In Utero and Lactational Exposure of F1 Adult Pups to Ethanol and Nicotine: Alteration of The Reproductive System and Epigenetic Modifying Enzymes of Sperm Cells?

Co-use of alcohol and nicotine is a common habit which could affect reproductive system. The present study was designed to investigate the effect of in-utero and lactational exposure of offspring to alcohol, nicotine, and combinations on the reproductive system and epigenetic alterations of male gamete. Pregnant mice and lactating NMRI pups randomly received nicotine, ethanol, and combinations during gestational days 1 until weaning. Sperm and testes were collected on postnatal day 90 for further experiments. Exposure to these substances, particularly, nicotine, highly affected testicular Johnsen’s score, sperm parameters including; number, motility, viability, DNA integrity and also serum MDA level. Interestingly, concurrent exposure to nicotine and alcohol somehow reversed the effects. Quantitative real-time PCR data showed an increase in the mRNA level of histone deacetylation 1 and 2 and a decrease in the level of DNA methyltransferase (DNMT) 3A, while no marked differences in the expression level of DNMT1 and 3B were observed between the exposed and non-exposed pups. Alleviating effects were observed in the epigenetic modifying enzymes transcripts of co-exposed pups compared with the exposure to nicotine or alcohol. Taken together, our ndings determined that exposure of offspring in-utero and during lactational period to these substances could result in lasting epigenetic changes in sperm cells. However, unexpectedly co-exposure of pups to nicotine and alcohol lessened these negative effects.


Introduction
Despite the well-known hazards and repeated warnings about smoking and alcohol drinking during pregnancy, some mothers still continue to use them. Considering rapid transmission of these substances through placental membrane 1,2 and secretion in the milk 3,4 , a signi cant population yet face with these substances during in utero life and also in breastfeeding period.
Each puff of cigarette is a complex mixture of more than 1000 chemicals. Of them, nicotine, a very toxic alkaloid, is one of the well-established dangerous substances. Sometimes, nicotine replacement therapy (NRT) is recommended as an effective method for smoking cessation during pregnancy 5 . Although developing fetus to some extent is protected from exposure to various chemicals and carcinogens in the cigarette, however, nicotine exposure, by itself is a crucial issue which affects various body organs including reproductive system. Alcohol intake, especially at higher doses is also associated with symptoms such as growth retardation, facial dysfunction and brain damage 6 . A growing body of evidence, both in rodents and humans, suggests that maternal exposure to nicotine and alcohol during key times of the reproductive system development and germ cell formation could result in abnormalities in male reproductive system, embryonic gonadal germ cells 7 , hormonal status, sperm quality [8][9][10][11] , and subsequently may negatively affect fertility potential of adult offspring. Nevertheless, there are some contradictory reports 12,13 . Epidemiologic studies have shown that the simultaneous consumption of nicotine and alcohol is more common 14,15 . Approximately eighty percent of people who consume alcohol are smoker 16 . It is well-known that consumption of any of these substances increase desire for the other toxicologic studies focusing on the adverse effects of nicotine and alcohol and their combination, have been conducted so far, yet very few records have addressed the cumulative effects of in utero exposure to alcohol and nicotine on developing reproductive system of male offspring. A previous study has shown harmful effects of maternal exposure to nicotine, alone or concurrently with ethanol, during prenatal day 9-12 on embryo development, although this effect was spared in the ethanol-exposed embryos 19 .
Regardless of the well-established developmental toxicity of alcohol and nicotine on male offspring, their intrauterine programming mechanism is not well-studied. Exposure to environmental factors such as alcohol and nicotine during early life could induce modi cations of speci c genes expression without altering their sequence via aberrant epigenetic alterations in various tissues of offspring [20][21][22][23][24][25][26][27] . These alterations have permanent adverse developmental and functional effects. DNA methylation and histone modi cations are one of the major mechanisms of epigenetic changes 28 . These modi cations occur by activity of modi cating enzymes including DNA methyltransferases (DNMTs) and histone acetylates (HAT) and deacetylases (HDACs) 29 . Several studies have evaluated the effects of maternal exposure to nicotine and alcohol, alone on a range of epigenetic changes in different tissues such as somatic and germ cells of male offspring 24,26,29 . Hypomethylation of placenta 30 , whole fetal genome 31 , and also imprinting genes such as H19 32 as well as decrease of DNMTs activity 31 were reported.
Despite the co-use of alcohol and nicotine in the world's population is more common, the effect of concurrent exposure to these substances during embryofoetal and lactation periods on male offspring's reproductive system is not yet evaluated. Hence, in order to bring experimental evidences nearer to the actuality of human life, the main aim of the present study was to assess the sperm and testicular characteristics of offspring born from mothers whom received alcohol, nicotine and their combination during pregnancy and lactation periods. Moreover, we further evaluated whether such alterations could affect the expression level of the epigenetic modi cation enzymes in sperm of offspring.

