In this study, we investigated the levels of SOD, CAT, GSH-PX and MDA in lung, liver, kidney, diaphragm, testis and stomach tissues of rats to determine the effects of TQ and/or DOX treatment.
Doxorubicin, an anthracycline antibiotic isolated in the 1960s from a pigment of Streptomyces Peacetius and now chemically synthesized, is a drug widely used in chemotherapy for the treatment of cancer. The main problem with the drugs used in chemotherapy is that they target tumor cells as well as other cells and cause the same damage not only to abnormal cells but also to normal cells. For this reason, chemotherapy is applied to patients who do not respond to other treatment methods and are not suitable for primary radiotherapy or surgery (Sausville et al. 2009).
It is stated that acute (nausea, vomiting, myelosuppression and arrhythmia) and chronic (liver, kidney damage) side effects occur after the use of DOX, which has been used in cancer treatment for a long time (Chen et al. 2007; Minotti et al. 2004; Quiles et al. 2006). Considering the oxidative metabolism and antioxidant defense mechanism of these damaged organs, it is seen that they are more susceptible to the damage caused by free radicals. Although low levels of oxidant species are necessary for the normal process, high levels are known to cause pathological conditions (Liou and Storz 2010; Pop-Busui et al. 2006). Antioxidant enzymes (such as SOD, CAT, and GSH-Px) form the antioxidant defense system of the cell. It is well known that antioxidants inhibit oxidative damage caused by free radicals (Harzallah et al. 2010). SOD is a superoxide radical scavenger that can convert superoxide radicals to hydrogen peroxide (H2O2). GSH-Px has an important role in the clearance of H2O2. CAT catalyzes the decomposition of H2O2 into molecular oxygen and water. Disruption of the balance between ROS and antioxidants causes oxidative stress. It has been shown that the production of free radicals and oxidative stress are closely related to the toxicity of DOX. ROS are produced mostly in the mitochondria. Enzymes that produce ROS in the mitochondria convert DOX to a semiquinone free radical via an electron reduction of the quinone moiety and this radical reacts readily with oxygen to form superoxide anions (O2−). Superoxide anions are converted to relatively low-toxic and stable H2O2 by SOD, and ROS are produced by the generation of toxic and highly reactive hydroxyl radicals (OH∙) via Fenton reaction. The generated ROS then reacts with surrounding mitochondrial biomolecules, mainly proteins, lipids and nucleic acids. It is known that DOX reacts with mitochondrial DNA (mtDNA) to form adducts that impair normal protein expression, mitochondrial function and lipid oxidation (Eder and Arriaga 2006). DOX leads to redox imbalances and hence oxidative stress by increasing the production of free radicals and significantly reducing the levels of endogenous antioxidants (Sangomla et al. 2018).
In recent years, natural products (herbal medicines) play an important role in the treatment of many diseases. Combined therapies are also prominent in cancer treatment due to the side effects that occur due to drugs. TQ is frequently used in combined treatments. Studies show that TQ combined with clinical drugs increases the therapeutic index of drugs used in cancer treatment and/or reduces their cytotoxicity on healthy tissues (Gali-Muhtasib et al. 2006; Goyal et al. 2017).
Thymoquinone, the most important bioactive component of Nigella sativa seed oil, is used as an anti-inflammatory (Wang et al. 2015), anti-tumor (Woo et al. 2012), and antioxidant agent (Ojha et al. 2015). TQ is a powerful anti-oxidant due to its scavenging activity against many ROS such as superoxide anion, hydroxyl radical and singlet molecular oxygen (Mansour et al. 2002; Nagi and Mansour 2000). By means of these properties, TQ antagonizes the negative effects of elevated ROS levels in various diseases. In addition, TQ has been shown to strongly inhibit iron-dependent microsomal lipid peroxidation. TQ has a high antioxidative potential due to the redox properties of the quinone structure of the molecule and its radical scavenging effect by easily accessing subcellular compartments. It is also known that TQ induces the expression and/or activity of antioxidant enzymes (Badary et al. 2003).
The liver is one of the important organs of the body, which regulates various processes (such as detoxification of toxic substances and drugs, protein and lipid metabolism, defense mechanism of the body) (Wang et al. 2005). The liver is very sensitive to oxidative stress and damage by free radicals. Scavenging free radicals and/or increasing antioxidant defense may result in the elimination of negative effects on liver tissue (Tsukamoto and Lu 2001). In our study, compared to the control group, liver tissue SOD, CAT and GSH-Px values of DOX-treated rats decreased and MDA values increased. The decrement in antioxidant enzyme levels and the increment in lipid peroxidation in the liver tissue are also in agreement with previous studies (Kuzu et al. 2019; Wali et al. 2020). Administration of DOX to rats increased lipid peroxidation, which was associated with the increased free radical formation in liver tissue. However, in accordance with the earlier study (Akin et al. 20221), when compared with the DOX-administered group, it was seen that antioxidant enzyme levels increased and MDA values decreased in the DT group. Our results determined that TQ both increased antioxidant enzyme levels and decreased lipid peroxidation by inducing antioxidant defense mechanism.
