Cursory analyses with a cutoff of 1.5 shows apoptotic pathways as being significant for TSC exposure as well.It is important to note that the marijuana used for this study was obtained from a contracted supplier that provides marijuana for therapeutic use in Canada.It is grown under strictly controlled and documented conditions.Although this study has only examined smoke condensate from a single lot of marijuana,the quality control measures would be expected to minimise differences between marijuana harvests.The TSC used in this study was generated from cigarettes containing Virginia flue-cured tobacco,the type of tobacco typically contained in Canadian cigarettes.This is distinct from the mixed tobacco blends typically found in American cigarettes.Our earlier toxicogenomic examination of TSC from three Canadian cigarette brands containing either Virginia flue-cured or mixed tobacco blends failed to show any appreciable brand-driven differences in gene expression profiles elicited by in vitro exposures.Therefore,we contend that the similarities and difference between MSC and TSC noted in this study can be cautiously extended to other types of tobacco.Nevertheless,it should also be noted that sometoxicogenomic studies have shown that cigarette brand can have a significant effect on gene expression signatures elicited by in vitro CSC exposures,and moreover,many aspects of cigarette design and smoking method have been shown to influence the composition and toxicological activity of TSC.Cannabis sativa is popularly known as marijuana and smoking is the commonly used form of this drug.Based on unofficial estimates of drug consumption conducted by the United Nations,it is the most abused drug in the world,with 140 million consumers.Users are young,and exposures occur during their reproductive age.Moreover,among pregnant women,it appears more frequently in self-reported questionnaires of drug use during gestation.Most of the studies on the toxicity of marijuana use during pregnancy have evaluated the neuro-behavioral effects.Epidemiological evidence has shown that marijuana impairs the growth trajectory of the fetus,vertical grow system resulting in low birth weight,intrauterine growth retardation and congenital malformation.
Maternal health is also negatively affected; marijuana-using mothers present higher prevalence of dysfunctional and precipitous labor,as well as meconium-stained amniotic fluid.Other potential adverse effects of smoking marijuana during pregnancy are lesser known.The majority of the toxicological knowledge about the effects of Cannabis sativa on the reproductive tract and fetal development comes from animal studies.In these studies,exposures are done primarily by gavage of marijuana extract orD9 -THC i.p.Regardless of the route of administration,findings of these studies demonstrate increased resorption rate and reduced fetal weight in both mice and rats.None of the studies have reported fetal malformations.Abel et al.reported that pregnant rats exposed to different doses of Cannabis extract throughout gestation presented reductions in weight gain and food consumption.Birth weight was reduced only in those groups exposed during the third week or during the whole gestation.Charlebois and Fried evaluated the effects of pre-gestational and gestational exposure to Cannabis smoke on rats fed low,normal,or high protein diet.They observed that Cannabis exposure lengthened the gestation period and increased the occurrence of stillbirths and litter destruction.When exposure was coupled with a high protein diet,these effects were attenuated.Furthermore,evaluation of the outcomes of the groups exposed both before and during gestation suggested a degree of tolerance to the drug effects.THC and its metabolites are able to cross the placental barrier and reach the fetus.The endocannabinoid system has an important role in reproduction,from the earliest stages of ontogenic development to parturition,including fertilization,embryo implantation,and placentation.The endocannabinoid system is present in different organs where it plays multiple physiological roles.It is composed of the cannabinoid receptors,CB1 and CB2,which are G protein-coupled receptors that are differentially distributed in the organs,and endogenous molecules derived from arachidonic acid: anandamide and 2-arachidonoylglycerol.Marijuana’s D9 -THC can also bind to CB receptors and activate multiple intracellular signal transduction pathways.Studies in humans have many confounding factors that make it difficult for interpretation and establishment of a causal relationship between smoking marijuana and poor gestational outcomes.Furthermore,toxicological studies conducted in animals use intraperitoneal injections or oral gavage of D9 -THC to perform the exposures,which exclude the interaction of compounds present in the smoke that could also contribute to pregnancy disorders,and the doses used are far beyond the dose commonly experienced by humans.