Animals
A total of 25 adult NMRI mice (15 females and 10 male) aged 8-10 weeks and weighting 25-30 g were used for breeding. These animals were obtained from animal house of Afzalipour faculty of Medicine, Kerman. They were housed in polypropylene cages and were kept under standard condition of animal house with a temperature of 23+2 C and constant 12-hours light/dark cycle with free access to drinking water and standard pellet food.

Experiment design
Three female and one male mice (proportion of three females per male) were housed in one cage overnight and the vaginal plug was checked in the following morning. Presence of a vaginal plug was considered as gestational day (GD) 1. The pregnant mice were randomly allocated into 5 groups (n= 8) and treated daily as follows: Vehicle group (Veh): the pregnant animals daily received 0.3 ml normal saline by intraperitoneal (i.p.) injection.
Nicotine + Ethanol group (Nic+Ethn): the pregnant mice daily received the same doses of nicotine and ethanol, simultaneously 68 .
Treatments started from GD1, continued throughout the pregnancy and lactation periods until offspring weaning (postnatal day; PND 21). The male offspring were maintained in separate cages under the same controlled conditions until PND 90. In order to prevent from any gene homogeneity, only 2 male offspring of each dam were randomly assigned for each experimental group (n=8).

Blood collection
At PND 90, 8 pups of each group were anesthetized using ketamine ((5-10 mg/kg) /xylazine (5 mg/kg), and their blood was collected from left ventricle. Blood samples were centrifuged at 2500-3000 rpm for 10 minutes and the serum removed and held at -20 ˚C for further evaluations.

Testis and sperm collection
The anesthetized animals were authonized by cervical dislocation, their abdomen wall was cut and the left cauda epididymis of each mice were carefully removed, and were transferred into a petri dish containing 1 mL pre-warmed HTF media supplemented by 15 mg/mL bovine serum albumin. It was cut by a pair of scissors, to release sperm cells. The petri dishes were incubated at 37 C for 15 minutes.
Meantime, the left testes were removed and weighted by a digital scale. Testes dimensions including length, width and thickness, were measured using a standard digital caliper and then xed by dipping in Bouin's solution for 48 hours.

Sperm motility
After incubation of sperm cells for 15 minutes, the suspension was collected, quietly pipetted, 10 μL of sperm suspension was deposed on the pre-warmed slide, covered by coverslip and then the motility of at least 200 sperm cells was evaluated under a light microscope at × 200 magni cation in 10 random elds.
The motility rate was expressed as the number of motile sperm cells to total counted sperm cells ( × 100 83 .

Sperm number
Five μL of sperm suspension was mixed with 5 μL of sperm xative solution (formalin/sodium bicarbonate). The mixture was pipetted and the solution was placed on an improved Neubauer hemocytometer and the sperm heads were counted under a light microscope (Olympus BX 51, Tokyo, Japan) at × 200 magni cation. The sperm number was expressed as million per one mL of suspension 84 .

