Here we show that fish exposed to Al presented an anxiety-like behavioral phenotype with reduced social interaction. These behavioral changes are probably related to AChE decrease and indicate neurotoxicity. In the antioxidant enzymes, probably Al interacted with SOD, CTL, and GST resulting in an oxidative stress condition³². When the fish were exposed to Al and oils, the negative effects of the metal does not occur, suggesting a neuroprotector and antioxidant effect of the CBD- and THC-rich oils. In our previous work⁸, we observed that the CBD- and THC-rich oils were able to reduce oxidative stress and impair the acetylcholinesterase activity and behavioral biomarkers in the invertebrate animal model organism Caenorhabditis elegans; now we show similar effects in the zebrafish model.

To understand the kinetics of the compounds with Al, as a stressor, we quantified the concentration of THC and CBD in the aquaria water, using HPLC. Here we observed that the concentrations of THC in the THC-rich oil were higher than in the other oil and the same observation was for the CBD quantification in the CBD-rich oil. The presence of CBD in the THC-rich oil as well as the presence of THC, in the CBD-rich oil, is normal once for the oil extraction they use specific strains of Cannabis sativa which produces different amounts of each compound. To produce a THC-rich oil (APEPI) they use a transgenic strain of Cannabis, that produces a higher amount of THC compared to other cannabinoids. In this way, the higher production of THC does not completely avoid the production of CBD and other cannabinoids and it can explain the presence of other compounds in lower concentrations in a specific rich oil³³.

An interesting result observed was the reduced concentration of THC and CBD in the groups treated with the oils + Al. In plants, terpenoids are produced to protect them against oxidative stress, toxicants, and physical predation³⁴. Many of the terpenoids have a great antioxidant effect also in animals³⁵. Both THC and CBD have considered specific terpenoids called cannabinoids and they can interact with toxic compounds reducing the toxicity³³. In our study, we observed that in the presence of Al, both terpenoids (THC and CBD) had the concentration reduced in the aquarium water. It means that had occurred an interaction between the compounds and the Al which probably leads to the conjugation of the compounds for neutralizing the Al-toxicity. In the body, CBD and THC may work in a similar way reducing the toxicity induced by hazardous compounds³⁶. In addition, the fish observed an increase in the antioxidant enzymes which suggests an increased oxidative resistance of the body to pro-oxidative compounds such as Al.

Our results show in the test of SPT that the Al reduced the interaction with the conspecifics. When Al is applied concomitantly with the THC-rich oil the social interaction is re-established. Related to behavior, a preserved behavioral repertoire is very important for species perpetuation³⁷. Behavioral alterations are responses that came from internal (physiological) and external (environmental) interactions. The zebrafish (Danio rerio) is a social specie, that needs to interact with its conspecifics³⁸. These findings in our study show the deleterious interaction of the Al in social behavior an important biomarker even for species perpetuation as well as for physiological and neural normal function. However, CBD- and THC oils would help in these observed changes.

The impact on cholinergic signaling and behavior was in part reversed by the THC-rich oil once the social preference was reestablished after THC administration. However, the anxiety-like pattern was not reversed after THC- or CBD-rich oils administration. Taken together, reduced social interaction and increased anxiety-like patterns exert a clear deleterious effect. Both observed behaviors originated from central nervous alterations such as cholinergic signaling. Another important behavioral biomarker is the anxiety pattern, which in humans is marked by intense, excessive, and persistent worry and fear of everyday situations³⁹. In fish, this feeling-like condition is defined by reduced locomotion and reduced exploratory behavior⁴⁰. A possible explanation for this discrepancy in the re-establishment of the social behavior but not the anxiety-like behavior after THC-rich oil administration might be due to the different behavioral paradigms used in these two experiments. THC is already known by causing anxiogenic effects when presented in high doses^41,42, and the observed effect of increased anxiety-like behavior is related to anxiogenic episodes. However, the social behavior reestablishment might be related to the CBD presence even in low concentrations of this oil other studies have shown that at low concentrations of CBD^41,43. In addition, CBD at low concentrations is responsible for decreasing the aggressiveness in mammalians by activating the 5-HT1A and CB1 receptors¹⁰, which can be linked with increased social behavior. In the environment, the resumption of the natural behavior of the species to gather in shoals is very important since the shoals have a clear advantage of protection, search for food, and reproductive success^44,45. On the other hand, the anxious behavior observed here (staying at the bottom) seems to be the clearest defense strategy when one is alone without even seeing the conspecifics, such as the situation imposed for the NTT assessment.

