The use of synthetic macromolecules for biological and medicinal applications has always been a popular approach (Yin et al., 2021). Accordingly, several studies have demonstrated the non-toxic and biocompatible nature of synthetic cucurbiturils (CBs) (Uzunova et al., 2010; Zhang et al., 2018). CBs are molecular containers comprising two hydrophilic carbonyl-lined portals with a central hydrophobic cavity. They have been synthesized in different sizes (Figure 1 for cucurbit[7]uril [CB7]) (Lee et al., 2003). Because of these properties, the bioactivity of the CB7 host–guest complexes (Cheng et al., 2018) and their clinical applications in vitro and in vivo have been confirmed by several studies. For example, a previous study revealed that CB significantly decreased the hepatotoxicity caused by nitidine chloride (Li W. et al., 2017) as well as trazodone and its metabolite m-chlorophenylpiperazine (Huang et al., 2018a). Further, the antidotal activity of CB7 against paraquat, a toxic pesticide, was observed both in vitro and in vivo (Zhang et al., 2019a,b). Another study reported that a high affinity between CB7 and sorafenib, an anticancer drug (Yang et al., 2017), and bedaquiline (Kuok et al., 2018) and clofazimine (Li et al., 2016), antituberculosis drugs, enabled the host to decrease the cardiotoxicity of these drugs without affecting their activity in vitro and in vivo in zebrafish models (Yang et al., 2017). In the same year, it was reported that CB reduced the neurotoxicity of pentylenetetrazol in zebrafish and mouse models (Huang et al., 2018b). Additionally, acyclic CB derivatives were shown to have antidotal activity against methamphetamine in rats (Ganapati et al., 2017).

Although several macrocyclic hosts have been used to reverse the effects of poisonous substances (Yin et al., 2021), CB7 as an antidote to the toxicity of cyanobacterial crude extract (CCE) has been validated in in vivo studies. Cyanobacteria produce a wide range of cyclic peptide hepatotoxins, such as microcystins and nodularin. These secondary metabolites of cyanobacteria can be detected both in freshwater and marine environments. Figure 1 presents the chemical structure of microcystin-LR (MC-LR), a predominant toxin in CCE, as an example (Dittmann and Wiegand, 2006). Cyanobacteria are responsible for wildlife fatalities and adverse health effects in humans in countries where drinking water supplies are contaminated with these microorganisms (Al-Jassabi, 2005). Alarmingly, some of these cyanobacterial toxins are powerful promoters of liver tumor and potent inhibitors of protein phosphatase 1 (PP1) and PP2A catalytic subunits (PP1c/PP2Ac) that are critical for cytoskeletal integrity (Goldberg et al., 1995). During cyanobacterial bloom lysis in aquatic ecosystems, a mixture of toxins and other cyanobacterial and bacterial components are released in the water, which affects various aquatic organisms.

In this study, we analyzed the effects of CCE on liver function in mice with and without CB7 co-administration. Previous studies reported that the maximum tolerated dosage of CB7 is 250 mg/kg in mice, which is at least three-fold less than its LD₅₀ (Uzunova et al., 2010). The LD₅₀ of CCE in this study was 35 mg/kg body weight intraperitoneally (i.p.). International guidelines permit the analysis of doses at least 20-fold less than the LD₅₀ of CB7 to evaluate its protective activity, which justifies the use of CB7 as an antidote agent at a dose of 35 mg/kg body weight.

Twenty-four hours after the i.p. injection, a significant increase in the liver mass was observed in group 3 (Figure 2 and Table 1) compared with group 1 (1.44 vs. 2.77 g; P < 0.05). This increase was attributable to massive intrahepatic hemorrhage and the pooling of blood in the liver (data not shown). CB7 alone (Table 2) did not significantly affect the liver mass (P > 0.05). However, co-administration of CB7 and CCE significantly nullified the increase in liver mass that was seen in group 3 (1.88 vs. 2.77 g; P < 0.05), which implies a protective effect of CB7 on CCE-induced liver enlargement.