Sperm viability
Ten μL of sperm suspension was mixed with an equal volume of 1% eosin Y/ 5% nigrosin staining solution in a microtube. After 2 minutes, a smear was prepared, air dried and at least 200 sperm cells were evaluated at × 1000 magni cation under immersion oil. (Figure 2-D). The sperm viability rate was calculated as the number of unstained sperm cells (alive) to total counted sperm cells (white + red)× 100 85 .
The sperm parameters were evaluated by an expert operator blinded to experimental groups.
Sperm DNA fragmentation assessment Sperm DNA fragmentation (SDF) was assessed by sperm chromatin dispersion (SCD) test based on our previous study with some changes 86,87 . Brie y, 30 μL sperm suspension was mixed with 70 μL 1% low gelling agarose, placed on a pre-coated slide with standard agarose (0.65%), covered by coverslip and transferred to refrigerator (4 C) to solidify. After 5 minutes, the coverslip was carefully removed and the slide was suspended in 0.08 N HCl solution for 17 minutes at room temperature in darkness. Next, the slide was incubated in two consecutive lysis buffer solutions; lysis buffer solution 1 containing 0.4 M Tris, 0.8 M 2-mercaptoethanol, 1% SDS and 50 mM EDTA (pH 7.5) for 20 minutes followed by lysis buffer solution 2 containing 0.4 M Tris, 2 M NaCl and 1% SDS, (pH 7.5) for 15 minutes. The slide was nally incubated in Trisborate-EDTA buffer (0.09 M Trisborate and 0.002 M EDTA (pH 7.5) for 12 minutes. The slide was washed and dehydrated in increasing concentration of alcohol in water. The slide was stained by Diff-Quick solution for 3 minutes and air dried. Under light microscopy at × 400 magni cation, 300 sperm cells were scored based on their halo size; big and medium-sized halo indicate sperm cells without DNA fragmentation, while small-sized halo and also sperm with no halo indicate sperm cells with DNA fragmentation. SDF rate was calculated as the number of sperm cells with small and no halo to total number of counted sperm × 100 88 .

Testicular morphometric evaluation
The xed testes were embedded in para n and sectioned by a rotary microtome (Leitz, Germany). Eight 5-μm thick sections of middle part of each testis was prepared at 10-μm intervals. The sections were stained by hematoxylin/eosin, the diameter and the area of 10 round seminiferous tubules in each section were evaluated. In order to evaluate the quality of spermatogenesis and maturity of germinal epithelium, Johnsen's score was used as a semi-quantitative method in 80 seminiferous tubules per animal 89 . The scores were summed up and their means were reported as Johnsen's score (JS). All morphometric assessment of testicular tissue was performed in a blind fashion by an expert operator.

Malondialdehyde (MDA) determination
MDA level as a nal product of lipid peroxidation in the serum of exposed pups (seven mice/ group) was assessed by thiobarbituric acid method using Nalondi™-Lipid Peroxidation Assay Kit-MDA (Navandsalamat Company, Urimia, Iran) based on the manufacture instructions. In short, 200 μL of serum was mixed with 1000 μL of working solution containing 2-thiobarbituric acid and trichloroacetic acid and then incubated at 95 °C for 45 minutes. The mixture was transferred on ice, incubated for 10 minutes and then, centrifuged at 1500 rpm for 15 minutes. Absorbance of the supernatant was read at 532 nm by a spectrophotometer (Alpha-1860, Thomas Scienti c, USA). MDA concentration was expressed as nM/mL.

Total RNA extraction and complementary DNA synthesis
Total RNA was extracted from sperm cells by RNX plus solution (Cinnagen, Iran) based on the manufacturer's instructions. In short, 1 mL of cold RNX plus solution was added to 1 mL of sperm suspension, and incubated at room temperature for 5 minutes. Then, 200 μL of cold chloroform solution was added, shacked vigorously and centrifuged at 12000 rpm, at 4 °C, for 15 minutes. The supernatant was transferred into a clean microtube, equal volume of cold isopropanol solution was added and the microtube was transferred to a -20 °C freezer, overnight. Next day, the mixture was centrifuged at 12000 rpm, at 4 °C for 15 minutes. The upper phase was then removed, the pellet was washed by 75% ethanol, and again centrifuged at 12000 rpm, at 4 °C, for 15 minutes. In the next stage, the supernatant was removed and the pellet was placed under a laminar-air ow hood to dryness. The dried pellet was resuspended in 20 μL diethyl pyrocarbonate (DEPC)-treated water. The integrity and quantity of total RNA were veri ed by 1% agarose gel electrophoresis and the absorbance ratio at 260/280 nm with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scienti c, Wilmington, DE, United States), respectively. One μg of total RNA was converted into complementary DNA (cDNA) with 1 μL of 200 U/μL SuperScript III Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Fermentas, Burlington, Canada), Four μL of 5X rst-strand buffer, 0.5 μL of 40 U/μL RNase inhibitor, 1 μL oligo (dT) primer and 1 μL 10 mM dNTP were mixed in a 20 μL reaction. The samples were incubated at 42 °C for 60 minutes, then the enzyme was deactivated at 70 °C for 10 minutes using a thermocycler (Biometra, Germany) 90 . The speci c primers were designed by the primer3 program. The primer sequences are presented in Supplementary table S1. Conventional reverse transcriptase (RT)-PCR was done for optimizing annealing temperature for each primer set. The RT-PCR products were evaluated by agarose gel electrophoresis (2%).

