Coenzyme Q10 supplementation inhibits elevated transaminases during MC-LR induced toxicity in mice

Algal blooms have emerged as a significant ecological concern on a global scale, leading to water pollution, eutrophication, and the discharge of various algal pollutants. These phenomena present risks to both human health and other organisms [1]. The existence of these bacteria has the potential to generate an assortment of toxins, including cyanotoxins, which pose a concern for public health and the environment. Through the food chain, cyanotoxins can accumulate in aquatic organisms and be transferred to higher trophic levels [2]. Microcystins (MCs) are the most common form of cell endotoxin among cyanobacterial toxins, displaying a broad spectrum of biological toxicities and having more than 100 isomers [3-5]. A well-known and very hazardous MC isomer, microcystin-LR (MC-LR), is chemically stable, difficult to break down, and may linger in natural water for months or even years [6, 7]. MC-LR has the potential to infiltrate the human body via various routes, including ingestion of aquatic products contaminated with MC-LR [8]. The liver is the main organ affected by MC-LR, causing significant hepatotoxicity. Additionally, MC-LR also demonstrates nephrotoxicity and carcinogenic activities [9]. As a result, MC-LR was categorized as an unconventional test substance in the updated Chinese Code of Hygienic Practice for Drinking Water, which was put into effect in 2001. This code set a safety limit of 1 ?g/L for the presence of MC-LR in drinking water [10, 11]. On the other hand, the concentration of MC-LR in natural water often surpasses 13 ?g/L [12].  At present, there are no efficient strategies to manage Microcystis blooms or to mitigate and address the risks associated with MCs. Hence, it is of paramount practical importance to carry out a comprehensive examination of the harmful processes of MC-LR in order to protect human and ecological well-being. Coenzyme Q10 (CoQ10) is an essential vitamin-like molecule that is fat-soluble and is found naturally in all cellular membranes [13]. Coenzyme Q10 is a kind of antioxidant that functions as a substance that may eliminate harmful free radicals [14]. Coenzyme Q10 also plays a role in the production of adenosine triphosphate in the mitochondrial respiratory chain, which is crucial for organs that need a lot of energy, such as skeletal muscle [15, 16]. CoQ10 is present in all tissues of the human body in both its reduced and oxidised forms. While the human body can synthesise it, the production of this substance declines as one ages. It is important to note that a lack of CoQ10 is strongly associated with several health issues such as diabetes, cardiovascular disease, and neurodegenerative disorders [17]. Aminotransferases, namely ALT and AST, are a class of enzymes that facilitate the conversion of amino acids and alpha-oxoacids by transferring amino groups (Figure 1) [18]. It has been proposed that these enzymes possess the most substantial clinical relevance. The intricate isoenzyme pattern of aspartate aminotransferase (AST), one of the most active enzymes in the cell, is tissue-specific and exists in both cytosolic and mitochondrial forms [19]. In addition to being present in liver cells, AST is also highly expressed in several non-hepatic tissues such as the heart, skeletal muscle, blood, kidney, pancreas, spleen, lung and erythrocytes [20]. The enzyme alanine aminotransferase (ALT) facilitates the transfer of amino groups in order to produce oxaloacetate, a hepatic metabolite [21]. The protein comprises 496 amino acids, the encoding of which is facilitated by a gene situated on chromosome 8's long arm. ALT is present in both cytosolic and mitochondrial forms. The liver has high levels of ALT, whereas the heart, muscle and kidney have comparatively lower levels [22]. This research aims to demonstrate the preventive and therapeutic benefits of coenzyme Q10 on MC-LR-induced damage in mice, specifically in relation to the liver, kidney and heart.
       
           
       

Chemicals

Coenzyme Q10 was purchased from Sanofi India Limited and MC-LR was obtained from Sigma-Aldrich Co., USA. ALT and AST kits were purchased from ERBA Diagnostics Pvt Ltd.

Experimental design

BALB/c strain mice of 12-14 weeks’ age weighing 25 to 30 g were used for the experiments. Mice were randomly divided into 3 groups (Normal, MC-LR and MC-LR+Q) with 3 mice in each group. The second group of mice (MC-LR) was treated with MC-LR (10 ?g/kg bw/day, ip) for 14 days and the third group (MC-LR+Q) was co-treated with MC-LR (10 ?g/kg bw/day, ip) and Coenzyme Q10 (10 mg/kg bw/day, im) for 14 days. Simultaneously normal control group mice were also treated with same volume of normal saline. This work was approved by Institutional Animal Ethics Committee (Approval No:379/CPCSEA/IAEC/202l/ll).

Preparation of homogenate

Following the conclusion of the treatment, mice were sacrificed from each group via cervical dislocation, and the intended organs were removed. The tissues were promptly cleaned using ice-cold normal saline and kept at -80°C until they were needed for biochemical analysis. The 10% tissue homogenate was produced by homogenizing it in ice-cold 20 mM Tris buffer (pH 7.4). The supernatant was collected by centrifugation at 20,000 x g for 40 minutes. The concentration of protein was ascertained by employing BSA as the standard [23].

