The growing use of THC-rich preparations in various forms, both recreationally or prescribed by medicinal professionals, raises issues regarding their safety and risks for users, especially in vulnerable subpopulations as cancer or chronically ill patients. Since THC is often used with other conventional drugs and/or therapies, any information regarding its toxicity profile and potential to interact or enhance toxicity of other compounds is important and potentially valuable. The genotoxic potential of THC in vivo has been poorly investigated so far. Knowing the genotoxic potential of a compound is an important prerequisite to predict other potentially detrimental effects, both at the level of cells and an organism. In a recently published review, Reece and Hulse [44] stated that low doses of THC (<5 µg/mL or <5 µmol/L, or <1 joint/day) are usually not associated with genotoxic adverse outcomes. However, the doses of THC used by many of the patients who experiment with self-medication are usually considerably higher. It is hard to predict the genetic risks associated with such exposure, since the available literature offers limited evidence regarding THC effects at DNA level. In their review, Li and Lin [45] discussed the potential genotoxicity of commonly abused substances, including cannabis, and singled out its detrimental effects at DNA level. The recent literature offers only one in vitro study [46], which used the comet assay to estimate the genotoxicity of marijuana smoke condensates in lung cancer cells. Without doubt, the experimental approach applied in the present study is a novel one, as there is a general lack of accurate data in this regard. With a previous pilot study [20], our group was the first to apply the alkaline comet assay to evaluate THC genotoxicity on a rat model. In our previous paper, we showed that continuous 7-day oral THC exposure produced low-level DNA damage in hepatocytes. This paper provides evidence that brain and white blood cells responded with significant increases of DNA damage much earlier than hepatocytes, at the same tested THC dose, which points to their greater genome susceptibility. What further distinguishes the present study from the preceding one is the fact that in the first study we investigated the DNA damaging effects of anticancer drug irinotecan (IRI) and THC when administered to rats as single compounds and in combination. Particular emphasis was also given to mutual interactions of IRI and THC and their harmful effects in the rat liver. Bearing in mind a general lack of the information regarding THC genotoxicity, after extensive evaluation of data obtained using the comet assay and biochemical assays in rats exposed to single THC, we prepared the present paper as a separate publication, which we believe brings sufficiently relevant and new evidence regarding the outcomes of acute and repeated THC exposure on other rat tissues at a dose found in illicit preparations. This study provides original and valuable information regarding the levels of DNA damage in blood and brain cells of THC-treated rats along with novel information regarding THC impact on oxidative stress markers and cholinesterase activity that have not yet been familiar or reported before in relevant literature sources.

The main aim of the present study was to investigate the genotoxic potency as well as the magnitude and dynamics of DNA breakage associated with THC treatment in vivo. The obtained results show the ability of both acute and repeated THC exposure to provoke measurable DNA damage in brain cells and white blood cells of rats. Increased DNA damage in brain cells of THC-exposed rats represents the most important finding of the present study and calls for concern, considering that it could impair viability of neurons, which ultimately might lead to their progressive loss and possible neurotoxic outcomes.

The observed differences in DNA susceptibility of white blood and brain cells might originate from the intrinsic differences between two tissues, and the efficacy of their inherent mechanisms which counteract damage. Since each of the tested tissues represents a specific matrix, the significance of obtained results will be discussed separately.

In this study, slides for the alkaline comet assay were prepared using the samples of whole blood. The use of whole blood in the DNA damage analysis using comet assay was confirmed previously in a study by Al-Salmani et al. [47]. Considering that the comet method relies on recording of DNA damage in single nucleated cells [32,35], measured levels of genotoxicity represent the sum of DNA lesions inflicted in all white blood cells. As known, leukocytes are a heterogeneous population of highly differentiated nucleated cells that is comprised mainly of neutrophils (up to 80%), followed by lymphocytes, monocytes and eosinophils. When discussing the results, we have to bear in mind potential differences known to exist between these cell types. For instance, DNA repair capacities of white blood cells vary according to cell lineage [48], which might influence their genome susceptibility and sensitivity to genotoxic agents as well. Interpretation of the results about possible time-dependent genotoxicity should also focus on life-spans of white blood cells. As known, life-spans of neutrophils and monocytes are measured in hours/days, compared to lymphocytes which might circulate through the body for up to several months [49,50,51].