Our data indicated that DOX induced a significant decrease of antioxidant enzyme levels and increase of lipid peroxidation in the lung tissue. Our results in lung tissue are correlated by earlier studies (Srdjenovic et al. 2010; Wali et al. 2020). The possible reason for these findings can be associated with the sensitivity of the lung tissue to oxidative stress in which the antioxidant defense mechanism is weak and has distinct oxidative mechanisms (Meadors et al. 2006). On the other hand, decreased antioxidant enzyme levels in lung tissue of DOX-treated rats returned back to the control levels with administration of TQ (DT group). When the literature is reviewed, although there is no study in which TQ and DOX applied simultaneously in lung tissue, many studies have shown that TQ increases antioxidant enzyme levels and decreases lipid peroxidation in lung tissue in cases such as Benzo(a)pyrene-induced lung injury and titanium dioxide nanoparticles induced toxicity. In this study, we obtained consistent results with previous studies (Alzohairy et al. 2021; Hassanein and El-Amir 2017).
Kidney tissue MDA level measured in DOX-treated group (D group) was significantly higher than those measured in the control group and antioxidants were found to be decreased. In the DT group, it was observed that TQ application decreased MDA levels and increased antioxidant levels. Our kidney tissue results are consistent with previous studies (Badary et al. 2000; Elsherbiny and El-Sherbiny 2014; Zidan et al. 2018). One of the main pathophysiologic mechanisms in nephrotoxicity caused by DOX is the increase in oxidative stress (Lahoti et al. 2012). According to our results, the increase in oxidative stress caused a decrease in antioxidant enzyme activities. It was observed that antioxidant enzyme levels increased and lipid peroxidation decreased in the kidney tissue in the DT group given TQ, which is explained by the protective antioxidant effect of TQ. Since TQ increases the antioxidant enzyme levels and is also a strong free radical scavenger, it can protect the kidneys from damage by cleaning the free radicals formed by DOX.
Our data showed that while lipid peroxidation increased in the testicular tissue in the DOX (D) group, it decreased in the DOX + TQ (DT) group. On the other hand, antioxidant enzyme levels decreased in the D group and increased in the DT group. Similar results were obtained in a previous study (Öztürk et al. 2020). In the literature, it is thought that testicular toxicity is caused by lipid peroxidation, oxidative stress and cellular apoptosis, although there is little information about testicular toxicity caused by DOX and its mechanism of action is not known exactly (Trivedi et al. 2011). In our study, it was determined that lipid peroxidation increased and antioxidant enzyme levels decreased in the group given DOX. However, in the group treated with DOX and TQ (DT group), it was observed that lipid peroxidation decreased and antioxidant enzyme levels increased, consistent with previous study (Öztürk et al. 2020). Due to free radical scavenging and antioxidant enzyme-inducing properties of TQ, it is thought that testicular tissue damage caused by DOX can be prevented by TQ treatment.
In our study, in which we measured the lipid peroxidation and antioxidant levels of the diaphragm muscle, we observed that lipid peroxidation increased and antioxidant enzyme levels decreased in the DOX-administered group (group D) compared to the control group. However, in the DT group, we found that lipid peroxidation decreased and antioxidant enzyme levels increased, and these results were consistent with previous studies (Hosseinzadeh et al. 2012; Montalvo et al. 2020). DOX binds to the cardiolipin molecule in the IMM (inner mitochondrial membrane) following administration. It is stated that this binding causes the formation of superoxide anions and then the production of ROS (Gorini et al. 2019). Increased ROS production causes disruption of mitochondrial structure, apoptosis and decrease in muscle functions (Min et al. 2015; Morton et al. 2019). Elimination of mitochondrial ROS caused by DOX can prevent dysfunction of the muscles (Min et al. 2015). The mechanism for the effects of TQ on the diaphragm muscle is unclear. However, the decrease in lipid peroxidation and the increase in antioxidant enzyme levels due to TQ administration in the DT group can be explained by the radical scavenging and protective antioxidant effect of TQ.
Similar to other tissues in our study, it was determined that DOX in stomach tissue increased lipid peroxidation and decreased antioxidant enzyme levels. In the DT group, TQ administration decreased lipid peroxidation and increased antioxidant enzyme levels. To the best of our knowledge, our study is the only study which DOX and TQ were applied together in stomach tissue in the literature. However, study showed that lipid peroxidation levels increased by creating ischemia/reperfusion (I/R) damage and decreased with TQ treatment (Magdy et al. 2012). The same study determined that the decrease in antioxidant enzyme levels caused by I/R damage increased to control levels with TQ treatment. In this study, in which the antioxidant and free radical scavenging properties of TQ come into prominence, it was determined that TQ protected the gastric tissue from lipid peroxidation and reduction of antioxidants caused by DOX. TQ non-enzymatically reacts with GSH, NADH and NADPH to form dihydro-thymoquinone, which is a more powerful free radical scavenger. This metabolite prevents lipid peroxidation by removing other free radicals, especially superoxide anions, and protects antioxidant defense molecules (Khalife and Lupidi 2007).
In our study, it was determined that lipid peroxidation increased and antioxidant enzyme levels decreased in the DOX group, whereas lipid peroxidation decreased and antioxidant enzyme levels increased in the group given TQ together with DOX. As we know as a result of our literature review, our study is the only study showing lipid peroxidation and antioxidant enzyme levels in liver, lung, kidney, testis, diaphragm muscle and stomach tissues. Since acute and chronic toxic effects occur due to DOX administration in cancer patient, we believe that TQ treatment can alleviate the toxic effects of this chemotherapeutic caused by lipid peroxidation. In addition, it is necessary to determine the most effective dose of TQ in terms of lipid peroxidation and antioxidant enzyme levels by applying different doses of DOX and/or TQ for each tissue. Besides, since the mechanism related to the effects of DOX and/or TQ treatment on lipid peroxidation and antioxidant enzyme levels is not clear in the literature, more comprehensive studies on the mechanism should be planned for further studies.