxiety-like behavior on the elevated plus maze is unchanged [125, 126].

6.4. Reward-related findings

Nicotine self-administration behaviors are unaltered in nicotine-sired offspring; however, male offspring show increased survival after toxic doses of nicotine and cocaine [125]. Thus, paternal nicotine exposure increases resilience to toxic nicotine and cocaine doses in male offspring. These findings may indicate that, in contrast to paternal cocaine studies which find cocaine-specific intergenerational effects, paternal nicotine exposure does not induce nicotine-specific reward responses in offspring.

6.5. Molecular and physiological findings

Paternal nicotine exposure alters nicotine and cocaine metabolism and signaling pathway involved in neural development. Male C57BL/6J nicotine-sired offspring had greater expression of genes involved with hepatic metabolism and nicotine clearance [125], as well as thalamic Wnt family member 4 mRNA levels [126]. The Wnt4 signaling pathway is an important regulator of neurogenesis and is associated with the pathophysiology of several neuropsychiatric disorders, including bipolar disorder and major depressive disorder [127, 128].

6.6. Epigenetic findings

Paternal nicotine exposure alters miRNA targeting the Wnt4 signaling pathway in offspring. Nicotine exposed sires have greater DNA methylation of mm-miR-15b in their sperm; hypermethylation of mmu-miR-15b was also maintained in the thalamus of offspring [126]. Changes in mmu-miR-15b methylation levels associate with greater locomotor activity, lower depression-like behavior, and thalamic Wnt family member 4 mRNA levels in offspring. Interestingly, viral-mediated overexpression of mmu-miR-15b induced hypoactivity and depression-like behavior in nicotine-sired offspring. Although a causal link has been demonstrated between paternal-nicotine exposure and mmu-miR-15b and the Wnt family member 4 signaling pathway, it would be useful to investigate whether this link mediates responses to nicotine reward in offspring.

7. Cannabinoids

7.1. Developmental findings

Few studies have examined the role of paternal cannabinoid exposure on developmental outcomes. Offspring of adult THC-exposed sires did not differ from control-sired offspring on litter size, sex ratio, or body weights when measured at birth and weaning. [129].

7.2. Learning and locomotor activity findings

Adolescent paternal exposure to the synthetic cannabinoid receptor agonist WIN55,212–2 (WIN) did not alter locomotor activity in adult SD offspring at baseline or after unpredictable stress [130]. Adult THC-sired offspring showed more rapid habituation of locomotor activity relative to control-sired offspring; this effect was not seen in adolescent offspring [129]. Additionally, episodic memory was unchanged in WIN-sired offspring relative to control offspring as measured by the object recognition test [130]. No effect of adolescent paternal THC exposure was seen on the novel object recognition test of non-spatial memory [129]. Although, THC-sired offspring do not show deficits on the 16-arm radial maze test of spatial memory, these offspring have impairments in sustained attention relative to control-sired offspring [129].

7.3. Affective findings

Paternal WIN exposure in adolescence alters stress-induced anxiety-like behaviors in offspring. Adolescent THC exposure in males did not alter anxiety-like behavior in offspring on the elevated plus maze [129]. While WIN- and control-sired offspring do not differ on open field anxiety measures, WIN-sired offspring show greater unpredictable stress-induced anxiety-like behaviors relative to control-sired offspring [130]. THC-sired offspring do not differ from control-sired offspring in fear response as measured by the novelty suppressed feeding task [129].

7.4. Reward-related findings

No studies found.

7.5. Molecular and physiological findings

Offspring of WIN-exposed sires show differential changes on DNMT’s and stress hormones prior to and after stress exposure [130]. Corticosterone levels do not differ between WIN- and control-sired offspring at baseline or after chronic unpredictable stress. Prefrontal DNMT1 mRNA levels are greater in WIN-sired offspring at baseline; however, no differences are seen between groups after stress exposure. Conversely, prefrontal DNMT3a mRNA levels do not differ between the WIN- and control-sired offspring at baseline, but after stress exposure, WIN-sired offspring have higher DNMT3a mRNA levels [130].

7.6. Epigenetic findings

Stress exposure in WIN-sired offspring enhances global DNA methylation levels in the prefrontal cortex [130]. Global DNA methylation levels do not differ between WIN- and control-sired offspring at baseline. However, when exposed to stress, WIN-sired offspring have greater 5-mc percentages compared to stressed control-sired offspring. Global DNA methylation levels correlate differentially with DNMT1 and DNMT3a mRNA levels in the prefrontal cortex. Specifically, global DNA methylation levels positively correlate with DNMT1 mRNA levels, but no relationship is evident between global DNA methylation and DNMT3a mRNA levels [130].