Even though the obtained results relied on measurements of three comet parameters, simultaneously calculated using the image analysis software, in this discussion we will place the main emphasis on tail intensity, as it is today considered the most useful parameter to describe the degree of DNA damage estimated by the comet assay. It points to the amount of DNA migrated in the tail and correlates with DNA break frequency. However, results obtained for two other comet parameters also contribute to the description of the overall THC genotoxicity, which could be characterised as being of low magnitude. As known, at low levels of damage comet tail length is also deemed a useful parameter [32]. Since it depends on the DNA loop length, after the comet tail is established, its length rapidly reaches a maximum and tends not to change [32,33]. The third comet parameter, i.e., total area, represents the overall surface area of the comet. Although not used as frequently as other comet parameters, its values can provide additional information on the extent of DNA damage that could further strengthen the significance of the comet assay findings.

In the present study, we evaluated THC genotoxicity at three different time-points and found the highest level of DNA damage, in terms of tail intensity, after the 1-day exposure (Figure 1). Since such a short exposure period covers the life-span of all leukocytes, the mean tail intensity recorded after 24 h refers to the total DNA damage measured in all nucleated cells. However, explanation of DNA damage levels measured in blood samples taken after 3-, and 7-day exposure is somewhat more complex. Within this period, the lymphocyte pool cannot entirely renew, in contrast to rapid neutrophil/monocyte turnovers which happen daily. Therefore, we could say that the overall levels of DNA damage measured after repeated 3-, and 7-day THC exposure were largely affected by the “cumulative” responses of the lymphocyte genome to the applied treatment. In contrast, levels of DNA damage measured in neutrophils and monocytes possibly persisted, irrespective of the treatment duration, considering their constant replacement by fresh cells, and daily administration of the same THC dose. This assumption, indeed, has to be proven in future studies on isolated cell subpopulations.

A slight decrease in the mean tail intensity measured in white blood cells after 3-day THC exposure (Figure 1) might be a result of DNA repair in lymphocytes. However, judged from the perspective of an increased mean tail intensity, and the significantly increased mean tail length recorded after 7-day THC exposure, it is possible that DNA repair processes in lymphocytes finally became saturated. This could be, at least in part, explained by the quiescence state of lymphocytes, which is characterised by lower metabolic and many other activities [52]. When speaking about the genome sensitivity of other types of white blood cells, monocytes are very vulnerable, due to a lack of key proteins that take part in base excision and DNA double strand break repair [50,53]. Neutrophils, due to their very short life-span mostly do not respond to DNA damage via repair, and are intrinsically predetermined to die by constitutive apoptosis [49,54,55]. Taken together, from the DNA damage pattern observed in white blood cells during this experiment it could be concluded that THC produced a low level of DNA damage in white blood cells of rats, in spite of the relatively high daily dose administered.

Results of the alkaline comet assay show that 1-day exposure of rats to THC at 7 mg/kg resulted in detectable primary DNA damage brain cells (Figure 1). The highest maximum values of both comet parameters (32.51% DNA in tail and tail length of 54.58 µm vs. control values of 6.15% DNA in tail and tail length of 31.67 µm) recorded at this time-point suggest that some brain cells acquired large amounts of DNA lesions. Such a finding also points to the possible occurrence of apoptotic cells as a response to treatment. This is highly likely since THC-related neurotoxicity and apoptosis were previously described in several studies on brain cell cultures [56,57,58]. Worth mentioning, in these studies neurons were shown to be more susceptible to THC than glial cells. After repeated exposure, neurons also tended to accumulate THC and its metabolites, which led to adverse effects [58]. Given that the comet assay’s scoring criteria do not recommend a capture of nucleoids outside the limits of the image analysis software [59], it is possible that the real DNA damage level in the brain cells of rats at this time-point was even higher than we measured. For us it is reasonable to assume that a certain proportion of highly damaged cells “escaped” measurement, either as a consequence of measurement with an automated image analysis system or due to the critical steps of the alkaline comet assay (lysis, denaturation, electrophoresis), which contribute to the “wash out” of their fragmented DNA from the agarose microgel. That is why apoptotic cells often are lost during automated scoring if they are not recorded and scored visually as an addition to image analysis.