Quantitative real time PCR (qRT-PCR)
Relative mRNA levels were quanti ed by real-time PCR with SYBR Green Master Mix (Genaxxon bioscience, Ulm, Germany) using a Light Cycler Real-Time PCR System (MIC (Queensland, Australia) for the following genes: DNMT1, DNMT3A and DNMT3B, HDAC1 and HDAC2. Beta actin was used as the housekeeping gene to normalize the qRT-PCR reaction. For the ampli cation reaction,1 μL synthetized cDNA solution was mixed with a 1 μL speci c primer and 10 μL SYBR Green I Master in a 20 μL reaction. The thermocycler cycling conditions was as follows: 95 °C for 15 minutes (1 rep), followed by 40 cycles of 95 °C for 15 seconds, 60-62 °C for 60 seconds and 72 °C for 30 seconds. Melting curves were checked for the validation of ampli cation of a single PCR product. The 2 −ΔΔ cycle threshold method was used to assess gene expression levels.

Statistical analysis
Data analysis was conducted by using SPSS software (version 22). At rst, data was analyzed for normality by using Kolmogorov-Smirnov test. Differences in parametric data were determined by One-way analysis of variance (ANOVA) test followed by Tukey post hoc test, while differences in non-parametric data were determined by using Kruskall-Wallis test. The data were presented as Mean ± SEM. The Graphpad Prism software was used for graphing.

Study Approval
Animal research was approved by the Animal Experiment Committee of the Kerman University of Medical Sciences of Iran (approval no.: EC/97-10/KNRC) and con rming that all animal study methods were conducted in strict accordance with the relevant guidelines and regulations. All experimental procedures were carried out in accordance with the ARRIVE guidelines.

Data Availability
All data generated or analyzed during this study are included in this published article (and its Supplementary Information le).

Biometric analysis: testis weight and dimension
The values of offspring's testes parameters are shown in Table 1. Exposure to nicotine and ethanol, either alone or in combination, during pregnancy and breastfeeding periods had no marked effects on testis weight, length, width and diameter in comparison to the unexposed vehicle group. Histopathological observations of testicular sections of unexposed animals in the vehicle group ( Fig. 1-a) showed a typical architecture of seminiferous tubules with clear lumens and active spermatogenesis. However, the seminiferous tubules of nicotine-exposed mice ( Fig. 1-b-e) revealed severe destructions such as atrophy, vacuoles and sloughing germinal epithelium with a small volume of sperm cells in the lumen. Also, in the ethanol ( Fig. 1-f) and concurrent -exposed animals ( Fig. 1-g), a perfect spermatogenesis event was observed in almost all the tubules, however several vacuoles were observed and yet sperm cells in the lumen were less than the unexposed mice.

Seminiferous tubules area
The results of measuring seminiferous tubules area in the testes of 90 day-old offspring did not exhibit a signi cant statistical difference in any of the exposed groups when compared to that of the vehicle group (Table 2).

Semi-quantitative analysis of spermatogenesis by Johnsen's score
The result of Johnsen's score evaluation is illustrated in Table 2. The average of Johnsen's score for the seminiferous tubules in the Veh group was 9.66 ± 0.04 and reduced signi cantly to 8.98 ± 0.09 (p= 0.000), 9.24± 0.08 (p= 0.003) and 9.23 ± 0.08 (p= 0.002) in the Nic, Ethn and Nic+Ethn groups, respectively, relative to the Vehicle's score.

Sperm quantitative analysis
Sperm number The sperm number was signi cantly reduced in the Nic group (6.36 ± 1.10, p= 0.01) and slightly in the Ethn (14.45 ± 2.14, p=0.80) and the Nic+Ethn (16.31 ± 3.11, p=0.99) groups in comparison to that of the Veh (17.32 ± 2.41) group. However, a signi cant difference in the sperm number was observed between the Nic and Nic+Ethn groups as well (p= 0.02), (Fig 2-A).