The effects of CCE on various biochemical markers of hepatic function were also determined in the presence and absence of CB7 (Figure 3A and Table 1). Group 2 did not exhibit a significant increase in ALT activity; however, group 3 exhibited a robust increase in ALT activity compared with that in the control group 1 (1950 vs. 550 U/L; P < 0.05; Figure 3A and Table 1). This activity was significantly diminished by co-treatment with CB7 (1485 U/L; P < 0.05) (Figure 3A and Table 1). Similarly, GST activity in the livers was significantly lower in group 4 than in group 3 (10.34 vs. 12.22 U/L; p < 0.05) (Figure 3B and Table 1). CB7 alone did not significantly affect GST activity (Figure 3B and Table 2).

We then assessed LPO by measuring the number of TBA-reactive substances. Compared with the control group 1, a marked increase in LPO was observed in group 3 (0.067 vs. 2.152 μM; P < 0.001; Figure 4). However, group 4 showed reduced LPO compared with group 3 (1.877 vs. 2.152 μM; P < 0.05). Furthermore, LPO was not significantly affected in group 2 compared with the control (0.072 μM; P > 0.05).

Figure 5 and Table 1 present the spectrophotometric determination of PP1 activity for the four groups. The injection of CCE alone (group 3) significantly diminished PP1 activity. This decrease was partially but significantly reversed in group 4. Surprisingly, CB7 alone (group 2) also decreased PP1 activity, though not as markedly as CCE.

The safety and toxicity of CB7 in mice were further tested and validated. The results revealed the absence of any significant changes in various biochemical markers of hepatic function (Table 2).

The present study illustrated that CB7 can serve as a chemoprotectant and can effectively diminish CCE-induced liver damage in mice. This is the first report that demonstrated the protective role of CB7 against CCE-induced liver damage. Our results show that CCE induced liver enlargement resulting from massive intrahepatic hemorrhage, which is consistent with previous findings that MC-LR inhibits PP1, a serine/threonine phosphatase (Honkanen et al., 1990). Inhibition of PP1 is known to increase cytoskeletal protein phosphorylation (Ohta et al., 1992), thereby disrupting the liver cytoskeleton (Hooser et al., 1989, 1991). Our observation that CB7 alone reduced PP1 activity without affecting liver size suggests that the hyperphosphorylation of cytoskeletal proteins was not sufficiently robust to significantly disrupt the integrity of hepatic cells. However, the detailed mechanism of such interactions remains to be investigated, especially considering that the crude extract contains several other components. In fact, the low water solubility of the crude sample precludes a nuclear magnetic resonance measurement that is necessary to determine the site or mode of interactions. It is also notable that CB7 is not entirely soluble in organic solvents (Uzunova et al., 2010). Therefore, regardless of the chemical tool used, the binding patterns can only be studied by monitoring the interaction of CB7 containers with standard MC-LR (Harada et al., 1990).

ALT is a routinely used clinical indicator of hepatocellular injury. The present study illustrated that CB7 protects mice against CCE-induced liver damage by reducing ALT activity upon the coadministration of CB7 and CCE. Similar results were observed for GST, a phase II detoxification enzyme, which was also revealed to detoxify MC-LR (Pflugmacher et al., 1998). Taken together, these observations clearly indicate the protective role of CB7 against CCE-induced liver damage.

The pharmacokinetic profile of CB7 has already been studied both in Balb/c mice and in Sprague Dawley rats after i.p. or i.v. administration, respectively. The i.p. plasma clearance in mice fits a 2-compartment model with exponential decay profile, where clearances of t_1/2α = 17.0 min and t_1/2β = 270.7 min are noted. Likewise, in Sprague Dawley rats, i.v. administration appeared to follow a multiphasic clearance model. Bio-distribution of CB7 in mice following i.p. administration show detection in the spleen, liver and brain 10 min post-administration, while activity in the kidneys occurred in two phases, at around 20 and 180 min. Due to this significantly higher activity in the kidneys (compared to that in liver), it was suggested that CB7 is excreted from the kidneys unchanged. It is worth mentioning that low levels of CB7 accumulate in the spleen and liver compared with the plasma and kidneys, while the brain accumulates the least activity, likely indicating that CB7 in unlikely to be able to cross the blood brain barrier (Li F. et al., 2017).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Animal house unit of Yarmouk University.

NS created the idea and wrote the final draft. NS and SA-J conducted all experimental work. AE and WN evaluated the final draft and added more references. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.