Recently, plant-derived natural compounds or phytochemicals are gaining increasing importance (due to their wide availability, low toxicity, and safety) to analyze their protective effects against drug-induced toxicity. In the present study, experiments were performed to screen whether Apigenin has any potential preventive effects against the Sorafenib-induced toxicity. Sorafenib has been used to treat liver and renal cancer for years. Despite its excellent treatment efficacy, Sorafenib-induced toxicity is becoming a key issue limiting its use by oncologists. Frequent exposure to Sorafenib is associated with severe toxicities, including hepatotoxicity and diarrhea, which reduce patients' quality of life, leading to the discontinuation of the treatment. In fact, in 44% of Sorafenib-treated patients in Phase II trials of HCC, the treatment was discontinued due to severe toxicities such as hepatotoxicity and gastrointestinal adverse events (Llovet et al. 2008). Therefore, the use of adjuvants that might work synergistically to reduce toxicity and increase treatment efficacy is highly recommended. To the best of our knowledge, this is the first study investigating the protective effects of a dietary flavonoid Apigenin using both in vitro and in vivo conditions against the Sorafenib-induced toxic effects.
Absorption studies showed a hyperchromic effect associated with non-covalent interactions, implicating an electrostatic mode of binding between compounds (Apigenin, Sorafenib, and a combination of Apigenin with Sorafenib) and ct-DNA. The electrostatic binding mode was further substantiated by fluorescence studies, which showed an enhanced fluorescence-emission intensity indicating the strong interaction of the compound firmly with DNA. Further, quenching experiments with E.B. displayed a decrease in emission intensity of the DNA-EB, which also supports the electrostatic interaction between compounds and ct-DNA.
The liver weight, body weight, and liver-to-body weight ratio in the control and Apigenin treated group showed no significant differences suggesting that Apigenin does not affect the animal's health. The genotoxicity assays revealed that Sorafenib had a statistically significant mutagenic effect on the chromosomes, indicating an increase in the mean number of chromosomal aberrations and micronuclei. However, the combination treatment with Apigenin and Sorafenib showed a preventive and protective effect of Apigenin as indicated by a decrease in the mean number of chromosomal aberrations and micronuclei, thus confirming the anti-genotoxic/protective effects of Apigenin. Though Apigenin alone showed insignificant mild genotoxic effects, it demonstrated improved therapeutic/anti-genotoxic effect when given in combination with Sorafenib, possibly targeting different pathways (Das et al. 2013; Singh et al. 2019). 8-OHdG is one of the common adducts formed due to oxidative DNA damage and is a reliable marker for DNA damage (Chao et al. 2021). High serum levels of 8-OHdG are associated with mutagenic effects, and mutations associated with 8-OHdG also reflect the total ROS-mediated damage in DNA. The current study revealed a high level of serum 8-OHdG in mice treated with Sorafenib, which indicates test chemical-induced DNA damage, reduced when the animals were treated with a combination of both the chemicals.
Various studies and growing evidence report that elevated ROS levels help maintain the oncogenic phenotype of cancer cells via acting as the secondary messenger in the intracellular signaling cascade and promote many aspects of carcinogenesis (Liou and Storz, 2014). Further, elevated ROS level is accompanied by the suppression of anti-oxidant enzymes, resulting in malignant transformation via different molecular targets (Bousquet et al. 2019). In addition, the diet rich in anti-oxidants scavenges different kinds of ROS, thereby preventing cancer development (Storz., 2005). In our study, the Sorafenib treated group also revealed maximum ROS intensity depicting the Sorafenib-induced toxicity mediated via the formation of ROS. Multiple experiments demonstrate that ROS-mediated toxicity appears to be alleviated by Apigenin via an anti-oxidant mechanism that protects against oxidative damage. NO is known to be cytotoxic and involved in cell-killing mediated by a necrotic process, either itself or via the formation of peroxynitrite with superoxide anion (Islam et al. 2015). Our results show that the NO concentration is higher in the Sorafenib-treated group. Interestingly, our data also showed the protective role of Apigenin against the Sorafenib-induced NO production as indicated by the reduction of NO concentration in the combination-treated group.
In the Sorafenib treated group, the weight of the liver and body along with the liver-to-body weight ratio was insignificantly/slightly lower when compared with control. This might be due to the toxic effects of Sorafenib on the liver, which were confirmed in the present study by the histopathological examinations and oxidative stress markers. In general, chemotherapy-induced liver toxicity is one of the most common adverse effects in cancer patients. As we know, Sorafenib is primarily metabolized in the liver and undergoes oxidative metabolism mediated by cytochrome P450 (CYP3A4) and glucuronidation through UDP-glucuronosyl transferase (UGT) 1A9 (Tao et al. 2020). The administration of Sorafenib activates CYP34A, which results in ROS production and subsequently generates oxidative stress and liver injury. Our study revealed an increased level of serum liver function markers, thus suggesting the hepatotoxic effects of Sorafenib. The enhanced marker levels might also be due to cell membrane leakage and loss of hepatocytes (Macgill, 2016).
Further, administration of Apigenin resulted in recovering the levels of these markers suggesting the possible hepatoprotective role of Apigenin which can prevent the liver cells from chemotherapy-induced injury and relieve the severity of liver damage. Further, the liver and kidney's histopathological analysis also revealed the protective effects of Apigenin against the Sorafenib-induced hepato-renal toxicity. These results agree with the study done by Wang et al. (2017), where Apigenin exerts its hepatoprotective effects against alcohol-induced liver injury and cisplatin-induced nephrotoxicity in mice (Hassan et al. 2017). In conclusion, our findings suggest the potential protective role of Apigenin against Sorafenib-induced genotoxic, oxidative, hepatotoxic, and renal toxic effects. With more evidence and extensive studies, it appears to have the potential to be developed as a dietary supplement or as an adjuvant with chemotherapeutic agents.