Analysis of aspartate aminotransferase and alanine aminotransferase

In accordance with the International Federation of Clinical Chemistry (1976), the activity of aspartate aminotransferase (AST) was determined using a commercially available reagent from ERBA Diagnostics Mannheim, Germany [24, 25]. The tissue extract was appropriately diluted and then combined with the AST reagent, which consists of L-aspartate, malate dehydrogenase, 2-oxoglutarate, LDH, NADH, ethylenediaminetetraacetic acid, and Tris-buffer at pH 7.5. The mixture was well mixed in a cuvette and incubated at a temperature of 37 °C for a duration of 5 minutes. Using a UV spectrophotometer, the decrease in absorbance was measured at 340 nm against a blank at intervals of 60 seconds for 5 minutes. The activity of AST was represented as IU/L. In addition, the measurement of alanine aminotransferase (ALT) was conducted following the guidelines set by the International Federation of Clinical Chemistry in 1976 [24, 26]. The tissue extract, diluted to the appropriate concentration, was combined with the ALT reagent, which consists of lactate dehydrogenase, nicotinamide adenine dinucleotide (NADH), 2 oxoglutarate, L ?alanine, and Tris-buffer at pH 7.5. The mixture in the cuvette was well mixed and then incubated at a temperature of 37 °C for a duration of 5 minutes. Using a UV spectrophotometer, the absorbance decrease against blank was measured at 340 nm for 5 minutes at intervals of 60 seconds. ALT activity was represented as IU/L.

Statistical analysis

The experimental results were presented as mean ± standard deviation. The student's t test was performed to determine the degree of significance between the control and experimental groups. p-values < 0>

RESULTS

Coenzyme Q10 restored the declined level of AST and ALT in liver, kidney and heart of MC-LR mice

Transaminases are enzymes that participate in the breakdown of amino acids and facilitate the conversion of a pair of amino acids and a pair of ?-keto acids via catalytic processes. Aspartate aminotransferase (AST) serves as an interface between carbohydrate and protein metabolism. Aspartate aminotransferase (AST) is an enzyme present in several tissues of the body, with particularly high levels in liver, heart, and skeletal muscle cells. The level of AST increased considerably in the MC-LR group during toxicity induced by MC-LR, in comparison to the normal control mice (p<0>
       
           
       

Figure 2. Aspartate aminotransferase level in the (A) liver, (B) kidney and (C) heart of normal (N), MC-LR, MC-LR mice treated with coenzyme Q10 (MC-LR + Q). Values represent mean ± SD, where n = 3. ^###p < 0>^???p < 0>

Similarly, alanine aminotransferase (ALT) enzyme facilitates the transfer of amino groups from L-alanine to ?-ketoglutarate, resulting in the production of L-glutamate and pyruvate. ALT is widely distributed throughout the human body, being present in several organs such as the kidney, heart, skeletal muscle, brain, pancreas, spleen and lung. The level of ALT increased considerably in the MC-LR group during MC-LR toxicity, in comparison to the normal control mice (p<0>

DISCUSSION

Aminotransferases facilitate the transfer of nitrogen atoms between amino acids and their corresponding oxoacids, which play a role in protein metabolism and gluconeogenesis. The transamination process is the first step for both the breakdown of amino acids and the production of these compounds. An amino group is moved from an acceptor ?-keto acid to a donor amino acid. Reversible transamination reactions take place within hepatic cells and serve as clinical indicators of hepatocyte injury when the liver is exposed to pathological conditions [27]. Amino transferases are enzymes that are reliant on pyridoxal phosphate and are responsible for its catalytic activity in transamination processes. As a result of the ?-amino nitrogen that is produced from the breakdown of amino acids being transported by glutamate, glutamate is an essential source of nitrogen for both biosynthesis and excretion [28].
       
           
       

Figure 3. Alanine aminotransferase level in the (A) liver, (B) kidney and (C) heart of normal (N), MC-LR, MC-LR mice treated with coenzyme Q10 (MC-LR + Q). Values represent mean ± SD, where n = 3. ^###p < 0>^???p < 0> ^??p < 0>

A less common kind of oxidative deamination occurs when an amino acid is converted into its equivalent ?-keto acid. The assessment of liver damage is based on the measurement of hepatic transaminase enzyme levels. Enzymes such as glutamate pyruvate transaminase (GPT/ALT), glutamate oxaloacetate transaminase (GOT/AST), lactate dehydrogenase, arginase and ?-glutamyl transaminase discharge out of the cell following damage to liver parenchymal cells or membrane permeability. This leads to an increase in the number of enzymes in the blood compared to normal [25, 29]. Since aminotransaminases have long been used to indicate liver damage and it has also been shown that elevated aminotransferases (ALT and AST) are linked to systemic control of metabolic processes and human illnesses [30]. Coenzyme Q10 supplementation effectively improved liver function, as seen by the reduction in AST and ALT levels [31, 32]. Analysis of the heart and kidney tissue revealed an increase in the levels of ALT and AST enzymes. This elevation can be attributed to the AST/ALT ratio (Figure 4), which serves as a reliable biomarker for non-liver diseases such as cardiovascular disease, different types of cancer and type 2 diabetes mellitus (T2DM) [33]. Modulation of AST/ALT ratio may reflect a markedly impaired kidney and cardiac function, which may be associated with acute kidney injury (AKI) and coronary artery disease (CAD) [34-36]. As seen by our results Coenzyme Q10 quite effectively ameliorated the level of AST and ALT in the kidney and heart of mice.