Sperm DNA fragmentation (SCD)
The result of sperm chromatin dispersion test is listed in Figure 3 A-B. In utero and lactation period exposure to nicotine (58.5 ± 6.44, p= 0.007) and alcohol (47.71 ± 8.84, p= 0.06), either alone or concomitant (32.5 ± 5.23, p= 0.756) has led to a rise in the mean percentage of the sperm DNA fragmentation.in addition, there was a signi cant difference between the Nic and Nic+Ethn groups considering SCD parameter (p= 0.05).
The level of malondealdehyde (MDA) as an oxidative stress biomarker in all exposed pups was found to be signi cantly higher than those of the vehicle' serum (p=0.000) (Fig. 3C).

Quantitative RT-PCR analysis for the expression of DNA Methylation regulators
The expression level of DNMTs (1, 3A and 3B) in the sperm cells of F1 pups was measured and compared to the values of the vehicle group. The results are shown in Figure 4. In the sperm cells of nicotine (p=0.05) and ethanol (p=0.247), alone and concurrently (p=0.01), DNMT3A expression was decreased compared to the vehicle pups. Furthermore, the expression level of DNMT3B in the sperm cells of all exposed mice increased and the nicotine group showed a higher level than those of other groups (p= 0.07), nevertheless it did not reach a signi cant level. Based on the DNMT1 expression, there was no marked alteration in the sperm cells when compared to the vehicle (p=0.869, 0.343 and 0.666 compared to the Nic, Ethn and Nic+Ethn groups, respectively).
In the sperm cells of F1 pups, changes in the expression of HDACs (1 and 2) genes were also measured ( Figure 5). In comparison with the unexposed mice, the expression levels of HDAC1 and HDAC2 were markedly increased by maternal exposure to nicotine (p=0.001, p=0.002) and ethanol (p=0.000, p=0.04), either alone or concomitant (p=0.003, p=0.178), respectively. However, concurrent-exposed pups showed alleviated changes in mRNAs expression of histone hypoacetylation regulators compared with nicotine or alcohol-exposed pups, so that a signi cant difference in the expression level of HDAC2 was observed between the Nic and Nic+Ethn groups (p= 0.026) (Fig. 5).