Cholinergic signaling is responsible for learning, and locomotion, and is important in several processes, such as memory, neuronal development, and participation in the reward system⁴⁶. Even more alterations in central systems are observed to understand and explain different disorders⁴⁷. Al is considered a neurotoxic compound once is related to cholinergic damage in animals ¹⁵. In animals, Al interacts with AChE reducing its activity and causing neurotoxicity^48,49. AChE is responsible for the breakdown of the synaptic ACh in the synaptic cleft. Reductions of this enzyme are responsible for longer allowance of the neurotransmitter ACh agonizing the cholinergic receptors¹⁵. This increased period of receptor agonization is responsible for a hyperexcitation of the cholinergic nervous system leading to individual changes and finally death. In our study, Al exposure reduced AChE accordingly to findings in the literature but just the THC-rich oil could reestablish the normal AChE activity.

In the central nervous system, several neurotransmitters seem to be interacting among different nervous systems. Both tested major compounds present in the oils are important neurotransmitters of the endocannabinoid system (ES). The THC and CBD are linked to agonizing the receptors CB1 and CB2 from the ES. The distribution and function of each one differ while the receptor CB1 is found predominantly in the central nervous system, as well as in the lungs, liver, and kidneys, the CB2 receptor is expressed mainly in the immune system and in hematopoietic cells⁵⁰. Cannabinoids are synthesized on demand in the body or acquired exogenously. Neurotransmitters will be released upon activation by a process called retrograde neurotransmission. In this process, membrane receptors signal calcium channels, which cause cannabinoids to bind to receptors, generating a cascade of reactions, such as inhibition of adenylate cyclase, decrease in cyclic AMP levels, and opening of potassium channels, which will decrease the transmission of signals that will generate an inhibition of the release of neurotransmitters, such as GABA and glutamate⁵¹.

Overall, AChE reduced activity seems to be linked just with social preference alterations. It suggests that the observed change in social preference is reestablished by AChE modulation. As it was mentioned, the cholinergic nervous system is responsible for learning, cognition, and memory, for fish recognize themselves and the conspecific seems to be linked with cognition. In this way, the modulation of AChE activity is linked with the reestablishment of functional and favorable conditions for cognition. Regarding the anxiety-like observed profile, which was not reconstructed after oils administration it seems to be linked with another pathway of interaction. Generalized anxiety disorder is linked to a disbalance among the main neurotransmitters dopamine, serotonin, and glutamate³⁹. The interaction between the endocannabinoid system and other neurotransmitters is still not well understood, but here we observe that even though cannabis oils can improve the cognition of fish it does not seem to improve a clear profile for reducing the anxiety induced by neurotoxicity.

At least, another important biomarker that is considered the basis of many diseases is the antioxidant system. The antioxidant system is composed of two groups with antioxidant proprieties called non-enzymatic antioxidant systems, composed of compounds with antioxidant activities e.g., carotenoids, flavonoids, phenols, anthocyanins, polyphenols, and others⁵². The second group is composed of enzymes with antioxidant activity such as superoxide dismutase (SOD), catalase (CTL), and glutathione-S-transferase (GST). These enzymes work to reduce toxic compounds to less toxic or non-toxic compounds seeking to reduce the deleterious effect of free radicals on the cell⁵³.

We show that the exposure to Al alongside the THC- and CBD-rich oils increased the activity of SOD and that this increase was enough to avoid cellular damage once the CTL in these same groups was not increased. SOD and CTL work in a tandem manner, while SOD reduces the superoxide anion (O₂^-) to hydrogen peroxide (H₂O₂), the enzyme CTL uses the H₂O₂ produced by SOD to reduce until water and oxygen⁵⁴. Other ways of H₂O₂ are presented in the animal body and can actuate right before or after CTL such as glutathione peroxidase (GPx)⁵⁵. Here, the increased SOD and the non-increase in CTL represent that the produced amount of H₂O₂ produced was transformed by another pathway and finally indicates that probably the oils were able to avoid cellular damage. Differently from this, in the group treated with Al was no observed increase in SOD activity, but the enzyme CTL was largely increased. The activation of the CTL indicates that Al continuously produced H₂O₂ which can be dangerous for cells. The non-activation of SOD in the group treated just with Al might be linked with metal inhibition of the SOD. This enzyme has a metallic center that interacts with other metals such as Al, iron, copper, and zinc³².

The enzyme GST is responsible for the detoxication of toxic compounds from the cells and the body. It plays an important role in adding glutathione groups to lipophilic compounds transforming them into hydrophilic facilitating the elimination from the body⁵⁶. In our study was observed that Al increased the GST activity highlighting the toxic pattern of Al on cells. When Al is applied with THC- and CBD-rich oils the GST activity is reduced characterizing a cellular protector effect of the oils.