Most of the published reviews have acknowledged that there are several uncertainties on the effects of maternal marijuana use on gestational and fetal outcomes.There is lack of information on biological mechanisms,whether fetal developmental disruptions occur indirectly,directly,or as a combination of both,and alterations in placental function,changes in hormonal balance,on sex-specific effects,effects on organogenesis of the kidney,lungs,spleen,and thymus.These aspects and the spreading legalization of recreational use of Cannabis sativa point out that there is an urgent need of further toxicological studies to better recognize the effects and elucidate the mechanism involved in this association.In the present study,we developed an experimental murine model to study the effects of recreational use of marijuana during pregnancy to mimic human “real world” exposures in terms of dose and use to evaluate the effects on gestational and fetal outcomes.The exposure system is composed of a pump that blows air through a HEPA filter into a pulse dumper.The airflow is split into two directions that pass across the valves that control the flow.One flow goes to the smoking chamber and the other directly to the mixture chamber.The smoking chamber is a 1-L sealed box with an aperture for the airflow,creating a positive pressure that forces the airflow to pass through the cigarette,and consequently the smoke flows to the mixture chamber.The mixture chamber is a 1-L sealed box with a bulkhead to promote the mixing process.Air-smoke flow was controlled at 1.2 L/min.The mixed smoke-air goes to the manifold where mice are arranged in individual tube-type holders.The prepared marijuana cigarette lasts for 5 min of exposure in this system.An identical system was built to conduct exposures to the control group.Fetal development was evaluated during pregnancy using ultrasound biomicroscopy.On GD 10.5 and 16.5,morphological evaluation of all pregnant mice was performed,with two fetuses per dam,where the crown-rump length,biparietal diameter,abdominal anteroposterior diameter,abdominal transverse diameter,placental diameter,and placental thickness were assessed.The analyses were performed with the aid of a high-frequency ultrasound imaging system with a 40-MHz transducer by MMV who was blind to the group.The limited time for the animal’s isoflurane sedation and examination duration was set at 30 min.Furthermore,a random sample of the fixed fetuses from each group was selected and the organs were dissected and weighed to verify the fetal development.
Fetal-to-placenta weight ratio was calculated as an indicator of fetal–placental dysfunctions.This measure is expected to increase as pregnancy progresses; if abnormally low or high,it could be indicative of poor fetal outcomes.In this study,marijuana smoke inhalation negatively affected the gestational and fetal outcomes in the Balb/C mice model of exposure.Five minutes of daily exposure during pregnancy resulted in reduced birth weight,and litter size was not altered; however,the number of male pups per litter was higher.Besides,placental wet weight was increased and fetal- to-placental weight ratio was decreased in male fetuses,showing a sex-specific effect.Although not significant,the difference in the post implantation loss index was two times higher in marijuana smoking female mice.Exposure procedures and animal handling and manipulation did not affect maternal food intake in both the control and Cannabis groups.At the end of gestation,females from the Cannabis grow supplies group presented reduced maternal net body weight gain,despite a slight increase in their daily food intake compared to control group.Most of the available experimental studies employ intraperitoneal injections or oral gavage of D9 -THC to perform exposures and access the toxic effects of Cannabis sativa on pregnancy and fetal outcomes.This means that in this study animals were exposed not only to D9 -THC,but to the whole smoke produced by marijuana pyrolisis.For this,we developed an inexpensive,efficient and reproducible exposure chamber to conducted controlled exposures of marijuana smoke.Smoke inhalation exposures using experimental animals are rarely used,due to many difficulties to control and standardize the exposure process.Our exposure was conducted based on Lichtman’s et al.work.In their study mice were exposed,via inhalation,to burning of marijuana cigarette during 5 min in a controlled air-smoke flow.Our exposure model has already proven successful,once it was confirmed the presence of THC–COOH in murine urine.Moreover,it also proved to be safe and not stressful method,causing no deaths during the exposure process and no changes in feeding behavior.Recent reviews highlight the importance of the endocannabinoid system for implantation and placentation,and although the mechanisms are not completely understood,data suggest that controlled concentration of endocannabinoids is essential for successful embryo development.