Our results show that repeated THC exposure resulted in a time-dependent increase of tail intensity in the brain cells (Figure 1). In view of this, it is reasonable to conclude that THC at the tested dose and exposure conditions was genotoxic for rat brain cells in total, since the comet assay slides were prepared from dissected brain tissue that contained neurons, astrocytes and other types of glial cells. The existing literature suggests that neurons and glial cells respond differentially to genotoxic agents and oxidative stress [60,61] and possess different capacities for DNA repair [30,48]. Although neurons retain the same DNA repair systems as other eukaryotic cells (nucleotide excision, base excision, mismatch and double strand breaks repair), the repair of lesions in their DNA is much slower than in dividing cells [62]. Accumulation of unrepaired lesions might impair transcription and protein synthesis, which finally may trigger cell death.

To obtain more specific explanations regarding the susceptibility of a particular cell type, the comet assay procedure might be coupled with cell separation and characterisation. However, as such procedures could represent a source of additional DNA damage detectable by the comet assay, to avoid false positive results, such an approach was not used in the present experiment.

Since the present study was a pioneer in the assessment of potential THC genotoxicity on rodent white blood and brain cells using the comet assay, it is not possible to draw a parallel between our findings and other literature sources. We can only mention the conclusions of our previous comet assay study where the same THC dose was used [20]. They suggest that continuous 7-day oral exposure of rats to THC caused low-level DNA damage in liver cells.

Considering that the alkaline comet assay identifies a wide array of lesions, we cannot define the quality and types of DNA lesions produced by THC treatment, or mechanisms behind the detected genome damage in white blood and brain cells of treated rats. However, without a doubt, the extent of DNA damage observed in the THC-treated rats was influenced by the exposure design. During the study, rats repeatedly received THC for seven days. This treatment approach led to a continuous delivery of new quantities of the tested chemical, which is extensively metabolised into many metabolites with a different half-life, and subjected to slow turnover and clearance from the organism [2,3,6,7,63]. Due to high lipophilicity, a part of the delivered THC also tends to accumulate in body fat, and slowly releases leading to “reintoxication” [64], producing potentially DNA-reactive compounds. We propose that the level of DNA damage measured after treatment is a sum of the lesions caused by mutual direct and indirect effects of parent THC, all of its metabolites, different reactive free radicals and secondary ROS. In addition, a part of induced DNA damage also depends on DNA repair processes, which might produce a certain quantity of further damage. Besides, after seven days a kind of balance between the formation and repair of DNA damage can be reached, which also influence the values detected by the alkaline comet assay.

Since our study was limited by the choice of a single THC dose, the possible harmful effects of THC at DNA level should be further investigated on a wider range of doses. Taking into account all of the facts acknowledged in the available literature sources and our own observations, we believe the issue of THC genotoxicity—due to controversial and inconsistent results—has to be further studied. Information provided in the present study could also be potentially interesting to other researchers, especially those experienced in the application of a comet assay.

The outcomes of the comet assay motivated us to further investigate the potency of the tested THC dose to provoke oxidative stress in blood and brain cells. According to the literature, this is likely high, as some studies [65,66,67,68] associated THC exposure with impaired mitochondrial function, which results in enhanced production of hydrogen peroxide. Previous studies about THC (or C. sativa) effects on oxidative stress markers in rodents relied on various free radical-related parameters and revealed inconsistent results [8,20,65,69,70,71,72].

Considering the detected levels of DNA damage after 1-day THC exposure, we decided to assess the changes of oxidative stress biomarkers only at this time-point. Although this could be a potential limitation of the present study, a similar experimental design on a rat model was previously successfully applied by Costa and Colleoni [65], who tested an even higher THC dose (10 mg/kg), but using another array of assays.