Discussion
The ndings of this study revealed that exposure of F1 pups to nicotine, alcohol and their combination during in-utero and locational period could change postnatal sperm and testicular parameters as well as germ cell epigenetic modifying enzymes expression. Interestingly, co-administration of these substances to F1 pups alleviated the negative effects of nicotine and alcohol alone.
In the current study, Johnsen score's data showed a decrease of spermatogenesis activity in the testes of alcohol and particularly nicotine-exposed pups which are in line with previous studies documenting the transgenerational adverse effects of these substances on spermatogenesis 8,33 .Another study has also reported that exposure to ethanol during late prenatal and breastfeeding periods could delay the onset of spermatogenesis in young 33 and adult male offspring 34 . Disturbed spermatogenesis in the nicotineexposed offspring is due to complex mechanisms such as a decrease in the expression level of Sertolicell-speci c desert hedgehog gene 35 , dysmorpho-function of Sertoli cells; nursing cell of spermatogenic cell lines 36 , increase of gonocyte and meiotic germ cell apoptosis 36 , reduction of the spermatogonial stem cell population 36 as well as de cits of seminiferous tubule organization 8 . In regard to in utero exposure to alcohol, fetal Sertoli cell development disruption 37 38 and a fewer number of primordial germ cells in tsetse of neonates 33 may be partially explained the offspring's disturbed spermatogenesis. In addition to, in an another study 33 suggested that direct harmful effect of ethanol on developing reproductive system may lead to spermatogenesis disruption.
A lower sperm number, especially in the nicotine-exposed offspring, was observed in our study. Several previous studies showed a signi cant decrease of sperm number after prenatal exposure to nicotine and ethanol 8,13 . We also observed a lower sperm motility in all exposed offspring, particularly, in the ethanolexposed pups. Consistent with decreased motility, analysis of serum MDA level; as a nal lipid peroxidation (LP) product and oxidative stress marker showed a signi cant increase in all exposed offspring. Oxidative stress occurs when there is an imbalance between reactive oxidizing species (ROS) production and antioxidant defenses. This imbalance was reported in the pregnant mothers 39,40 and infants 39-42 that were exposed to ethanol and smoke. A previous study has 43 reported that maternal smoking could reduce antioxidants levels such as vitamin E and A in milk, therefore, the antioxidant status of newborns is also diminished. However, among sperm parameters, it seems that sperm motility 44 and DNA integrity 45 are more vulnerable to non-physiologic levels of ROS. Sperm membrane is rich in polyunsaturated fatty acids. This characteristic makes it vulnerable to ROS-induced LP. This process can cause structural changes of sperm membrane and motility apparatus housed within the sperm agellum, as well as alteration of biophysical features such as diminished activity of sodium/potassium ATP; an essential pump for motility 46 . Moreover, since normal mitochondria function is in a part, necessary for sperm motility, and ROS-mediated disturbed mitochondria membrane integrity 47 could affect its proper function, hence mitochondrial dysfunction in the nicotine-exposed-F1 male 8 could be one of the possible reasons for decreased sperm motility. Also, a decrease in the expression level of genes involved in sperm agellum formation and development, or the other structures related to motility e ciency might be another mechanisms 36 .
Oxidative stress could affect genome integrity 48 . In agreement with a previous report 8 , we observed high frequencies of sperm DNA fragmentation (SDF) in the all exposed animals, being highest in the nicotineexposed offspring. Since, high level of ROS production and diminished activity of antioxidant molecules could lead to oxidative stress, the highest SDF level in the nicotine-exposed offspring, in comparison to other exposed mice, may be a result of a lower level of antioxidant defense activity 40,49,50 . We assume that oxidative stress resulting from these toxins could affect germline lineage genome during early life, leading to disruption of gamete DNA integrity of adult males. Moreover, we observed a less deleterious effect in the ethanol-exposed mice in comparison with nicotine. Since the nicotine in fetus bloodstream has higher level than that of smoking mother 51 , hence this may be a causal explanation for more permanent effects of nicotine rather than ethanol. On the one hand, the ethanol effects were applied in a dose-dependent manner 52 ; the dosage used in our experiments was equivalent to that of serum after moderate drinking 53 , so it appears that ethanol, at this dose, had a slighter negative effect than nicotine.
Also, it is reported that alcohol at low and moderate doses could increase heat shock proteins 54,55 . These proteins could inhibit apoptosis and facilitate cell recovery via inhibition of the caspase cascade and proapoptotic proteins activity 56 , which could limit in ammation as well as cell death. Taken together, the decreased sperm parameters and spermatogenesis changes may lead to a lower fertility potential in the F1 offspring after exposure to nicotine or alcohol. However, for the rst time, we observed mildly effects of concurrently exposure to these substances on F1 male reproductive properties in comparison with exposure to each one, alone as co-exposure to these substances showed a lighter reduction of sperm motility. In line with our ndings, a previous study reported an improvement of cognitive impairment in alcohol drinking subjects by smoking 57 . Nicotine is considered to be responsible for such alleviating effects 58,59 . Another study also showed that alcohol toxi cation is lower in chronic smokers compared to that of the non-smokers 60 . Recently, Bhattacharya and colleagues 61 showed that prenatal ethanol exposure (10% v/v) could increase ROS production, LP, caspases activities as well as decrease spatial memory in F1 rats, while simultaneous exposure to ethanol and nicotine (6 mg /kg/day, by mini pump) could alleviate the adverse effects of ethanol on oxidative stress and hippocampal memory. However, they exposed pregnant rats to nicotine and ethanol at higher and lower doses, respectively compared to our study. Although the exact