Since the D9 -THC can bind to the same receptors as anandamide,which is an endogenous cannabinoid that participates in the control of many reproductive functions such as fetal implantation and development,the THC could act on the system equilibrium or its control,thereby affecting the embryo development and placentation.The levels of endogenous cannabinoids decrease during pregnancy progression ; low systemic levels are necessary for a healthy progression of pregnancy,and any increase in the levels of anandamide impacts pregnancy,increasing the risk for abortion,pregnancy loss,and growth restriction in humans and mice.Besides,AEA increases the activity of NOS,which acts as a potent local vasorelaxant that is important for the maintenance of low vascular resistance in the fetoplacental circulation.Depending on the route of administration,dose,time of exposure,the animal model,and how Cannabis toxicity is tested,the results from the studies vary.Joneja,Harbison and MantillaPlata,and Mantilla-Plata et al.noted that depending on the gestational period of exposure to D9 -THC,the outcomes are different.Exposures during initial stages are associated with increases in fetal reabsorptions and pregnancy loss,depending on the dose,and exposures in mid gestation lead to fetal growth retardation.In contrast to Abel that pointed out that administration of cannabinoids reduces food and water consumption,food intake was not altered in our study.Studies reviewed by Abel used gavage to administrate the drug and this may have caused some discomfort and thus could have depressed the food intake.Trezza et al.and Campolongo et al.used gavage to treat pregnant rats from GD 15 to the end of pregnancy,and no difference in maternal weight gain and fetal birth weight was observed; however food intake was not monitored.However,the effects observed in our study should be interpreted and attributed to inhalation of the smoke produced by the pyrolysis of Cannabis sativa and not to any specific components.There are several noxious chemicals present in marijuana smoke,NO,NOx,CO,hydrogen cyanide,aromatic amines,ammonia,toluene,benzene,and polycyclic aromatic hydrocarbons,with potential effects on maternal and fetal health.The observation of higher rates of post implantation losses and increased number of male pups per litter in Cannabis group allowed us to suggest another sex-specific endpoint: early in pregnancy female embryos are more susceptible to the effects of smoke.
Male and female embryos present differences not only in sex chromosomes but in their metabolism due to differences in sex and autosomal-related genes expression and this may consequently determine gender susceptibility.Regarding differences in placental weight,our data indicate that inhalation of Cannabis smoke during pregnancy compromised placental efficiency.Placentas from the Cannabis group were heavier,although fetal weight was reduced,compared to control group.This implies that more grams of placental/gram fetal tissue were needed to support development.This effect was marked in males,but with a borderline significance in females; the mechanisms involved need to be investigated,although hypoxia and maternal metabolic changes might be involved.Many mechanisms could underline the compromised fetal development observed in our study.Reduced weight at birth and restricted intrauterine growth are effects commonly associated with exposure to Cannabis sativa during pregnancy ; however,no previous study has reported increased susceptibility of male fetuses to this effect.Two studies found similar results of increased number of male pups per litter of exposed dams.In our study,estimation of the ponderal index [3 100)] for the Cannabis group indicated that fetal growth is restricted at the end of pregnancy.There were some limitations in this study.Our evaluation of the presence of THC–COOH in murine urine is an indicator of exposure and not dose.The small volume of urine that we could sample after the exposures,and the low-dose exposure that we adopted in this study,imposed many difficulties to determinate its quantities in this biological matrix.In the study of Lichtman et al.,mice were exposed,via inhalation,to smoke produced by burning 50,100,and 200 mg of marijuana,containing 3.4% of D9 -THC,during 5 min.The estimated D9-THC doses were 2.0,3.5,and 5.6 mg/kg,respectively.Our exposure was conducted in a very similar manner,we burnt 200 mg of marijuana,during 5 min,however our sample contained 10 times lessD9 -THC,which allows us to suggest a dose of exposure equivalent to 0.5 mg/kg.In summary,our results indicate that smoking marijuana during pregnancy even at low doses can be embryotoxic and fetotoxic,increasing implantation failures and compromising fetal development.