We found that acute exposure of rats to 7 mg/kg of THC resulted with a detectable increase in the concentration of TBARS in the brain. In spite of the potential lack of specificity of the applied TBARS assay, this result is an indication of possible lipid peroxidation.

Considering the essential role of glutathione in the cellular defence, our study also measured its levels after THC treatment. The obtained results show that acute THC exposure elevated brain GSH level (Table 1). Based on the existing knowledge on glutathione metabolism and cycling, the observed rise in brain GSH level in THC-treated rats, compared to their controls, could be associated with either treatment-related GSH production in brain or the uptake of GSH from plasma. The former is quite possible, since plasma GSH levels in THC-treated rats slightly lowered compared to controls (Table 1). As known, plasma GSH plays an important role in brain GSH homeostasis [73].

After single oral exposure to THC we found a slightly increased catalase activity and decreased SOD activity in brain. According to the literature, both SOD and catalase activities in rats tend to increase with longer exposure, and also after THC delivery via intraperitoneal route [72].

FRAP values, which remained within the control levels both in plasma and brain after a single administration of THC (Table 1) indicate that compensatory homeostasis among the different cellular antioxidants efficiently counteracted potential imbalances caused by acute treatment with the tested compound.

The results regarding oxidative stress markers could be, at least in part, explained by the oral route of THC delivery which was used in the present study. It is well known that oral administration of THC leads to its slow absorption. This type of delivery also allows for a greater removal of THC by the liver and more abundant formation of the active metabolite, THC-OH, which easily crosses the blood-brain barrier. As the brain extracts a large fraction of the drug present in the circulation, brain levels of the metabolite tend to gradually rise, while at the same time its plasma levels decline [7]. This largely explains the discrepancies between plasma and brain TBARS levels we found following oral THC delivery. In a similar study, Costa and Colleoni [65] found that a single intraperitoneal administration of THC at 10 mg/kg enhanced the energetic brain metabolism in rats, which resulted with increased lipid peroxidation. They associated the observed phenomena with increased mitochondrial oxygen uptake via the CB1 cannabinoid receptor.

The cellular damage by oxidative stress could have a resultant effect on loss of cognition and altered neurotransmitter system [74]. It is also known that acute THC effects are related to disturbed functioning in performance and cognitive tasks (reaction time, motor coordination and attention, learning perception) [75,76]. Since the central cholinergic neurotransmission in the brain is crucial for cognitive functions including learning and memory formation [77], we used ChE as a potential biomarker towards the evaluation of its acute toxic potential. The main effects of THC in the central nervous system are mediated through cannabinoid receptors CB1 densely concentrated at brain regions [78] but it has also been revealed that THC reduced the synthesis of the neurotransmitter acetylcholine in the hippocampus pointing to its negative effects on cognitive processes [79,80]. However, literature reports make it clear that chronic exposure of old animals to low THC doses resulted with an improvement of neurological deficits. Inhibition of acetylcholinesterase (AChE) by THC is considered to be one of the reasons for this improvement [80]. Based on high lipophilicity and a fused tricyclic structure of THC, some authors hypothesized that it could bind to AChE. Eubanks et al. [81] performed computational modelling of the THC-AChE interactions and demonstrated that THC binds in the allosteric peripheral anionic site (PAS) of AChE. Further studies focused on THC effects on cholinesterases reported inconclusive results. In an in vitro study reported by Srivastava et al. [82], a screening of the activity of human red blood cells (RBC) AChE resulted with no inhibitory effect of the C. sativa extracts. Ebuehi and Abey [83] reported a reduction of brain AChE activity in Sprague-Dawley rats after seven weeks of exposure to a diet containing 10% and 5% C. sativa chow while Abdel-Salam et al. [77] found increased brain AChE activity in Sprague-Dawley rats that were daily subcutaneously given C. sativa extract resin rich on THC for six weeks. At the same time, serum BChE activity was markedly inhibited.

In our study, the application of a single THC dose (7 mg/kg) did not affect cholinesterase activities, either in the plasma, or in brain tissue, 24 h after treatment (Table 2).