8. Conclusions and future directions

Paternal drug exposures induce a wide range of developmental, emotional, physiological, and epigenetic consequences. Over the last few decades, paternal alcohol exposure has received more attention relative to other substances. However, given the increased rates of dependency on prescription opioid drugs, the popularity of electronic cigarettes, and decriminalization of marijuana in several U.S states, further investigation of other drug classes is warranted. Furthermore, studies vary widely in methodology across and within drug classes. Age, dose, duration and route of administration of paternal drug exposure are all important factors which may contribute to a lack of consilience between investigations. In many instances, paternal drug effects occur in a sex-dependent manner in offspring, findings that likely indicate complex interactions between sire-induced epigenetic modifications and the organizational and activational effects of gonadal hormones. In a similar vein, behavioral effects might also occur selectively in male offspring due to undetected paternal drug-induced epigenetic modifications on the Y chromosome that can modify other genes via epistasis [131].

There is also a bourgeoning literature on paternal drug-induced effects on drug reward in offspring. There is preclinical evidence of a protective effect of paternal drug exposure on drug consumption in offspring that conflicts with studies in humans that demonstrate familial transmission of SUDs [132–136]. Conversely, many of the findings discussed above are in line with a recent longitudinal investigation that focused on paternal drug use on risk of alcohol use disorder in offspring. Maternal, but not, paternal SUD increased the risk of alcohol use disorder in offspring. Although, paternal SUD is not shown to be protective against alcohol use disorder, it does not enhance risk [137]. Looking forward, it would be beneficial if preclinical and human work parallel each other when investigating the role of paternal drug consumption. For instance, the amount of drug consumed is not a criterion for a SUD; thus, there is heterogeneity in drug intake within and across drug classes. Whenever possible, it is important to measure clinical features of SUDs in sires, such as drug motivation. Rat studies showed that males with high motivation for cocaine had offspring that self-administer greater amounts of cocaine [114]. Thus, paternal motivation for a drug, coupled with high drug intake, may predispose offspring to develop addiction-like behaviors.

The role of maternal behaviors has also received little attention. This is unfortunate given that many paternal treatments reviewed above continued into the mating period. Furthermore, paternal environment can alter maternal behavior. For example, paternal housing conditions can alter a dam’s licking and grooming behaviors toward their offspring [138], supporting findings that females adjust maternal care depending on paternal quality across several species [139–141]. Additionally, some paternal effects disappear after in vitro fertilization [70] and embryo transfer likely because these effects are buffered by maternal behaviors [142]. These findings highlight complex maternal-paternal interactions that may contribute to offspring phenotype. Notably, some paternal drug studies show that maternal behavior is unaltered [10, 109, 119] or use a cross-fostering protocol [126].

Paternal-drug induced epigenetic modifications in offspring are an understudied area. Future studies can focus on how epigenetic modifications may be facilitating the biological and behavioral changes observed because of paternal drug exposure. Given that some short non-coding RNA’s may mediate DNA methylation processes (i.e. piRNA’s) [143, 144], it would be beneficial to elucidate their role in the transgenerational effects of paternal drug use. Additionally, no studies examined interactions between genetic and epigenetic marks. DNA methylation commonly takes place in an allele-specific manner across the genome [145]. Stress-induced epigenetic modifications can also occur in an allele-specific manner [146–148]. Thus, it is likely that the intergenerational consequences of paternal drug use rely on complex interactions between genetic and epigenetic marks. For example, an allele that inactivates alcohol dehydrogenase 2 (ALDH2) reduces risk for developing alcohol use disorder [149]. Such variants may interact with epigenetic processes to moderate predisposition to certain alcohol drinking phenotypes. In summary, paternal drug exposure, even during periods prior to conception, can have a long-lasting impact on future generations. Further work in this area will identify novel mechanisms that underlie the paternal contribution to addiction; such findings may lead to the development of more effective prevention and treatment strategies for substance use disorders.

Highlights.

• Epigenetic studies involving paternal drug exposures are in their infancy

• Paternal drug exposure has long-lasting consequences in offspring

• Epigenetic changes in the germ line associate with behavioral abnormalities

• Information about epigenetic processes may yield novel therapeutic approaches