alleviating mechanism still is not fully understood, Hurley and co-workers 62 explained that the dose and also co-use of these substances could change the adverse effects of each, alone 63 by alteration of peripheral metabolism of the other 64,65 . Several mechanisms such as modulation of calcium, anti-apoptotic mechanism and also nitric oxide signaling 58 were reported to improve the effects between these two determents. Yet, some studies determined that this counteracting effect was only observed in acute exposure dose 66 and in contrast, some reported a synergistically contribution of these drugs 67 in impairment even in acute dose exposure. In contrast to our ndings, Sabzalizadeh and co-workers 68 revealed that these two substances could synergistically impair cognition in pups. However, it is yet unknown that whether such a different effect is dependent on the developing tissue; so that the interaction of these substances during embryofoetal life might differently affect cells in developing reproductive system rather than the nervous system. Con rmation of this hypothesis need further investigations.
On the other hand, the widespread reprogramming of male gamete in in utero development period 69 makes it sensitive to environmental and chemical factors. Many studies have investigated the effects of nicotine and ethanol, alone, on the epigenetic signature of developing fetus and neonates as well as male gamete. However, the ndings are controversial and the effect of concurrently maternal and lactational exposure to these substances has not yet been elucidated. Therefore, we examined the possible epigenetic modi cations by the expression level assessment of a diversity of modifying enzymes; DNMTs and HDACs in the exposed offspring's sperm cells. Our data provides evidences that in utero and lactational exposure to alcohol and nicotine markedly decrease DNMT3a transcripts and signi cantly increase HDACs (1 and 2) transcripts of the sperm cells. Interestingly, we noted that alcohol and nicotine co-exposure had less obvious effect on these enzymes levels compared with alcohol and nicotine, alone.
Previous studies have reported that exposure of fetus to alcohol could change chromatin structure and epigenetic signature in GD 17 developing fetal brain of mice 70 , global DNA hypomethylation of fetus genome 31 , obvious diminished DNMT activity 30,31 , hypomethylation of imprinted gene; H19, elevated histone 3 acetylation in lung 71 , and cardiac cells 72 as well as reduced HAT activity in cerebellum 73 .
These alterations suggested a strong correlation between this epigenetic signatures with congenital malformations' attendance. A recent study 74 exposed pregnant Wistar rats to alcohol (4 mg/kg/daily, 9-20 GD days) to evaluate the epigenetic mechanism involved in testicular function of F1 pups. They showed that testicular dysplasia due to elevated corticosterone level was associated with low-functional programming of 3β-hydroxysteroid dehydrogenase (3β-HSD), increase of HDAC2 expression level and also decrease of histone 3 lysine 14 (H3K14ac) acetylation. We also observed a marked increase in the HDACs transcripts in the ethanol-exposed offspring's sperm cells. Gangisetty and co-workers 75 demonstrated that in utero alcohol exposure could lead to pituitary changes via epigenetic mechanism.
Although, in agreement with our data, they observed a signi cant increase in HDAC2, but they also reported a signi cant increase in pituitary mRNA level of DNMT 1 and 3b, while we did not observe such transcripts changes in sperm cells of exposed pups. In some studies, oxidative stress after acute ethanol use 76 , and also reduction of methyl donors such as s-adenosylmethionine (SAM) for DNMTs following its chronic use 77 are explained as some of ethanol mechanisms as a modi er of DNA methylation signature in sperm cells which represents into global DNA hypomethylation.
Meanwhile, multiple experimental and clinical studies have reported the effects of maternal exposure to smoking on epigenetic alterations in various tissue of exposed fetus, newborns, children and adolescent.
However, for the rst time, we found the changes of sperm cell's mRNA level of HDAC1 and 2. A previous study 78 determined that second-trimester fetuses of active smoker mothers had different DNA methylation state as well as reduction of neuronal proportion in the developing brain prefrontal cortex. They suggested that nicotine, is responsible for such effects. Another recent study revealed that in utero exposure to smoking led to differentially DNA methylation in 10,381 CpGs sites of cord blood CD41 positive cells of newborns 20 as well as 69 CpGs sites 29 in 36 genomic regions of adult offspring blood cells. Moreover, Rehan and collagenous 79 observed that prenatal exposure to nicotine (1 mg/kg) could increase DNA methylation and H3 and H4 acetylation in F1 offspring's testicular cells. They showed a transmission of germline epigenetic marks to F2 offspring. However, based on our results, it appears that exposure to these substances may affect histone deacetylation level of sperm cells more than DNA methylation. Histone alterations in sperm cells might cause abnormal further generation development, gene expression and phenotype 80,81 . Besides that, in line with almost our entire dataset, simultaneous exposure to these detrimental substances had a slighter effect on the expression level of epigenetic modifying enzymes in comparison to exposure to these substance, alone.
In conclusion, the present study revealed that in utero and lactational exposure to ethanol and nicotine could trigger changes in the sperm properties and interestingly, for the rst time, presented an unusual alleviating effect of co-exposure to these substances on F1 offspring's reproductive system. However, there are still many questions about how the interaction of these drugs could counteract the negative effects of one drug by the other. Therefore, further studies especially at the molecular level are needed.
Moreover, our ndings revealed changes in the expression level of epigenetic modifying enzymes included DNMT3A, HDAC 1 and 2 in the sperm cells of F1 offspring exposed mice. These transcripts changes could associate by aberrant germline epigenetic alterations. What make these ndings even more important, is the potential of transgenerational transmission of these epigenetic changes via exposed germline to the next generation. Declarations